Poster presentation

Transcription

Poster presentation

The 4th Symposium of the Polish Society for
Biology of Reproduction
and
Joint Polish-Japanese Seminar
September 22-24, 2005
Book of Abstracts
Krakow, Poland
ORGANIZERS
University of Agriculture, Krakow
Jagiellonian University, Krakow
National Research Institute of Animal Production, Balice
Institute of Animal Reproduction and Food Research of the Polish
Academy of Sciences in Olsztyn
The Symposium of the Polish Society for Biology of Reproduction
and the Joint Polish-Japanese Seminar were held under the auspices
of the Polish Academy of Sciences
and the Japanese Society for Promotion of Science
Andrzej Sechman
(Computer composition – Department of Animal Physiology,
University of Agriculture, Krakow, Poland)
Drukowano w Zespole Wydawnictw I Poligrafii IZ, 2005
The 4th Symposium of the Polish Society for
Biology of Reproduction
and
Joint Polish-Japanese Seminar
„Cutting-edge Reproductive Physiology - Regulation of Ovarian
Function"
under the agreement on scientific cooperation
between the Japanese Society for Promotion of Sciences
and the Polish Academy of Sciences
22-24 September, 2005
BOOK OF ABSTRACTS
Krakow, Poland
The contents of the abstracts are entirely
the responsibility of the contributors.
Contents
Plenary Sessions ……………………………………..
Epler Piotr. The new trends in fish reproductive physiology…………………………………
Koyama Koji, Akiko Hasegawa. Follicular dysfunction induced by autoimmunity to zona
pellucida…………………………………………………………..……………
Kurpisz Maciej. Immune regulation of male reproductive function…………………………
Okuda Kiyoshi: Regulatory mechanism of luteolysis in cattle – possible roles of tumor necrosis
factor α……………………………………………………………………………
Paczoska-Eliasiewicz Helena. Leptin and avian ovarian function……………………….
Sato Eimei. Regulation of follicular development and atresia in the porcine ovaries by VEGF and
GDF-9 gene fragments…………………………………………………………………………
Zięcik Adam. Inhibition of luteolysis and embryo-uterine interactions during maternal recognition of
pregnancy in the pig……………………………………………………………
Thematic Sessions ……………………………………..
Session I - Follicular growth, function and atresia …………….
Oral Presentation……………………………………..……………
Kamiński Tadeusz. Opioids as local regulators of follicular steroidogenesis in the pig
Kaneko Hiroyuki. Growth of antral follicles in normal and abnormal reproductive conditions
of cattle
Manabe Noboru, Fuko Matsuda-Minehata, Yasufumi Goto, Toshikatsu Matsui, Akihisa
Maeda, Yuan Cheng, Junyou Li. Follicle atresia and its regulation: granulosa cell,
a type II apoptotic cell, regulates follicular atresia in pig ovaries
Miyano Takashi. Artificial oocyte growth in domestic species
Słomczyńska Maria. Porcine ovary and its steroid hormone receptors
Poster Presentation…………………………………..
Błaszczyk Barbara, Tomasz Stankiewicz, Jan Udała, Dariusz Gączarzewicz, Bogdan Lasota,
Beata Seremak. Transrectal ultrasonography and cervico-vaginal mucous electrical
resistance measurements as an aid for the diagnosis of the ovarian follicular cysts in
goats
Brodowska Agnieszka, Maria Laszczyńska, Andrzej Starczewski. Apoptosis in ovarian cells
in peri- and postmenopausal women.
Chrostowska Małgorzata, Wojciech Barański, Włodzimierz Markiewicz, Tomasz Maślanka,
Jerzy Jaroszewski, Tomasz Janowski. Influence of meloxicam on the ovulation in
heifers.
Galas Jerzy, Maria Słomczyńska, Maria Szołtys. Expression of androgen receptor and
3β-hydroxysteroid dehydrogenase in the ovaries of neonatal rats.
Kątska-KsiąŜkiewicz Lucyna, Hannelore Alm. Early antral bovine follicles after growth
culture – oocyte survival, maturation and fertilization.
Kimura Naoko, Mari Suda, Yumi Hoshino, Eimei Sato, Kiyoshi Totsukawa. Cellular features
of oocytes associated with reproductive aging in mice.
Maeda Akihisa, Yasufumi Goto, Fuko Matsuda-Minehata, Yuan Cheng, Takafumi Sai and
Noboru Manabe. Cellular flice-like inhibitory protein in granulosa cells was upregulated by interleukin-6 during follicular atresia.
Miyake Yuko, Hiromichi Matsumoto, Woro Anindito Sri Tunjung, Naoko Kimura, Hiroshi
Sasada and Eimei Sato. The changes of CD44 post-translational modification in atretic
follicles during follicular atresia in pig ovaries.
Moniruzzaman Mohammad, Takashi Miyano. Growth of primordial oocytes from adult and
newborn pigs in xenografts.
Słomczyńska Maria, Małgorzata Duda, Małgorzata Burek, Katarzyna Knapczyk, Zbigniew
Tabarowski, Jerzy Galas, Marek Koziorowski. The presence of 3β-HSD in the ovary
of the pregnant swine.
Staszkiewicz Jaroslaw, Tadeusz Kaminski, Gabriela Siawrys, Bartlomiej E. Krazinski,
Mariusz T. Skowronski, Jadwiga Przala, Stanislaw Okrasa. Expression of
proenkephalin gene in porcine theca interna and granulosa cells.
Session II - Central regulations of reproduction……………
Oral Presentation……………………………………
Kochman Kazimierz. Role of intracellular and external facrors in the ovulatory release
of GnRH
Martyńska Lidia, Ewa Wolińska – Witort, Magdalena Chmielowska, Jolanta Polkowska,
ElŜbieta Wasilewska – Dziubińska, Wojciech Bik, Bogusława Baranowska. Effects
of intraventricular infusion of orexin-A on GnRH-nerve terminals and plasma
gonadotrophins levels in immature female rats
Misztal Tomasz. Regulation of prolactin and LH secretion by melatonin in the sheep on the
level of the pituitary gland
Tomaszewska-Zaremba Dorota, Franciszek Przekop. The role of GABAA and GABAB
receptors in the regulation of GnRH release in ewes
Wasilewska – Dziubińska ElŜbieta, Magdalena Chmielowska, Ewa Wolińska – Witort, Lidia
Martyńska , Wojciech Bik, Bogusława Baranowska. The effects of β2 adrenergic
agonist – fenoterol and neuropeptides VIP and PACAP38 on progesterone release from
cultured rat ovarian granulosa cells
Zięba Dorota, Gary L. Williams. Effects of leptin on the hypothalamic-pituitary axis in
domestic animals.
Ciechanowska Magdalena, Magdalena Łapot, Tadeusz Malewski, Krystyna Mateusiak,
Franciszek Przekop. Effect of stress on the expression of GnRH gene and GnRH
receptor (GnRH-R) gene in the preoptic area-hypothalamus, and on the GnRH-R gene
in the stalk/median eminence and anterior pituitary gland in the ewes during the
follicular phase of the estrous cycle.
Ciechanowska Magdalena, Magdalena Łapot, Tadeusz Malewski, Franciszek Przekop.
Expression of GnRH gene and GnRH receptor (GnRH-R) gene in the preoptic area
hypothalamus and GnRH-R gene in the stalk/median eminence, and anterior pituitary
gland of anestrous ewes.
Górski Konrad, Tomasz Misztal, Katarzyna Romanowicz. Effect of intracerebroventricular
infusion of genistein or estradiol on FSH and GH secretion in ovariectomized ewes.
Kruczek Małgorzata. Mechanism of sexual selection in bank voles, Clethrionomys glareolus.
Łapot Magdalena, Magdalena Ciechanowska, Tadeusz Malewski, Krystyna Mateusiak,
Franciszek Przekop. Effect of stress on the expression of GnRH gene and GnRH
receptor (GnRH-R) gene in the preoptic area-hypothalamus, and of the GnRH-R gene
in the stalk/median eminence and the anterior pituitary gland of anestrous ewes.
Molik Edyta, Tomasz Misztal, Katarzyna Romanowicz, Edward Wierzchoś, Maciej Murawski
Kinga Szwiertnia. Effect of melatonin on LH secretion in prepubertal sheep.
Pierzchała-Koziec Krystyna. Modulation of hypothalamo-pituitary-gonadal axis by opioids
and glucocorticoids.
Pierzchała-Koziec Krystyna, Barbara Kępys, Joanna Zubel, Marcin Śmigielski. The role
of steroid hormones in the modulation of opioid peptides release from hypothalamopituitary axis in sheep.
Pierzchała-Koziec Krystyna, Joanna Zubel, Olaf Katarzyński, Janusz Rząsa, Andrzej DroŜdŜ.
The effect of prolonged progesterone treatment on the opioid-like peptides in the sheep
brain.
Smolińska Nina, Gabriela Siawrys, Tadeusz Kamiński, Jadwiga Przała, Alina Gajewska,
Kazimierz Kochman, Stanisław Okrasa. Long form leptin receptor in the hypothalamus
and pituitary of pregnant pigs.
Wańkowska Marta, Anna Wójcik-Gładysz, Jolanta Polkowska. Patterns of storage and
synthesis of gonadotrophic hormones in the pituitary cells during early adulthood
of female sheep……………………
Wąsowska Barbara, Jarosław Całka, Jolanta Chłopek, Stanisława Stefańczyk-Krzymowska.
Direct effect of boar pheromone 5α-andostenol on Gn-RH and oxytocin release from
porcine hypophysis………………………………………………
Zięba Dorota A., Beata Klocek, Izabela Derecka, Maciej Murawski, Katarzyna Romanowicz.
Seasonal and dose-dependent effects of leptin on LH secretion by ovine
adenohypophyseal cells………………………………………….
Session III - Regulation of corpus luteum function ……………
Acosta Tomas. In vivo studies on the vascular function in the bovine ovary: determination
of blood flow and hormonal secretion in the follicle and corpus luteum
Jaroszewski Jerzy. Nitric oxide as a regulator of the blood flow and secretory function of the
ovary
Kotwica Jan. Autoregulation of luteal function in cattle
Nishihara Masugi. Regulation of functional luteolysis in rodents.
Sugino Norihiro. Regulation of apoptosis in the human corpus luteum
Korzekwa Anna, Izabela Wocławek-Potocka, Jerzy J. Jaroszewski, Mamadou M. Bah,
Beata Barszczewska, Dariusz J. SkarŜyński. Effect of prostaglandin F2α on bovine
corpus luteum depends on cell composition and contact.
Pilawski Wojciech, Katarzyna M. Deptuła, Katarzyna K. Piotrowska, Anna Korzekwa,
Dariusz J. SkarŜyński. Analogues of prostaglandin (PG)F2α differently regulate the
function of bovine corpus luteum (CL).
Rękawiecki Robert, Ewa Liszewska, Jan Kotwica. Regulation of apoptosis in bovine luteal
cells.
Ryosuke Sakumoto, Kiyoshi Okuda. Possible roles of interleukin-4 and interleukin-6
in regulating luteolysis in pigs.
Torii Ryuzo, Hideaki Tsuchiya, Norio Okahara, Junko Narita, Takahiro Nakagawa, Tatsuyuki
Takada. Synchronization of ovarian cycle in cynomolgus (Macaca fuscicularis) and
Japanese (Macaca fuscata) monkeys.
Wąsowska Barbara, Jolanta Chłopek. The effect of steroid hormone infusion into the ovarian
artery on inhibin concentration in porcine follicular fluid.
Wąsowska Barbara, Stanisława Stefańczyk-Krzymowska, Jolanta Chłopek. Retrograde
transfer of testosterone to the porcine ovary in luteal and follicular phases of the estrous
cycle in vivo.………………………..
Session IV - Andrology I ……………
Ginter-Matuszewska Barbara, Kamila Kusz, Anna Spik, Katarzyna Matylla, Jadwiga
Jaruzelska. NANOS1 protein interacts with translation factors in human germ cells
Koyias Charles, Anna Spik, Jadwiga Jaruzelska. The role of OCT-4 transcription factor
in development of the human germ cells
Marchlewicz Mariola. Factors affecting male fertility.
Spik Anna, Sławomir Oczkowski, Maciej Kotecki, Piotr Formanowicz, Jacek BłaŜewicz,
Jadwiga Jaruzelska. SIAH1 is candidate mRNA regulated by NANOS1—PUMILIO2
protein complex in the human germ cells
Świder−Al−Amawi Małgorzata, Barbara Wiszniewska. Changes of surface of the epididymal
epithelial cells stimulated with LH/hCG in vitro
Kotula-Balak Małgorzata, Leszek Bablok, Stanisław Frącki, Barbara Bilińska. Androgen
receptors and aromatase in testes of patient with Klinefelter’s syndrome immunohistochemical study.
Kotula-Balak Małgorzata, Józefa Styrna, Jan K. Wolski, Barbara Bilińska. Morphological
analysis and immunoexpression of aromatase in the male gonads with impaired
spermatogenesis.
Spik Anna, Agata Olszak, Sławomir Oczkowski, Maciej Kotecki, Piotr Formanowicz, Jacek
BłaŜewicz, Jadwiga Jaruzelska. A search for mRNAs interacting with fertility protein
PUMILIO2 in the human germ line.
Session V - Nutrition and reproduction ……………
Gromadzka-Ostrowska Joanna. Dietary fat and androgens secretion and metabolism
Opałka Marek, Barbara Kamińska, Luiza Dusza. Effects of phytoestrogens on the
hypothalamus and pituitary gland in mammals
Wójcik- Gładysz Anna, Jolanta Polkowska. Neuropeptide Y – a neuromodulatory link
between nutrition and reproduction on the central nervous system levels
Zięba Dorota, Gary L. Williams. Regulatory roles of leptin in ruminant reproduction.
Jabłońska Olga, ElŜbieta Olczak and Joanna Gromadzka-Ostrowska. Dietary fat type and
different dietary vitamin e level effects on gonadal steroids and leptin concentration
in rats.
Korzekwa Anna, Izabela Woclawek-Potocka, Anna M. Rogozińska, Dariusz J. SkarŜyński.
Equol and para-ethyl-phenol modulate cytokines action on bovine corpus luteum (CL).
SkarŜyński Dariusz J., Anna Korzekwa, Izabela Woclawek-Potocka, Aleksandra Bober.
Intracellular mechanism of phytoestrogenes action on bovine corpus luteum.
Wolińska-Witort Ewa, Lidia Martyńska, Magdalena Chmielowska, ElŜbieta WasilewskaDziubińska, Wojciech Bik, Bogusława Baranowska. The effect of leptin administration
on the activity of the somatotrophic and thyreotrophic axis in peripubertal female rats.
Wójcik-Gładysz Anna, Marta Wańkowska, Tomasz Misztal, Jolanta Polkowska. The effect of
intracerebroventricular infusions of leptin on the immunoreactivity of NPY and GnRH
neurons in the hypothalamus of prepubertal sheep in conditions of acute undernutrition.
Session VI - Toxicology of reproduction ……………
Demska-Zakęś Krystyna, Jan Glogowski, Małgorzata Jankun, Zdzisław Zakęś. Effect of
4-nonylphenol on fish reproduction
Pawlak Andrzej L. Defects of vascular development related to decreased signaling of AhR
gene are rescued by gestational exposure to dioxin.
Petroff Brian K. Chronic exposure to the aryl hydrocarbon receptor agonist 2,3,7,8tetrachlorodibenzo-p-dioxin accelerates reproductive senescence in the female rat
Ptak Anna, Gabriele Ludewig, Hans-Joachim Lehmer, Ewa Ł. Gregoraszczuk. Effect of parent
compounds (PCB3) and its metabolites (4-OH-PCB3 and 3,4-diOH-PCB3) on
follicular cell function
Strauss Ewa, Ewa Florek, Andrzej L. Pawlak. The cigarette smoke exposure improves the
rearing capacity of the AhR bd C57BL congenic female mice
Augustowska Katarzyna, Ewa L Gregoraszczuk. A mixture of dioxins and furans acts on CYP
1A1 activity but does not induce apoptosis of placental human choriocarcinoma JEG-3
cells.
Kuthanová Zuzana, Jaroslav Petr, Radko Rajmon, Michal Ješeta, Eva Chmelíková, Markéta
Sedmíková. The influence of zearalenone on the meiotic maturation of pig oocytes.
Lee Hwi-Cheul, Keitaro Yamanouchi and Masugi Nishihara. Effects of perinatal exposure to
dbp, dinp and deha on hypothalamic gene expression and sexual behavior in rats.
Marchlewska-Koj Anna , Marta Labocha, Zbigniew Burgieł. Effect of a fungicide (Funaben T)
on reproduction of bank voles (Clethrionomys glareolus).
Młynarczuk Jarosław Janusz, Jan Kotwica. Effect of polychlorinated biphenyls (PCBs)
on oxytocin secretion from the ovarian cells in cow. Possible participation of
glucocorticoid receptors.
Nynca Anna, Olga Kraszewska, Maria Słomczyńska, Renata Ciereszko. Phytoestrogens action
in granulosa cells derived from small ovarian follicles in pigs: steroidogenesis,
proliferation and expression of estrogen receptors.
Nynca Anna, Magdalena Macewicz, Maria Słomczyńska, Barbara Kamińska, Renata
Ciereszko. Genistein action in granulosa cells derived from large ovarian follicles in
pigs: steroidogenesis, proliferation and expression of estrogen receptors.
Sechman Andrzej, Anna Hrabia, Janusz Rząsa. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
affects progesterone secretion by chicken preovulatory follicles.
Sechman Andrzej, Marcin W. Lis, Anna Hrabia, Jerzy Niedziółka, Janusz Rząsa. Effect of
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on estradiol level in chicken embryos.
Socha Magdalena, Mirosława Sokołowska-Mikołajczyk, Paweł Szczerbik, Piotr Epler. Action
of two different PCBs mixure (Delor 103 and Delor 106) on LH secretion by dispersed
pituitary cells incubated in vitro.
Szczerbik Paweł, Tomasz Mikołajczyk, Mirosława Sokołowska-Mikołajczyk, Magdalena
Socha, Jarosław Chyb, Piotr Epler. Influence of some environmental stressors on
common carp (Cyprinus carpio) pituitary cell functions.
Wójtowicz Anna, Anna Stankiewicz , Ewa Ł. Gregoraszczuk. Effect of 2,2’,4,4’,5,5’hexachlorobiphenyl (PCB 153) on the FSH-stimulated estradiol secretion and
LH-stimulated progesterone secretion by pig ovarian follicular cells.
Wróbel Michał, Krzysztof Kamiński, Jan Kotwica. Influence of polychlorinated biphenyls
(PCBs) on the contraction of bovine uterus in vitro.
Session VII - Andrology II ……………
Gancarczyk Monika, Magdalena Kuklińska, Jerzy StrzeŜek, Barbara Bilińska. Relationship
between aromatase immunoexpression and antioxidant ability in LH, IGF-I and PRL treated bank voles
Kuklińska Magdalena, Monika Gorazd, Agata Nowacka, Kazimierz Kosiniak-Kamysz, Jerzy
StrzeŜek. Antioxidant system of boar and stallion seminal plasma – comparative
studies.
Marchlewicz Mariola, Barbara Wiszniewska. Antioxidative enzymes protecting the
epididymal spermatozoa
StrzeŜek Jerzy, Paweł Wysocki, Władysław Kordan, Magdalena Kuklińska, Marzena
Mogielnicka, Daniel Soliwoda, Leyland Fraser. Proteomics of boar seminal plasma –
current studies and possibility of their application in biotechnology of animal
reproduction.
Bilińska Barbara, Amalia Carpino, Monika Gancarczyk, Anna Hejmej, Małgorzata KotulaBalak. The presence of functional enzyme, P450 aromatase, in rodent and human
testicular cells.
Ciereszko Andrzej, Halina Karol, Mariola Kotłowska, Grzegorz J. Dietrich, Mariola
Wojtczak. Optimization of comet assay for boar, bull, and turkey spermatozoa.
Dziekońska Anna, Magdalena Kuklińska, Anatoly S. Erokhin, Jerzy StrzeŜek. Effect of
synthetic antioxidant addition on biological properties of boar spermatozoa following
liquid storage at 5º and 16ºC.
Fraser Leyland, Jerzy StrzeŜek. Comparisons of DNA integrity of spermatozoa from whole
semen and rich fractions of boar ejaculate following freezing-thawing in different
extenders.
Fraser Leyland, Jerzy StrzeŜek. Dialysis of boar semen and its effect on post-thaw sperm
quality.
Gancarczyk Monika, Anna Hejmej, Barbara Bilińska. Morphological changes within
seminiferous tubules in immature bank voles treated either with 17β-estradiol or a pure
anti-estrogen ICI 182,780.
Kolasa Agnieszka, Mariola Marchlewicz, Lidia Wenda-RóŜewicka, Barbara Wiszniewska.
Immunoexpression of Cu/Zn − SOD in epididymal epithelial cells of rats with DHT
deficiency.
Kondracki Stanisław, Dorota Banaszewska, Anna Wysokińska, Joanna Chomicz. Estimation
of differences in sperm morphology of cattle and domestic pig males.
Kondracki Stanisław, Anna Wysokińska, Dorota Banaszewska, ElŜbieta Woźniak. Variability
of males spermiogram in domestic pigs in relation to different breeds and crossbreeding
variants.
Kordan Władysław, Jerzy StrzeŜek, Anna Kolendo. Effects of supplementation of platelet
activating factor (PAF) on selected motility characteristics and plasmalemma integrity
of boar spermatozoa following liquid storage.
Kosiniak-Kamysz Kazimierz, Monika Gorazd, Zenon Podstawski, Anna Bittmar. Comparative
analysis between alkaline phosphatase activity and other components in stallion semen.
Krzymowski Tadeusz, Stanisława Stefańczyk-Krzymowska, Przemysław Gilun, Michał
Radomski, Marek Koziorowski. A local pathway for increase of testosterone supply
from the testis to the epididymis, vas deferens and sex accessory glands of rat.
Kurowicka Beata, Genowefa Kotwica, Alina Gajewska, Anita Franczak, Agnieszka
Oponowicz. Effect of warm-rearing and warm-acclimation on reproduction in male
rats.
Laszczyńska Maria, Marian Wylot, Małgorzata. Piasecka. Proliferative activites in the
epithelial cells of the rat prostate lobes.
Lecewicz Marek, Jerzy StrzeŜek, Paweł Kaźmierczak. Properties of seminal plasma protein
complexes with lipoproteins extracted from bird egg yolk.
Mogielnicka Marzena, Jerzy StrzeŜek. Use of two-dimensional (2-D) electrophoresis to
identify heparin-, Zn2+- and phosphorylcholine (PCH)- binding proteins of boar seminal
plasma.
Słomczyńska Maria, Małgorzata Duda, Jerzy Galas, Monika Gancarczyk, Barbara Bilińska.
Expression of the StAR protein in ovarian and testicular cells of the pig and bank vole,
respectively.
StrzeŜek Rafał, Leyland Fraser, Jerzy StrzeŜek. Osmotic effects on motility and plasma
membrane integrity of spermatozoa from rich fractions of dog ejaculate.
Trybek Grzegorz, Agnieszka Kolasa, Mariola Marchlewicz, Barbara Wiszniewska. The
expression of androgen receptor in the epididymis of rats with dihydrotestosterone
(DHT) deficiency.
Wysocki Paweł, Jerzy StrzeŜek. Biochemical characteristics of epididymal molecular forms
of acid phosphatase isolated from boar seminal plasma.
Session VIII - Progress in treatment of infertility ……………
Głąbowski Wojciech. Importance of basic science for improvement of culture of human
oocytes and embryos in IVF programs.
Kurzawa Rafał. Immunological infertility – facts and myths
Piasecka Małgorzata. Diagnostic evaluation of sperm mitochondria
Starczewski Andrzej, Agnieszka Brodowska, Maria Laszczyńska. The effectiveness of
reconstructive oviduct surgery depending on the medical and physiological factors
Session IX - Immunology of reproduction ……………
Harada Kayoko, Hiroyuki Tanaka, Yoshiyuki Tsuji and Koji Koyama. Analysis of the
relationship between ureaplasmal infection and the outcome of pregnancy
Komori Shinji, Hiroyuki Kasumi, Kazuko Sakata, Koji Koyama. Analysis of androgenic effect
during spermatogenesis in testis
Rozwadowska Natalia, Dorota Fiszer, Piotr Jedrzejczak, Włodzimierz Kosicki, Maciej
Kurpisz. Quantitative analysis the interleukin 1 gene system in normal or impaired
human spermatogenesis and testicular tumors.
Sanocka Dorota, Maciej Kurpisz. A male factor: link between innate immunity and oxidative
stress
Chruściel Marcin, Aneta Andronowska, Katarzyna Jankowska, Teresa Doboszyńska.
Immunoreactivity of PDGF and PDGF receptors in the porcine paraovarian vascular
plexus in different stages of the oestrous cycle.
Deptuła Katarzyna, Dariusz SkarŜyński. The role of cytokines in regulation of secretory and
motor function of oviduct.
Dzerzhinsky Mykola, Andriy Pustovalov. Influence of immunisation, melatonin and serotonin
treatment, and pinealectomy on hypothalamic-gonadal axis in bird.
Jankowska Katarzyna, Aneta Andronowska, Teresa Doboszyńska, Hubert Niewęgłowski,
Anna Postek, Marcin Chruściel. ET-1 immunoreactivity in the endothelium of
subuterine precollector lymphatics in the ligamentum latum uteri in the cow.
Maj Tomasz, Alicja Halberstadt, Jarosław Pająk, Marian Stanisław Gabryś, Anna
Chełmońska-Soyta. Examination of CD25 antigen on decidual mononuclear cells
(DMC) in the cases of spontaneous abortions.
Piotrowska Katarzyna, Anna Korzekwa, Michiyo Tanikawa, Mamadou M. Bah, Tomas.
J. Acosta, Kiyoshi Okuda, Dariusz J. SkarŜyński. Regulation of prostaglandin (PG)
synthesis by interleukin-1 (IL-1) in bovine endometrium.
Session X - Biotechnology of reproduction ……………
Bochenek Michał, Zdzisław Smorąg, Tomasz Herjan. Sex regulation in mammalians by
separation of X and Y spermatozoa
Karasiewicz Jolanta, Jacek A. Modliński. Embryonic stem cells – the hope of biotechnology ?
Skrzyszowska Maria, Marcin Samiec. Somatic cell cloning in mammals – present possibilities
and limitations
Słomski Ryszard, Daniel Lipiński, Joanna Zeyland, Wojciech Juzwa, Andrzej Pławski, Robert
Kalak, Marlena Szalata, Karolina Wielgus, Jacek Jura, Maria Skrzyszowska, Zdzisław
Smorąg, Marek Pieńkowski. Expression of gene constructs in animal transgenesis.
Demianowicz Wiesław, Zygmunt GiŜejewski, Heriberto Rodriguez-Martinez, Radosław
Kowalski, Jan Glogowski. Cryopreservation of European bison (Bison bonasus)
epididymal spermatozoa: a preliminary results.
Gajda Barbara, Lucyna Kątska-KsiąŜkiewicz, BoŜenna Ryńska, Michał Bochenek, Zdzisław
Smorąg. Survival and cell cycle analysis of vitrified bovine somatic cells.
GiŜejewski Zygmunt, Jan Glogowski, Wojciech Bielecki, Wiesław Demianowicz.
Characteristics of the vestigial masculine uterus of the European bison /Bison bonasus
L./ - preliminary results.
Gogol Piotr, Mirosław Cegła. Iron induced luminescence of fresh, equilibrated and frozen ram
spermatozoa.
Grzmil Paweł, Agnieszka Dziuba, Paweł Chomiak, Józefa Styrna. Identification of a new gene
on the mouse Y chromosome.
Jura Jacek, Zdzisław Smorąg, Barbara Gajda, Ryszard Słomski, Daniel Lipiński, Robert
Kalak. Production of F1 generation of transgenic pigs suitable for xenotransplantation –
effectivenes of transgen transmission.
Jura Jacek, Ryszard Słomski, Daniel Lipiński, Barbara Gajda, Zdzisław Smorąg. Selection
of transgenic pig embryos for xenotransplantation project – the use of GFP gene
marker.
Kareta Wiesław, Kazimierz Korman, Mirosław Cegła. Ovulation rate and prolificacy in ewes
differing in age, birth type, and proportion of prolific breeds in genotype.
Kozioł Katarzyna, Przemysław Gilun, Bartosz Jagusztyn, Marek Koziorowski. Steroid
receptors in domestic pig and wild boar crossbreed (pig-boar) uterus during the luteal
phase in estrous cycle in different seasons of the year.
Koziorowski Marek, Przemysław Gilun, Bartosz Jagusztyn, Katarzyna Kozioł, Ewelina
Kowal. Seasonal changes in insulin-like growth factor-I (IGF-I) concentration in
peripheral blood in gilts of domestic pig and wild boar crossbreed (pig-boars).
Koziorowski Marek, Bartosz Jagusztyn, Przemysław Gilun, Katarzyna Kozioł. Seasonal
changes in thyroid hormones concentration in peripheral blood in gilts of domestic pig
and wild boar crossbreed.
Koziorowski Marek, Ewelina Kowal, Katarzyna Kozioł, Bartosz Jagusztyn, Przemysław
Gilun. Seasonal changes in total antioxidant activity (TAA) in blood plasma of
domestic pig/wild boar crossbreed (pig-boar) gilts.
Koziorowski Marek, Ewelina Kowal, Katarzyna Kozioł, Bartosz Jagusztyn, Przemysław
Gilun. Seasonal changes in Cu-Zn SOD concentration in red-blood cells in domestic
pig/wild boar crossbreeds (pig-boar) during estrous cycle.
Koziorowski Marek, Katarzyna Kozioł, Agnieszka Sajdak, Bartosz Jagusztyn, Przemysław
Gilun. Seasonal changes in leptin concentration during the estrous cycle in peripheral
blood plasma in gilts of domestic pig and wild boar crossbreed.
Koziorowski Marek, Stanisława Stefańczyk-Krzymowska, Przemysław Gilun, Anna TabęckaŁonczyńska, Katarzyna Kozioł, Bartosz Jagusztyn. Seasonal changes in steroid
hormone concentration during estrous cycle in peripheral blood in gilts of domestic pig
and wild boar crossbreed (pig-boar).
Panasiewicz Grzegorz, Adriana Cegiełka, Jacek Rutkowski, BoŜena Szafrańska. Biodiversity
of red deer populations examined by amplicon-length polymorphism of the pregnancyassociated glycoprotein genes.
Samiec Marcin, Maria Skrzyszowska. The live-DNA diagnostics of early apoptotic death in
porcine adult ear skin-derived fibroblasts selected to somatic cell nuclear transfer.
Szafrańska BoŜena, Grzegorz Panasiewicz, Karol Bociek, Beata Seremiak, Małgorzata Sulik.
Advantageous application of porcine pregnancy-associated glycoprotein gene family
(pPAG) for discovery of the PAG-like amplicons in the Chinchilla lanigera genome.
Szczęśniak-Fabiańczyk Barbara, Zdzisław Smorąg, Waldemar Kowalski. Effect of sodium
hyaluronic acid added to boar semen extender on the survival time and fertility.
Tombarkiewicz Barbara, Krzysztof Pawlak, Jerzy Niedziółka. Effect of geomagnetic
disturbances caused by metal elements on selected parameters of rats reproduction.
Warzych Ewelina, Karolina Błajecka, Martyna Łabusińska, Dorota Lechniak Apoptosis and
developmental competence of bovine embryos derived from oocytes matured in TCM
199 medium supplemented with FBS, fafBSA or PVP40.
Wierzchoś Agnieszka. The parthenogenetic development of rabbit oocytes as an effect of
electric field.
Zagórska-ŚwieŜy Katarzyna, Maria Nowogrodzka-Zagórska, Olga Szeleszczuk, Piotr
Niedbała, Janusz Bigaj, Adam Miodoński. Morphology and ultrastructure of raccoon
dogs (Nyctereutes procyonoides, Gray) spermatozoon.
Session XI - Cancers of reproductive organs……………
Milewicz Tomasz, Ewa Ł. Gregoraszczuk, Katarzyna Augustowska, Ewa Stępień, Janusz Ryś,
Józef Krzysiek. The sex steroid receptor phenotype of female breast cancer explants
influence the action of hGH and IGF-I on their proliferation and apoptosis
Milewicz Tomasz, Ewa Ł. Gregoraszczuk, Krystyna Sztefko, Katarzyna Augustowska, Józef
Krzysiek, Janusz Ryś. Lack of synergy between estrogen and progesterone on local
IGF-I, IGFBP-2 and IGFBP-3 secretion by both hormone dependent and hormone
independent breast cancer explants in vitro. Influence of tamoxifene and mifepristone
Pityński Kazimierz, Marcin Opławski, Krzysztof Okoń, Antoni Basta. Isolated tumor cells and
micrometastasis in sentinel lymph nodes in squamous uterine cervical cancer.
Pityński Kazimierz, Marcin Opławski, Krzysztof Okoń, Antoni Basta. The expression of
vascular growth factors A, C and D (VEGF - A, VEGF - C, VEGF - D) in squamous
uterine cervical cancer stage Ia2-Iia.
Pityński Kazimierz, Marcin Opławski, Krzysztof Okoń, Antoni Basta. The investigation over
lymphangiogenesis in squamous uterine cervical cancer.
Session XII - Reproduction of lower vertebrates ……………
Biegniewska Anna, Julian Świerczyński, Frans Ollevier, Edward F. Skorkowski. Spermatozoa
motility and adenylate energy charge of two teleost fish (Cyprinus carpio and Clarias
gariepinus).
Drąg-Kozak Ewa, Ewa Łuszczek-Trojnar, Włodzimierz Popek. Influence of melatonin on the
release of dopamine from hypothalamic aminergic nuclei in immature, maturing and
mature carp female (Cyprinus carpio L.).
Glogowski Jan, Grzegorz Dietrich, Wiesław Demianowicz, Radosław Kowalski, Roman
Kujawa, Adam Rzemieniecki, Mariola Wojtczak, Mariola Kotłowska, Eugeniusz
Bogdan, Andrzej Ciereszko. Fertilizing capacity of cryopreserved sperm of siberian
sturgeon and sterlet after short-time exposure of milt to mercury and cadmium
Sokołowska-Mikołajczyk Mirosława, Magdalena Socha, Piotr Epler. The effects of
testosterone on LH secretion in response to naltrexone (opioid receptor antagonist)
in common carp (Cyprinus carpio L.).
Wojtczak Mariola, Grzegorz J. Dietrich, Andrzej Ciereszko. Transferrin and antiproteases major proteins of common carp seminal plasma.
Poster Presentation………………………………….
Chyb Jarosław, Magdalena Socha, Mirosława Sokołowska-Mikołajczyk, Tomasz
Mikołajczyk, Piotr Epler. Differential effects of inhibin on LH secretion in male and
female common carp: an in vitro study.
Dietrich Grzegorz, Wiesław Demianowicz, Radosław Kowalski, Mariola Wojtczak, Stefan
Dobosz, Andrzej Ciereszko, Jan Glogowski. Fertilizing capacity of cryopreserved
sperm of rainbow trout after short-time exposure of milt to mercury and cadmium.
Jagusztyn Bartosz, Alicja Boroń, Dorota Juchno, Marek Koziorowski. Ovarian puberty
of the nase Chondrostoma nasus (L.) (Pisces, Cyprinidae) in the spring – autumn
season.
Kowalski Radosław, Wiesław Demianowicz, Beata Sarosiek, Stefan Dobosz, Jan Glogowski.
Sperm motility parameters of homo (XX, YY) and heterogametic (XY) rainbow trout
(Oncorhynchus mykiss Walbaum).
Sarosiek Beata, Radosław Kowalski, Jan Glogowski. The influence of heavy metals (mercury
and cadmium) on the activities of siberian sturgeon (Acipenser ruthenus) spermatozoa
enzymes.
Wojtczak Mariola, Jan Glogowski, Andrzej Ciereszko. Isolation and characterization of
α-1-antiproteinase from common carp seminal plasma.
Session XIII - Early pregnancy……………
Andronowska Aneta, Marcin Chruściel, Teresa Doboszyńska. The activity of nitric oxide
synthase in the porcine uterus during early pregnancy.
Chełmońska-Soyta Anna. Pleiotropic activity of IFN-TAU during peri-implantation period
of pregnency in ruminants.
Siawrys Gabriela, Tadeusz Kamiński, Nina Smolinska, Alina Gajewska, Stanisław Okrasa,
Kazimierz Kochman, Jadwiga Przała. Leptin and its role in implantation process.
Warzych Ewelina, Dorota Lechniak. Gene expression analysis as a tool for quality assessment
of bovine embryos.
Zygmunt Marek. Vasculo- und angiogenesis in the fetomaternal unit.
Poster Presentation………………………………….
Duras Magdalena, Jan Kotwica. Non-genomic effect of steroids on oxytocin-stimulated
intracellular mobilization of calcium and on prostaglandin F2α and E2 secretion from
bovine endometrial cells.
Franczak Anita, Izabela Wocławek-Potocka, Agnieszka Oponowicz, Beata Kurowicka,
Genowefa Kotwica. Effect of oxytocin on expression of enzymes of cyclooxygenase
pathway in porcine myometrial cells.
Kojs Marta, Marek Romek, Janusz Karasinski. Connexin43 gap junctions in prepubertal
porcine myometrium.
Oponowicz Agnieszka, Anita Franczak, Beata Kurowicka, Genowefa Kotwica. Oxytocin
receptor gene expression in porcine endometrium and myometrium during luteolysis
and early pregnancy.
Panasiewicz Grzegorz, Marta Majewska, Adriana Cegiełka, Aleksandra Romanowska, Jolanta
Dajnowiec, Janina Bukowska and BoŜena Szafrańska. Binding of semi-purified porcine
and bovine secretory chorionic ligands by gonadal and extragonadal gonadotrophin
receptors.
Skipor Janina, Andrzej Kowalik. Luteinizing hormone attenuates the vascular response to
norepinephrine.
Słomczyńska Maria, Małgorzata Duda, Małgorzata Burek, Jerzy Galas, Marek Koziorowski.
Immunolocalization of androgen receptor (AR) and cytochrome P450 aromatase in
porcine embryo tissues.
Stefańczyk-Krzymowska Stanisława, Jolanta Chłopek, Waldemar Grzegorzewski, Michał
Radomski. Local transfer of prostaglandin E2 into the ovary and oviduct and its
retograde transfer into the uterus in early pregnant sows.
Wacławik Agnieszka, Monika M. Kaczmarek, Agnieszka Blitek, Adam J. Zięcik. Expression
pattern of microsomal prostaglandin E2 synthase-1 in the porcine corpus luteum and
trophoblast.
Wocławek-Potocka Izabela, Anna Korzekwa, Dariusz J. Skarzyński. Precursors and enzymes
responsible for prostaglandin (PG) F2α synthesis in the bovine endometrium.
Session XIV - Avian reproduction ……………
Bednarczyk Marek, Paweł Łakota, Przemysław Czekalski, Mirosław Lisowski, Magdalena
Wawrzecka. Effect of egzo- and endogenous embryonic cells on selected reproductive
traits of birds
Hrabia Anna, Kiyoshi Shimada, Soichi Takagi, Janusz Rząsa. In vitro fertilisation in birds.
Olszańska BoŜenna, Rusłan Obłap, Paweł Majewski, Bogdan Lewczuk, Urszula Stępińska,
Anna Barańska, Krystyna Skwarło-Sońta. Melatonin, its synthesis and role during
early avian development.
Sechman Andrzej, Helena Paczoska-Eliasiewicz, Janusz Rząsa. Effect of thyroid hormones on
avian ovarian function.
Arhzaf Zine Laabidine. Avian biotechnology: maturation, ovulation and in vitro fertilization
of the Babcock300.
Błędniak Dorota, Maria Droba, Bogusław Droba..Acid glycosidases from hen oviduct.
Dzerzhynsky Mykola E., Igor M. Varenyuk, Nataliya V. Nuzhyna. Role of dopamine
receptors in realization of melatonin effects on hypothalamic-hypophyseal-gonadal system
of birds.
DŜugan Małgorzata. Distribution of acid glycosidases in the male genital tract of the pheasant.
Grzegorzewska Agnieszka, Tomasz Jacek, Marcin Lis, Helena E. Paczoska-Eliasiewicz.
Leptin as a regulator of chicken embryogenesis.
Gumułka Małgorzata. Sperm penetration of the perivitelline layer of the ovum after artificial
insemination of broiler breeder hens depending on age.
Jacek Tomasz, Helena Paczoska-Eliasiewicz. Leptin-induced thyroid apoptosis in immature
chickens.
Józefczyk Radosław, Maria Droba, Bogusław Droba, Andrzej Witkowski. Arylsulphatase during
post-natal development and involution of testes and epididymides from Japanese quail.
Kotłowska Mariola, Mariusz Olczak, Mariola Wojtczak, Jan Glogowski, Jan Jankowski,
Wiesław Wątorek, Andrzej Ciereszko. Isolation, characterization, and cDNA
sequencing of kazal-type proteinase inhibitor from seminal plasma of turkey (Meleagris
gallopavo).
Kugla–Owczarska Justyna, Helena Puchajda–Skowrońska, Marek Opałka, Luiza Dusza.
Semen quality evaluation in one-year-old Biłgoraj ganders.
Mróz Emilia, Aneta Orłowska, Katarzyna Michalak. Hatching value of turkey eggs stored for
longer periods of time.
Opałka Marek, Barbara Kamińska, Helena Puchajda-Skowrońska, Luiza Dusza. The influence
of phytoestrogens on testosterone secretion by Leydig cells in Biłgoraj ganders.
Pawlak Krzysztof, Barbara Tombarkiewicz, Jerzy Niedziółka. Comparison of cardiac work in
embryos of broiler chicken and dual purpose breed.
Plenary Session Abstracts
Krakow 2005
THE NEW TRENDS IN FISH REPRODUCTIVE PHYSIOLOGY
Piotr Epler
Department of Ichthyobiology and Fisheries,
ul. Spiczakowa 6, 30-199 Krakow, Poland
University
of
Agriculture,
1) Investigations of the functional diversity of GnRH molecules and receptors.
The research is focused on the presence of different forms of GnRH in fish brain and their
role in the control of reproduction and reproductive behaviour. So far 16 different GnRH
forms were found in fish brain. It has also become clear that each species possess two or three
different GnRH forms. More and more interesting is the role of extra-brain GnRH found in
gonads and in adrenal glands. The presence of three GnRH receptor types in the brain and
pituitary provides an interesting field of the research on the significance of these forms in the
regulation of fish reproduction.
2) Differential production (synthesis and secretion) of FSH and LH.
The problem of the duality of fish gonadotropins has already been established, however the
function and differential regulation of their secretion is not yet clear mainly because of the
lack of the specific methods of FSH level determination in other then salmonid species. The
recent investigations are carried out in order to reveal the role of (other than GnRH and
dopamine) factors that influence the synthesis and secretion of gonadotropins (gonadal
factors: sex steroids, inhibins, activins, follistatin) as well as the molecular mechanisms
involved in the control of FSH and LH synthesis and secretion. Another field of interest is
connected with the recent cloning of the gonadotropin receptors.
3). Research on sex determination in fish
Recent data show that estrogens play an important role in sex differentiation and that
a deficiency of estrogens is responsible for the testis development. The molecular mechanisms
of sex determination is investigated (experiments on the expression of sex determining genes
Sox9 and DMY).
4). Investigations on the control of fish spermato- and oogenesis.
The main interest is the determination of the molecular control of the sex steroid action on
these processes (characterisation and the expression of the specific genes coding for the
enzymes involved in the process of steroid biosynthesis in the different stages of
gametogenesis)
5). Biotechnology in the investigations of fish reproduction
The great progress in the field of molecular biology enables to conduct the sophisticated
experiments with the undifferentiated sex cells (primordial germ cells) as well as with the ripe
gametes. Germ cell transplantation from one to another species may serve as a tool for
transgenic animal production. There are also recent data on the production of biologically
active recombinant peptide hormones. In the case of fish gonadotropins such recombinant
hormones may serve as the factors inducing fish gonad maturation.
FOLLICULAR DYSFUNCTION INDUCED BY AUTOIMMUNITY TO ZONA
PELLUCIDA
Koji Koyama and Akiko Hasegawa
Department of Obstetrics and Gynecology, Hyogo College of Medicine; Laboratory of
Developmental Biology and Reproduction, Institute for Advanced Medical Sciences,
Hyogo College of Medicine, Japan
The zona pellucida, which is an extra-cellular matrix surrounding an oocyte consists of three
glycoproteins called ZP1, ZP2 and ZP3. zpA, zpB and zpC genes are coded for ZP1, ZP2 and
ZP3 respectively in various species. Currently, one more gene, zpB’ coded for ZPB protein
has been found in the human zona pellucida. The zona pellucida has been shown to possess a
strong immunogenicity thus inducing ovarian dysfunction. In our own findings, native zona
antigen caused oophoritis with follicle-like cell clusters in rabbits and hamsters. From a
clinical viewpoint, the ovarian histology of premature ovarian failure (POF) detected in some
infertile patients was quite similar to that of the immunized animals. However, it is still
unknown whether the anti-zona antibody detected in the patients’ sera is a real pathogen in
POF or not.
Initially, an experiment was conducted to identify the zona epitope sequence which causes
autoantibody production. A synthetic peptide comprising 18 amino acids of dog ZP2 was used
for immunization of dogs. The produced antibody recognized dog zona pellucida. Histological
examination showed severe ovarian damage including no normal follicles, impaired granulosa
cells and fibrosis of interstitial tissues. This result indicates that immunization with even
a small peptide can produce self-reactive antibodies and induce an ovarian impairment.
Secondly, as a clinical examination, we performed a dot-immunoassay with human zona
pellucida. Some POF patients’ sera showed higher positive reactions to the human zona
pellucida than the control group. This data strongly suggests that zona pellucida is
a pathogenic antigen in some POF patients.
Thirdly, to examine the effect of antibody on folliculogenesis, an in vitro experiment was
carried out using cultured mouse follicles. Growing oocyte-granulosa cell complexes (OGCs)
were isolated from mice and cultivated for 6 days in a medium containing antibody to zona
pellucida. Severe agglutination of granulosa cells and precipitates formation on the zona
pellucida were observed. This suggests that the antibody interferes with the normal growth of
granulosa cells, and subsequently follicle growth. This result also indicates that zona proteins
are secreted and dispersed into the inter-cellular spaces of oocyte and granulosa cells, because
zona proteins are synthesized only in the oocyte.
In addition, the importance of zona proteins was found for oocyte growth by RNA
interference. From these results we concluded that impairment of the zona pellucida by the
antibody possibly induce ovarian dysfunction such as POF in some infertile patients.
IMMUNE REGULATION OF MALE REPRODUCTIVE FUNCTION
Maciej Kurpisz
Institute of Human Genetics, Polish Academy of Sciience, 60-479 Poznan, Poland,
e-mail: [email protected]
Spermatogenesis appears as the particularly complex process of cell differentiation. Somatic
cells of human testis support the growth and differentiation of gametogenic cells ensuring the
most appropriate ones to reach the final stage, i.e. conversion into spermatozoa. Testis is
separated from the circulating immune elements through the blood-testis barrier. Next, active
immune tolerance is created through the action of Sertoli cells with immunosuppressive
abilities, Leydig cells paralyzing the cells of immune system, locally produced hormones, as
well as suppressive per se gametogenic cells. CD4CD25 cell subset actively regulates the
tolerance within immune cells. Inside of seminiferous epithelium a unique composition of the
a) histocompatibility antigens, b) cytokines, c) apoptotic factors, creates the semi-privileged
immunological zone. Testicular macrophages seems to be a major source of different
cytokines including both the pro-inflammatory ones (IL-1 beta and TNF alpha) which may
lead to apoptosis as well as anti- inflammatory ones (protective agents) as TGF beta family
members, IL-1 alpha - IL-18 complex or IL-10 of which presence within male gonad must be
yet unequivocally proven. FasL and TNF super family seem to be vigorously participating in
regulation of gametogenic cell number and quality, switching off the immune response from
the male gonad environment. There are reports suggesting the significant amounts of soluble
FasL form inducing apoptotic properties within this organ. Existing mouse strains with lossof-function mutations of Fas and FasL are characterized by uncontrolled accumulation of
lymphocytes which leads to autoimmune disease. There are, however, reports describing the
active role of spermatozoa themselves (in rodents) with active part of FasL on their surface.
This was not confirmed, however, in the human subjects. We have previously documented the
unique composition of the histocompatibility genes expression within seminiferous epithelium
assuring immunoprotective zone. This is composed of non-classical Class I antigens (genes),
namely HLA-E and –F presenting oligomorphism in adluminal compartment excluding the
cells from antigenic presentation. In turn, studied IL-1-IL-18 super family members suggest
compartmentalization of the most potent cytokines, IL-1 beta with the prevalent existence in
the interstitium and IL-1 alpha mostly located within seminiferous epithelium. IL-1 system is
a vital connection between proliferation and apoptosis including members of the both
pathways within its own system. IL-18 seems to be low expressed in physiological testis
while is increased during malignancy, perhaps guarding the possibility to combat this disease
through the other effector factors. A proportion between IL-1 alpha to IL-1 RA seems to be
crucial for the proper differentiation of gametogenic cells and testicular physiology. The
proportion between those factors is of vital prognostic value for subsequent pathology of
testis.
REGULATORY MECHANISMS OF LUTEOLYSIS IN CATTLE POSSIBLE
ROLES OF TUMOR NECROSIS FACTOR α
Kiyoshi Okuda
Laboratory of Reproductive Endocrinology, Graduate School of Natural Science,
Okayama University, 700-8530 Okayama, Japan,
E-mail: [email protected]
One of the most important phenomena regulating fertility and reproductive processes is
luteolysis. Regression of the corpus luteum (CL) is essential for normal cyclicity as it allows
the development of a new ovulatory follicle, whereas prevention of luteolysis is necessary to
establish and maintain pregnancy. Pulsatile release of prostaglandin F2α (PGF) from the uterus
in the late luteal phase and at the end of pregnancy induces regression of CL (luteolysis) in
many species including cattle. Recently, we suggest that tumor necrosis factor-α (TNF) plays
a role in the initiation of luteolysis via stimulation of uterine PGF in cattle, whereas TNF also
induced the production of luteotropic PGE2 (PGE) in bovine CL in vitro. To examine the
influence of TNF on the estrous cycle in cattle, Holstein heifers were treated on Day 14 of the
estrous cycle (Day 0=estrus) by infusion into the aorta abdominalis of saline (n=8, negative
control) or saline with different doses of TNF (0.1-50 µg per animal) with or without
indomethacin (INDO; 480 mg). One microgram of TNF increased PGFM and decreased
progesterone (P) levels in blood plasma, whereas higher doses of TNF (10, 25, 50 µg)
stimulated synthesis of P and PGE, and consequently prolonged the estrous cycle. Inhibition
of PG production by INDO blocked the effects of both the lower and higher doses of TNF on
CL function. These results suggest that low levels of TNF causes luteolysis, whereas high
levels of TNF activates CL function and prolongs the estrous cycle in cattle. Thus, depending
on concentration, TNF may play some crucial roles in both luteolysis and establishment of
pregnancy. To examine the regulatory mechanism of TNF during the establishment of
pregnancy, endometrial stromal cells were exposed to TNF (0.006-0.6 nM) and/or interferonτ (IFN) (0.03-30 ng/ml) in vitro. Although IFN did not alter basal PGF production, IFN
suppressed the stimulative TNF action in PGF production dose-dependently. These findings
suggest that one of the mechanisms of IFN action to ensure the establishment of pregnancy is
the inhibition of TNF action in PGF production in the uterus.
LEPTIN AND AVIAN OVARIAN FUNCTION
Helena Paczoska-Eliasiewicz
Department of Animal Physiology, University of Agriculture, Al. Mickiewicza 24/28,
30-059 Krakow, Poland, E-mail: [email protected]
Leptin, the protein hormone synthesized and secreted mainly by the adipose tissue has been
primarily found to control food intake and energy expenditure. It has been also widely
demonstrated that leptin is involved in the regulation of reproduction and mediates
undernutrition-induced deficits in reproductive functions. Exogenous leptin can rescue the
sterility of leptin-deficient ob/ob female mice and this action of leptin is independent from its
effect on weight loss since feed restriction alone fails to restore fertility. In rodents,
recombinant leptin can advance the onset of puberty or at least reverse the delay caused by
feed restriction while in women, leptin deficiency or resistance due to genetic defect in leptin
or leptin receptor prevents entering puberty. In contrast to mammals, the function of leptin in
avian reproduction has been hardly investigated and the only reports came from our and
French collaborating laboratory. Our studies were performed in chickens of layer strain and
addressed to examine the effect of recombinant leptin on: (i) the prepubertal development of
the ovary and the timing of sexual maturity, and (ii) the events occurring in the ovary of
mature laying hens subjected to fasting. Application of recombinant leptin to immature
chickens advanced the prepubertal rises of LH, estradiol and progesterone in blood plasma
and significantly accelerated the onset of puberty by attenuation of apoptosis in developing
follicles and enhancement of folliculogenesis. In mature laying hens, injections of leptin
during fasting affected ovarian steroidogenesis and attenuated atresia of yellow hierarchical
follicles by inhibition apoptosis which resulted in delayed cessation of egg laying. These
results indicate that leptin is involved in the regulation of prepubertal development of chicken
ovary as well as maintenance of its activity in mature birds. Furthermore, we have shown the
presence of leptin receptor mRNA and receptor protein at all sites of the hypothalamicpituitary-ovarian axis and in all compartments of the ovary. The effect of leptin on in vitro
steroids secretion by white nonhierarchical and yellow hierarchical follicles suggested that
leptin might act directly on the ovary. This suggestion has been recently substantiated by the
French group investigating the potential involvement of leptin and its receptor in ovarian
abnormalities observed in fast growing broiler breeder hens fed ad libitum and associated with
formation of excessive numbers of yellow follicles arranged in multiple hierarchies They
have demonstrated that ad libitum feeding dramatically up-regulated expression of leptin
receptor mRNA in the granulosa cells of four largest yellow hierarchical follicles, while feed
restriction reduced the overall level of expression and restored characteristic decrease in leptin
receptor gene expression observed during follicular maturation in slow growing broiler
breeder hens having no reproductive dysfunction. Most likely leptin controls establishment of
follicular hierarchy through the regulation of steroidogenesis.
REGULATION OF FOLLICULAR DEVELOPMENT AND ATRESIA IN THE
PORCINE OVARIES BY VEGF AND GDF-9 GENE FRAGMENTS
Eimei Sato
Laboratory of Animal Reproduction, Tohoku University, Sendai 981-8555, Japan,
Perifollicular angiogenesis is closely associated with ovarian follicular development and
atresia(1,2). To investigate whether additional induction of perifollicular angiogenesis would
support subsequent follicular development, we directly injected VEGF gene fragments into
the ovaries of miniature gilts at 7 days before eCG treatment. Injection of VEGF gene
fragments increased the level of mRNA expression of VEGF 120 and 164 isoforms in the
granulosa cells and VEGF protein contents in the follicular fluid. The Flt-1 mRNA expression
show a tendency toward increasing in the thecal tissue of antral follicles in the ovaries
injected with VEGF gene fragments. The number of preovulatory follicles and the capillary
density in the theca interna increased significantly in the ovaries injected with VEGF gene
fragments compared with those treated with eCG alone, indicating that the regulation of
perifollicular angiogenesis during follicular development is a very important factor in the
development of ovulatory follicles (3). GDF-9 is a growth factor secreted by oocytes in
growing ovarian follicles. We cloned porcine GDF-9 complementary DNA(cDNA), and then
injected its gene fragments into the ovary in gilts. The injection of porcine GDF-9 gene
fragments resulted in an increase in the number of primary, secondary and tertiary follicles,
concomitant with a decrease in the number of primordial follicles, indicating that exogenous
GDF-9 can promote early folliculogenesis in the porcine ovary (4). Our findings may offer an
innovative technique for enhanced induction of follicular development in the ovary through
gene and hormonal treatment, which may lead to prevention of infertlity caused by ovarian
dysfunction.
INHIBITION OF LUTEOLYSIS AND EMBRYO-UTERINE INTERACTIONS
DURING MATERNAL RECOGNITION OF PREGNANCY IN THE PIG
Adam J Ziecik
Institute of Animal Reproduction and Food Research of Polish Academy of Sciences,
Tuwima 10, 10-747 Olsztyn, Poland, E-mail: [email protected]
Inhibition of luteolysis and establishment of pregnancy in pigs results from oestrogen
secretion by conceptus and requires progesterone produced by corpus luteum. The integral
part of maternal recognition of pregnancy in the pig is redirection of prostaglandin (PG) F2α
secretion from endocrine to exocrine direction and an increase of PGE2 synthesis in
endometrium and conceptus. Estrogens and prostaglandins are critical components of
inhibition of luteolysis and maintenance of corpus luteum function during early pregnancy in
pigs. Expression of estrogen receptors in the uterus coincides with the secretion of estrogen
from the conceptus. Majority of proteins (growth factors, integrins, inhibitors, ect.) are
expressed simultaneously with estrogen secretion by conceptus (some of them are upregulated by estrogen) and take part in trophoblast elongation or attachment to the uterine
surface and in the process of implantation. Along with determination of prostaglandins E2
(PGES) and F2α (PGFS) downstream enzymes in endometrium and spherical/elongated
conceptuses it became evident of integrated role of uterine/conceptus PGES in inhibition of
luteolysis by changing PGE2:PGF2α ratio. New emerging concepts emphasize also the
autocrine and paracrine roles of luteal prostaglandins and estrogen in corpus luteum function.
The luteotrophic PGE2 action in pigs probably requires estrogen for inhibition of PGE2
9-oxoreductase in maternal and conceptus prostaglandins producing tissues. This can explain
negative results of luteal function maintenance in cyclic gilts by intrauterine infusions of
PGE2, since such treatment stimulated PGF2α release by the intact pig uterus.
The luteolytic or luteotrophic changes in the corpus luteum are synchronized with release of
the maternal pituitary and ovarian hormones. The presence of the uterine oxytocin and
luteinizing hormone receptors are important for luteolytic effect of PGF2α. The secretion of
estrogen from conceptus coincides with autocrine and paracrine dialogue between the
multiple conceptuses and uterine biological compounds and their receptors in trophoblast and
endometrium.
Thematic Session Abstracts
Krakow 2005
Session I
Follicular growth, function
and atresia
Oral presentation
OPIOIDS AS LOCAL REGULATORS OF FOLLICULAR
STEROIDOGENESIS IN THE PIG
Tadeusz Kamiński, Stanisław Okrasa, Gabriela Siawrys, Jarosław Staszkiewicz,
Mariusz Skowroński, Iwona Bogacka, Jadwiga Przała
Department of Animal Physiology, University of Warmia and Mazury in Olsztyn,
10-719 Olsztyn, Poland, E-mail: [email protected]
Endogenous opioid peptides (EOP) comprise of approx. 30 peptides deriving from one of
three precursor proteins: proopiomelanocortin (POMC), proenkephalin, and prodynorphin.
POMC is the origin of, among others, β-endorphin, proenkephalin − Met- and Leuenkephalin, prodynorphin − dynorphins A and B, α- and β-neoendorphin. EOP act through
three major classes of opioid receptors: β-endorphin preferentially through µ receptors,
derivatives of proenkephalin mainly through δ receptors, products of prodynorphin − κ
receptors. The effect of EOP on reproductive functions in different species, including pigs,
may be achieved indirectly − through their action on hypothalamic and pituitary levels or
directly − inside the ovarian structures, among others within the ovarian follicles. The latter
statement is based on finding of opioid receptors in the follicle, opioid local production and
their influence on follicular steroidogenesis. In pig ovarian follicles density of opioid
receptors decreases during follicular maturation. It was found that porcine follicular cells are
able to produce at least two opioids − β-endorphin and α-neoendorphin. Secretion of
β-endorphin by granulosa and theca interna cells is greatly stimulated by FSH and, to a lesser
extent, by LH, respectively. Ability to produce opioid peptides by pig follicular cells is also
strongly suggested by presence in these cells mRNAs for three major opioid precursors. The
gene expression is enhanced after stimulation of granulosa and theca cells with FSH or LH,
respectively. Short incubation of follicular cells with opioid peptides is followed by an
inhibition of steroidogenesis. The presence of LH in culture milieu generally changes the cells
response to opioids from inhibitory to stimulatory. Prolongation of exposure of the nonstimulated follicular cells to the opioids affects secretion of ovarian steroid hormones by the
cells − it causes either disappearance of opioid inhibitory effect or stimulation of steroid
output. It seems that the influence of opioids on porcine follicular steroidogenesis is achieved
through affecting at least two intracellular signal transduction pathways: adenylyl
cyclase/protein kinase A and phospholipase C/protein kinase C. Presented data suggest an
involvement of opioid peptides in the regulation of follicular functions.
GROWTH OF ANTRAL FOLLICLES IN NORMAL AND ABNORMAL
REPRODUCTIVE CONDITIONS OF CATTLE
Hiroyuki Kaneko
Genetic Diversity Department, National Institute of Agrobiological Sciences,
Tsukuba, Ibaraki 305-8602, Japan, E-mail: [email protected]
Growth and ovulation of antral follicles are regulated by the balance between effects of
gonadotropins from the pituitary and feedback effects of secretions from ovaries, such as
inhibin and steroid hormones. The development of specific immunoassay for inhibin A has
enabled us to determine circulating levels of inhibin A in various reproductive conditions.
This paper summarizes the relationship between profiles of inhibin A, FSH, and follicle
growth during the intact estrous cycle, and in cows with follicular cysts. During the bovine
estrous cycle, two or three waves of follicular development occur. Each wave is first
characterized by the development of a cohort of small follicles, then one follicle becomes
dominant and the remainder regresses. Circulating levels of inhibin A increased concomitant
with the emergence of each follicular wave. The highest levels of inhibin A were noted during
the growth phase of dominant follicles; however inhibin A levels declined as the dominant
follicles ceased to grow or ovulated. An inverse relationship between patterns of plasma
inhibin A and FSH existed during each follicular wave. Ovarian cysts in cattle are
characterized by the persistence of anovulatory dominant follicles in the absence of corpora
lutea, with interruption of normal estrous cycles. Ultrasonographic examination of the ovaries
clearly reveals that follicular waves with cysts are replaced with different waves by turnover
process, but the interwave interval is much longer than that in normal cows. Circulating levels
of inhibin A increased concomitantly with the appearance of follicular waves with cysts and
showed a negative correlation with FSH profile. The release of inhibin A from cystic follicles
was maintained for a longer period than that from normal dominant follicles. The above
results indicate that inhibin A released from dominant follicles determines the turnover of
follicular waves through regulation of FSH secretion in the normal and abnormal reproductive
conditions of cattle.
FOLLICLE ATRESIA AND ITS REGULATION: GRANULOSA CELL, A
TYPE II APOPTOTIC CELL, REGULATES FOLLICULAR ATRESIA IN PIG
OVARIES
Noboru Manabe, Fuko Matsuda-Minehata, Yasufumi Goto, Toshikatsu Matsui,
Akihisa Maeda, Yuan Cheng, Junyou Li
Research Unit for Animal Life Sciences, Animal Resource Science Center, The
University of Tokyo, 3145 Ago, Ibaraki-Iwama 319-0206, Japan,
Apoptosis in granulosa cells is initiated by death receptor(s) and plays crucial roles in
follicular atresia, but the intracellular regulating mechanism, especially the mitochondriondependent apoptosis signaling pathway, is still largely unknown. Firstly, we examined
whether the mitochondrial pathway is associated with granulosa cell apoptosis during atresia
in pig ovaries. The mRNAs and proteins of bit, caspase-9 and apoptotic protease-activating
factor-1 (Apaf1), which are major signal-transducing components in the mitochondriondependent pathway, were detected in granulosa cells prepared from healthy, early atretic and
progressed atretic follicles by RT-PCR and Western blot, respectively. No changes in the
expression levels of Apaf1 were seen during follicular atresia, but those of truncated bit (t-bit)
and activated caspase-9 increased during atresia. These findings indicate that the
mitochondrion-dependent signaling pathway, which is mediated by bit, Apaf1, caspase-9 and
cytochrome c, plays crucial roles in determining the fate of granulosa cells during atresia, and
we conclude that the porcine granulosa cell is a type II apoptotic cell, which has the
mitochondrion-dependent apoptosis signaling pathway. Secondary, we examined the changes
in the levels of cellular-Flice like inhibitory protein (cFLIP) expression in porcine granulosa
cells during atresia. cFLIP is the homologue of intracellular apoptosis inducer (procaspase8/Flice), and has two alternative splicing isoforms: cFLIP short form (cFLIPS) and long form
(cFLIPL). By competing with caspase-8, cFLIP inhibits apoptosis initiated by death receptors.
The changes in the levels of cFLIPS and cFLIPL mRNA and protein expression in granulosa
cells were determined by RT-PCR and Western blot, respectively. cFLIPL mRNA and protein
were highly expressed in granulosa cells of healthy follicles and decreased during atresia.
cFLIPS mRNA and protein levels in granulosa cells were extremely low and showed no
change among the stages of follicular development. Immunohistochemical and in situ
hybridization analyses demonstrated that the cFLIP mRNA and protein were highly expressed
in the granulosa cells of healthy follicles but weakly expressed in those of atretic follicles.
These data suggested that cFLIPL plays an anti-apoptotic role in the granulosa cells of healthy
follicles, and that cFLIP could be a major survival factor that determines whether growth or
atresia occurs in porcine follicles.
ARTIFICIAL OOCYTE GROWTH IN DOMESTIC SPECIES
Takashi Miyano
Laboratory of Reproductive Biology and Biotechnology, Faculty of Agriculture,
Kobe University, Kobe 657-8501, Japan, E-mail: [email protected]
Ovaries contain a huge number of growing and non-growing oocytes. Once non-growing
oocytes (pig and cow: 30 µm) in primordial follicles enter the growth phase, they grow
toward their final size (pig and cow: 125 µm) taking a long period of time. However, the
population of the oocytes that grow to the final size in the ovaries is quite small. Artificial
growing-up of small oocytes collected from ovaries could provide a new source of mature
eggs for livestock production and assisted reproduction in humans. Baby mice have been
produced by in vitro growth (IVG) culture of oocytes in primordial follicles (Eppig &
O’Brien, 1996). In large domestic species, baby calves have been produced from IVGcultured oocytes (Yamamoto et al., 1999; Hirao et al., 2004). However, the oocytes used in
the experiments were growing ones at the mid-growth phase (90-99 µm in diameter) from
early antral follicles of 0.5 mm in diameter. IVG systems have not been established for nongrowing oocytes of large domestic species. Xenotransplantation of small oocytes to SCID
(severe combined immune deficiency) mice is a substitute for an effective long-term IVG
culture. When bovine primordial follicles (40 µm in diameter, oocyte: 30 µm) and secondary
follicles (150-200 µm in diameter, oocyte: 55 µm) from slaughtered cows were transplanted
separately into SCID mice for 4-6 weeks, secondary follicles developed to the antral stage
with oocytes reaching their final size. Primordial follicles survived but did not grow in the
grafts (Senbon et al., 2003). Furthermore, primordial follicles from adult pigs did not develop
in xenografts after 2 months. On the other hand, primordial follicles from piglets developed to
the antral stage with oocytes reaching their final size. These findings show that secondary
follicles and their oocytes have already entered their growth phase and are able to develop to
the final stage in appropriate conditions, and that primordial follicles/oocytes especially from
adult animals require some additional factor(s) to enter the growth phase. IVG and
xenotransplantation systems enhance our knowledge about the oocyte growth as well as
follicular selection in mammalian ovaries.
PORCINE OVARY AND ITS STEROID HORMONE RECEPTORS
Maria Słomczyńska
Lab. Endocrinology and Tissue Culture, Dept. Animal Physiol., Inst Zoology,
Jagiellonian University, 30-060 Krakow, Poland, e-mail: [email protected]
The growth of ovarian follicles, ovulation and the formation of the corpus luteum are complex
processes that involve dramatic changes in the function of the follicle. The rapid switch from
the proliferative stage of granulosa cells of preovulatory follicles to the terminally
differentiated luteal cells could be observed. Cell cycle progression and proliferation are
controlled by positive and negative regulators as well as by hormones. Therefore, in order to
better understand the hormonal control of the ovary, the expression of steroid hormone
receptors (androgen receptor- AR, progesterone receptor-PR, estrogen receptor-ER) and some
steroidogenic enzymes (3β-hydroxysteroid dehydrogenase- 3βHSD and cytochrome
P450aromatase- P450arom) within porcine ovary (in estrous cycle and in pregnancy), was
analyzed using immunohistochemical technique. In the ovary of the cycling pig cyclic
changes of AR, ER, PR distribution have been demonstrated. AR was localized in the nuclei
of granulosa cells, in a few theca cells and in stroma. The intensity of staining decreased as
the follicle grew and matured.
Androgens are important regulators of the follicular function, and interact with various factors
to enhance granulosa cell differentiation. Androgens are products of progesterone
metabolism, intermediates in estrogen biosynthesis and local regulators of ovarian function.
ERβ was the main form of the estrogen receptor present in the porcine ovary. ERα appeared
just before ovulation in granulosa cells. PR was present in theca cells of preovulatory
follicles and gradually shifted to granulosa cells to achieve maximum intensity in the
preovulatory follicle. Both forms of PR (PR-A and PR-B) were investigated. Immunostaining
for 3βHSD was restricted to the theca interna cells whereas P450 aromatase immunostaining
intensity reached the highest level in the granulosa cells of the preovulatory follicles, shortly
before LH surge.
The ovary of pregnancy escapes regulation that was observed in the cycling animals. The
placenta overtakes production of many hormones. However, the ovaries still are necessary for
successful pregnancy. Therefore, the same study of steroid hormone receptors
immunolocalization was performed to specify the role hormones play in the ovary of
pregnancy. Obtained results in the ovary of pregnancy revealed different patterns of
immunohistochemical localization of steroid receptors and enzymes than those observed in
the ovaries of cycling swine.
Supported by the State Committee for Scientific Research as a Solicited Project PBZ-KBN084/PO6/2002.
Poster presentation
TRANSRECTAL ULTRASONOGRAPHY AND CERVICO-VAGINAL
MUCOUS ELECTRICAL RESISTANCE MEASUREMENTS AS AN AID FOR
THE DIAGNOSIS OF THE OVARIAN FOLLICULAR CYSTS IN GOATS
Barbara Błaszczyk, Tomasz Stankiewicz, Jan Udała, Dariusz Gączarzewicz, Bogdan
Lasota, Beata Seremak
Department of Animal Reproduction, Agricultural University of Szczecin, 71-466
Szczecin, Poland, E-mail: [email protected]
Ovarian cysts reduce reproductive efficiency and represent serious problems especially in
goats, because goats are seasonal breeders; those with ovarian cysts will be barren until the
following breeding season if left untreated.
Therefore, a study was conducted to evaluate the accuracy of utrasonographic evaluation of
ovarian follicular cysts in goats. Moreover, the aim of this study was to see if monitoring
estrous cyclicity by cervico-vaginal mucous electrical resistance measurements can be
a useful aid in the diagnosis of follicular cysts.
Ovarian changes determined by daily transrectal ultrasonic were scanned in 15 goats.
Furthermore, electrical resistance of their vaginal mucus was also measured daily. The study
was conducted during the mid-breeding season and included 4 interovulatory intervals for
each goat.
The electrical resistance in the vagina near the cervix uteri was significantly lower (P<0.01) in
the estrus stage compared with other stages of the estrous cycle. In all goats, a decline in
vaginal resistance (to <400 units) was closely associated with the onset of behavioral estrus.
The mean interestrous interval for the 13 goats was 21.16±2.95 days. Two goats showed
a longer interestrous interval, associated with absence of ovulation and development of
follicular cysts. In these two goats the periods with the lowest values of vaginal mucus
resistance were 24 and 26 days, and ranged between 260 and 400 units. In this time, ovarian
follicular cysts were detected by transrectal ultrasonography. The mean diameter of an
ovarian follicular cyst was 17.74±0.39 mm.
These results show that measurement of cervico-vaginal resistance could serve as an indicator
of the follicular development in goats. However, present of ovarian cysts should be as
confirmed by transrectal ultrasonography. Our results support the use of transrectal ultrasound
scanning as an aid for the diagnosis of the cystic ovarian disease in goats.
APOPTOSIS IN OVARIAN CELLS IN PERI- AND POSTMENOPAUSAL
WOMEN
Agnieszka Brodowska1, Maria Laszczyńska2, Andrzej Starczewski1
1
Clinic for Reproduction and Gynecology, 2Department of Histology and Embriology,
Pomeranian Medical University, Unii Lubelskiej 1, 71-252 Szczecin, Poland,
E-mail:[email protected]
During reproductive period apoptosis in ovaries of a woman is a physiological process,
however during peri- and postmenopausal period apoptosis is regarded as still not fully
investigated. In woman 5 years following menopause ovaries consist of fibrous tissue, blood
and lymphatic vessels, nerves and corpori albicans containing mainly fibroblasts,
macrophages and myofibroblasts.
The aim of study was to assess the apoptosis in ovaries of peri- and postmenopausal women.
Study was performed using ovaries of 15 women aged 45-69, operated for uterine myomas or
descent of the uterus.
In histopathological study these ovaries were assessed as a normal. Serum levels of folitropin,
luteinizing hormone, estradiol, testosterone and androstendione were evaluated for the
assessment of ovarian steroidogenesis. Hematoxylin (HE) and periodic acid Schiff (PAS)
methods were used for the assessment of morphology of ovaries. The TUNEL method
(ApopDETEK, and in situ detection system horseradish peroxidase-DAB, Enzo Diagnostic,
USA) was used for the histochemical detection of apoptotic cells in the ovary (in situ
localization of fragmented DNA). For immunohistochemical detection of caspase 3 in ovarian
cells DAKO EnVision System Alkaline Phosphatase (DAKO/AS, Denmark) was used. For
serum hormonal tests radioimmunoenzymatic methods (radioimmunoassay kits, Amersham,
UK) were applied.
In ovaries of perimenopausal women many corpori albicans, fibrous tissue (with a large
number of macrophages) and numerous blood vessels were found. In older women fewer
blood vessels, corpori albicans, fibrous tissue and macrophages were present.
In perimenopausal women the highest activity of apoptosis in the wall of blood vessels,
corpori albicans and fibrous tissue was noted. Most of the phagocitized macrophages were
found in the area of blood vessels.
In postmenopausal women apoptosis was weakly expressed. The single TUNEL positive cells
and the single cells with positive immunohistochemical reaction to caspase 3, localized
especially in the area of blood vessels and fibrous tissue were found.
INFLUENCE OF MELOXICAM ON THE OVULATION IN HEIFERS
Małgorzata Chrostowska1, Wojciech Barański2 Włodzimierz Markiewicz1, Tomasz
Maślanka1, Jerzy Jaroszewski1 Tomasz Janowski2
1
Department of Pathology and Pharmacology, 2Department of Clinical Science,
Faculty of Veterinary Medicine, University of Warmia and Mazury, 10-718 Olsztyn,
Poland; E-mail: [email protected]
Nonsteroidal anti-inflammatory drugs are one the most widely used of all therapeutic agents
in the human medicine and become more often used in veterinary medicine. These chemically
different drugs have anti-inflammatory, analgetic and antipyretic effects, which are
associated with inhibition of arachidonate cyclooxygenase responsible for prostaglandin
production. The inhibition of cyclooxygenase by nonsteroidal anti-inflammatory drugs
produces clinical benefits, however, it may also cause some adverse-side effects, including
disturbance in ovary function. It is suggested that nonsteroidal anti-inflammatory drugs are
responsible for formation of the luteinised unruptured follicle syndrome in primates.
Therefore, the aim of the present study was to determine the effect of meloxicam, a selective
cyclooxygenase-2 inhibitor, on follicular growth and ovulation in heifers.
Normally cycling crossbred Holstein/Polish Black and White (75%/25% respectively) heifers
(340-410 kg body weight; n=10), were injected intramuscularly with 25 mg of dinoprost, an
analogue of prostaglandin F2α (Dinolytic; Pharmacia & Upjohn, Belgium) during the luteal
phase, to induce luteolysis and estrus. Animals were randomly divided into 2 groups; in
Group I (control) and Group II heifers received 34-41 ml of saline or 0.5 mg/kg body weight
(34-41 ml) of meloxicam (Metacam®, Boehringer Ingelheim Vetmedica GmbH, Germany)
respectively, at 24 and 72 h after dinoprost administration. All heifers were subjected to
ultrasound analysis (Pie Medical Scanner 480, Holland) before the analogue of prostaglandin
F2α administration and thereafter since day 3 to day 9 of the subsequent cycle to study the
time of ovulation. In Group I rupture of the ovarian follicles and ovulation were finished
between day 4 and 5 after dinoprost administration. In Group II unruptured follicles were
observed in all heifers examined to day 9 after estrous cycle synchronization. Obtained data
suggested that inhibition of cyclooxygenase by meloxicam is able to cause significant delay in
ovulation and may also produce luteinised unruptured follicle syndrome in heifers.
EXPRESSION OF ANDROGEN RECEPTOR AND 3β
β -HYDROXYSTEROID
DEHYDROGENASE IN THE OVARIES OF NEONATAL RATS
Jerzy Galas, Maria Słomczyńska, Maria Szołtys
Laboratory of Endocrinology and Tissue Culture, Institute of Zoology, Jagiellonian
University, 30-060 Krakow, Poland, E-mail: [email protected]
Sequential changes in androgen receptor (AR) distribution have already been established in
rat ovarian follicles developing and maturing during the rat estrous cycle. The aim of the
present study was to investigate how early in the postnatal life AR appears in the rat ovaries,
and whether AR distribution in the particular types of early growing follicles is similar to that
characteristic of adult animals. Ovarian steroids contents were also estimated and
steroidogenic cells were identified by immunolocalization of 3β-hydroxysteroid
dehydrogenase (3β-HSD).
The animals were killed between day 1 and 7. Excised ovaries were subjected to steroid
analysis or they were submitted to immunohistochemical localization of AR or 3β-HSD on
deparafinized and rehydrated paraplast sections. AR was localized with the use of anti-AR
antibody (NCL-ARp: Novocastra) and 3β-HSD with the use of polyclonal antibody antirecombinant mouse 3β-HSD (a gift from prof. Anita H. Payne from Stanford University
Medical Center). Androgen, estradiol and progesterone contents were measured by
appropriate RIAs in homogenized ovaries.
Between postnatal day 1 and 2 AR was expressed in the ovarian epithelium and the oocyte
cytoplasm of primordial follicles, while the oocyte nuclei and the remaining ovarian structures
were AR-negative. During the consecutive days (3 - 7) AR immunostaining was limited to the
oocyte cytoplasm but as the oocytes grew, the intensity of AR immunoreaction in their
cytoplasm became gradually weaker. As previously established, in the ovaries of adult
animals such early growing follicles displayed oocyte AR immunostaining similar to that of
neonatal rats, but in contrast, a strong nuclear AR immunostaining exhibited also the
granulosa cells of small follicles. On day 1 3β-HSD-positive immunostaining was present in
the ovarian epithelium and the cells surrounding clumps of oocytes. During the consecutive
days a positive immunoreaction was observed in the ovarian epithelium and the granulosa
cells of early growing follicles. On day 7 3β-HSD immunostaining appeared also in some
theca cells of the secondary follicles.
Progesterone and androgen contents in ovarian homogenates showed a gradual rise until day
5, while estradiol concentration was almost negligible.
These findings indicate that the process of steroidogenesis starts very early in the neonatal rat
ovaries and at that time androgens most likely exert a hormonal action as the amount of
ovarian estradiol is very low. However, until day 7 AR is expressed only in the ovarian
epithelium and the oocyte cytoplasm.
Support by DS/IZ/UJ/2005.
EARLY ANTRAL BOVINE FOLLICLES AFTER GROWTH CULTURE –
OOCYTE SURVIVAL, MATURATION AND FERTILIZATION*
Lucyna Kątska-KsiąŜkiewicz1, Hannelore Alm2
1
Department of Biotechnology of Animal Reproduction, National Research Institute of
Animal Production, 32-083 Balice, Poland, E-mail:[email protected]; 2Dept. of
Reproductive Biology, Research Institute for the Biology of Farm Animals, 18-196
Dummerstorf, Germany, E-mail: [email protected]
It has been known that the mammalian ovary contains a huge number of small oocytes, of
which only a small part grows to the final size, matures, and ovulates. Since >99.9% of
ovarian follicles undergo atresia, it would be of great practical benefit if these follicles,
destined to become atretic, could be rescued by a long-term in vitro culture offering a large
pool of oocytes for in vitro maturation and fertilization.
The aim of the experiment was to develop methods for long term in vitro culture of oocytes
originating from early antral bovine ovarian follicles that allow obtaining mature ova suitable
for in vitro fertilization.
The morphological quality, meiotic competence and fertilizability of oocytes originating from
follicles with the diameter from 0.2 to 0.7 mm were evaluated following in vitro growth for 7
to 14 d and subsequent IVM and IVF. Long term growth culture was carried out in two
culture systems: 1) intact follicles (IF) were embedded into microdrops of collagen gel or
cultured in hanging drops of medium; 2) isolated cumulus-oocyte complexes surrounded by
parietal granulosa cells (COCGs) were embedded into microdrops of collagen gel. For growth
culture modified medium TCM 199 supplemented with 3% BSA, 4 mM hypoxanthine and
mixture of insulin, transferrin and sodium selenite was used. For maturation and fertilization
standard procedures were applied (Kątska et al., 1996, Anim. Reprod. Sci 44, 23-31).
It was shown that isolated COCGs created in culture follicle-like structures, oocytes could
reach meiotic competence and mature at above 2-fold higher rate than oocytes cultured into
IF. Significantly higher (P<0.01) recovery rate of COCs suitable for IVM was obtained from
COCGs embedded in microdrops of collagen gel (63.2%) compared to IF grown in
microdrops of gel (44%) or in the hanging drops (39.3%). Maturation and fertilization rates of
COCs growing in IF resulted in 8.5% and 23.1% respectively while following growth in
COCGs 20.0% of oocytes reached maturity and 27.6% were fertilized. The investigations are
being continued.
*
Research was supported by the State Committee for Scientific Research as a solicited
project PBZ-KBN-084/PO6/2002 and carried out as part of the program of the PolishGerman Cooperation in Agriculture Sciences.
CELLULAR FEATURES OF OOCYTES ASSOCIATED WITH
REPRODUCTIVE AGING IN MICE
Naoko Kimura1, Mari Suda1, Yumi Hoshino2, Eimei Sato2, Kiyoshi Totsukawa1
1
Laboratory of Animal Reproduction, Faculty of Agriculture, Yamagata University,
Tsuruoka 997-8555, Japan, E-mail: [email protected]
2
Laboratory of Animal Reproduction, Graduate School of Agricultural Science,
Tohoku University, Sendai 981-8555, Japan
Introduction: The present study aims to investigate the effect of maternal age on the meiotic
and developmental competence of oocytes and incidence of chromosomal anomalies in
oocytes from fertile mice.
Matearials and Methods: The quality of oocytes retrieved from ICR female mice after
exogenous ovarian stimulation at 6, 12, 18, 24, 36, 42, 48 and 60 weeks of age were analyzed.
Results and Discussion: Body and liver weight were steadily increased with female aging,
whereas ovarian weight was significantly increased from 6 to 24 weeks of age and was
decreased after 24 weeks of age. Mean number of ovulated oocytes reached a peak at 12
weeks (40.7±14.6) of age and was considerably reduced from 18 (31.6±10.1) to 42 weeks
(13.7±7.8) of age and reached the bottom after 42 weeks of age. The proportion of
morphologically abnormal oocytes (cellular fragmentation, shrinking, swelling) was raised in
24-48 weeks (34-44%) of age compared with 6-18 weeks (9-30%) of age. Female age had no
effect on numbers of cumulus cells that enclosed oocytes (2094±876-4580±1926 per oocyte).
Morphologically normal oocytes (containing one polar body) were examined meiotic
maturation and distribution of chromosomes in metaphase II (MII) plate. There was no
significant difference in the proportion of normal MII from 6 to 36 weeks (88-93%) of age.
Chromosome misalignments at MII stage were frequently found after 42 weeks (19%<) of
age. These results indicate that 1) while a peak of number of ovulated oocyte comes soon after
the onset of puberty, ovary weight shows maximally following the ovulation peak, 2)
morphological abnormalities in oocytes are observed even at young age and are gently
increased associated with aging, however, chromosome scattering at MII stage is
unexpectedly caused at the latter phase in a reproductive lifespan. Future studies about
behaviors of some microtubule motor proteins will be helpful in understanding the
mechanism of meiotic chromosome misalignments.
CELLULAR FLICE-LIKE INHIBITORY PROTEIN IN GRANULOSA CELLS
WAS UP-REGULATED BY INTERLEUKIN-6 DURING FOLLICULAR
ATRESIA
Akihisa Maeda, Yasufumi Goto, Fuko Matsuda-Minehata, Yuan Cheng, Takafumi Sai
and Noboru Manabe*
Research Unit for Animal Life Sciences, Animal Resource Science Center, University
of Tokyo, 3145 Ago, Ibaraki-Iwama 319-0206, Japan,
*E-mail: [email protected]
In mammalian ovaries, more than 99% of follicles undergo a degenerative process known as
atresia, and only a few follicles ovulate during ovarian follicular growth and development.
Follicular selection dominantly depends on granulosa cell apoptosis, but the molecular
mechanism of selective follicular atresia is still largely unknown. In the present study, we
examined whether interleukin (IL)-6 is involved in granulosa cell apoptosis during follicular
atresia or not. Both mRNAs of IL-6 and gp130 [a subunit of IL-6 receptor (IL-6R)] were
detected in granulosa cells prepared from healthy, early atretic and progressed atretic follicles
of porcine ovaries by quantitative real-time RT-PCR. The expression of these mRNAs
decreased during follicular atresia. IL-6 soluble receptor (IL-6sR) protein (a subunit of IL-6R)
that binds with gp130 was detected in follicular fluids of healthy, early atretic and progressed
atretic follicles by the ELISA method, and this protein level also decreased during follicular
atresia. Moreover, recombinant IL-6 up-regulates cellular FLICE-like inhibitory protein long
form (cFLIPL) in cultured cells derived from granulosa cells. These results indicate that IL-6
is dominantly produced in granulosa cells of healthy follicles in porcine ovaries and that IL-6
up-regulates cFLIPL and prevents apoptotic cell death.
THE CHANGES OF CD44 POST-TRANSLATIONAL MODIFICATION IN
ATRETIC FOLLICLES DURING FOLLICULAR ATRESIA IN PIG OVARIES
Yuko Miyake, Hiromichi Matsumoto, Woro Anindito Sri Tunjung, Naoko Kimura,
Hiroshi Sasada and Eimei Sato
Laboratory of Animal Reproduction, Graduate School of Agricultural Science,
Tohoku University, Sendai, 981-8555, Japan, E-mail: [email protected]
CD44 is expressed in granulosa cells, cumulus cells during cumulus expansion and
preimplantation embryo in mammals in vitro, these evidences suggest that CD44 has a
positive role in oocyte maturation and preimplantation development of embryos. The
distribution of CD44 in mammalian ovary has not investigated in vivo, and the exact
roles of CD44 in follicular development and atresia are still unclear. Recently, it has
been reported that CD44 is present on human macrophage that is involved in
phagocytosis, so the present study was conducted to investigate CD44 on macrophage
during atresia in porcine ovaries. Frozen sections were prepared and subjected to
examine immunohistological localization of CD44 and macrophages. High expression
of CD44 was detected in late atretic and disintegrated atretic follicles. CD44 was
expressed in macrophage at all stages, that was not detected inside of follicles in
healthy and early atretic follicles but invaded to inside in progressing atretic follicles.
Healthy, early atretic, and progressing atretic antral follicles having 2-6 mm in
diameter were dissected from ovaries, and inner cells of each follicle were analysed
by RT-PCR and western blotting. Accompanied with advance of atresia, the
expression of CD44 mRNA and protein were increased significantly. 90 kDa of CD44
in progressing atretic follicles, that were observed invaded macrophages, are heavily
glycosylated with polylactosamine. The post-translational modification
(glycosylation) of CD44 in atretic follicles was altered during atresia, caused by
invasion of macrophages. These results suggested that glycosylated CD44 on
macrophage was involved in macrophage-mediated phagocytosis of apoptotic
granulosa cells.
GROWTH OF PRIMORDIAL OOCYTES FROM ADULT AND NEWBORN
PIGS IN XENOGRAFTS
Mohammad Moniruzzaman and Takashi Miyano
Graduate School of Science and Technology, Kobe University, Kobe 657-8501,
Japan, E-mail: [email protected]
Mammalian ovaries contain a large number of primordial follicles that contain non-growing
oocytes (primordial oocytes). A limited number of these oocytes initiate growth and reach to
the final size to be matured for fertilization. The mechanisms for the initiation of oocyte
growth have not been understood well. The present experiment was conducted to compare the
growth of primordial oocytes from newborn and adult pigs in xenografts. Effect of the gender
of host mice on pig oocyte growth was also examined. Cortical slices containing only
primordial follicles were collected under a dissection microscope from the ovaries of 6month-old gilts (n = 8) and 15-day-old piglets (n = 6). Size of the slices was about 2 mm × 1
mm × 0.5 mm. Each slice was cut into 2 pieces; one was fixed for histological examination
and the other was transplanted. For transplantation, 6- to 8-week-old male and female SCID
(severe combined immune deficiency) mice were anesthetized, and the cortical slices were
inserted under the kidney capsules. After 2 months, in the xenografts of newborn pig ovaries,
21.3 ± 5.0, 14.4 ± 4.7 and 1.5 ± 0.6% follicles developed to the primary, secondary and antral
stage in male SCID mice, respectively. There was no antral follicle in the grafts in female
SCID mice. The proportions of oocytes with the diameter of 100-120 mm were 1.5, 0 and
1.0% in male, intact female and ovariectomized female SCID mice, respectively. Higher
number of non-developing primordial follicles was observed in intact female (85.3 ± 3.0%)
than in male (62.7 ± 8.1%) and ovariectomized (70.1 ± 6.0%) female SCID mice. On the
other hand, primordial follicles of adult pig ovaries survived in the xenografts but none of
those developed to the primary stage or beyond in any type of SCID mice. However, after
prolonged transplantation (6 months), 3.5 ± 2.9%, 0 and 3.1 ± 2.4% follicles developed to the
antral stage in totals of 350, 213 and 219 follicles in male, intact female and ovariectomized
female mice, respectively. These results suggest that the mechanism for primordial oocyte
growth in adult pigs might be different from that in newborns.
THE PRESENCE OF 3β-HSD IN THE OVARY OF THE PREGNANT SWINE
Maria Słomczyńska1, Małgorzata Duda1, Małgorzata Burek1, Katarzyna Knapczyk1,
Zbigniew Tabarowski1, Jerzy Galas1, Marek Koziorowski2
1
Department of Animal Physiology, Institute of Zoology, Jagiellonian University, 30060 Krakow, Poland, E-mail: [email protected]
2
Department of Physiology and Reproduction of Animal, University of Rzeszów,
Poland
Steroid hormones play a major role in the normal ovarian development as well as
maintenance of puberty. Both, the theca interna and granulosa participate in the follicular
estrogen synthesis. They are also believed to contribute to corpus luteum formation and
progesterone production after ovulation. The shift in ovarian steroid synthesis from follicular
estrogen to luteal progesterone occurs trough changes in steroidogenic enzymes expression.
Pregnenolone synthesis is considered to be rate-limiting in steroidogenesis and is controlled
by two enzymes: cytochrome P450 17α-hydroxylase (P450c17) and 3β-hydroxysteroid
dehydrogenase (3β-HSD). These two enzymes are the most important ones in the pathway
leading to estrogen or, alternatively, to progesterone synthesis. During pregnancy, follicles
escape from regulation by gonadotropins and many of them undergo the process of atresia.
Many reports indicate that androgen can inhibit follicular development by increasing
follicular atresia.
Therefore the aim of our study was to examine morphological transition and to localize the
cellular expression of 3β-HSD and apoptotic cells in the ovary of pregnancy. Porcine ovaries
were obtained from pregnant pigs on days 10, 18, 32, 71 and 90 post coitum (p.c.). The
sections were exposed to polyclonal antibody against recombinant mouse 3β-HSD (which was
a gift from prof. Anita H. Payne from Stanford University Medical Center) at dilution 1:1000.
The color reaction was performed using stable DAB solution. Apoptotic cells were identified
with APOPTAG kit (Roche Diagnostics GmbH Roche Applied Science, Mainheim).
Immunostaining for 3β-HSD was restricted to theca interna cells of the porcine follicle and
this enzyme appeared to be expressed by only a relatively small percentage of cells. However,
no 3β-HSD staining was detected in granulosa cells of any porcine follicle regardless of the
day of pregnancy. 3β-HSD expression was also observed in endothelial cells of some of larger
microvessels.
The pattern of the intensity of expression of 3β-HSD in corpora lutea (CL) did not change
dramatically during pregnancy progression. Immunostaining was intense in the majority of
the luteal cells and also persisted in a few blood vessels surrounding porcine CL. However, in
some CL (not dependent on the day of pregnancy) 3β-HSD staining was obvious primarily in
some of the elongated cells. The present study showed the dynamic nature of the ovary of
pregnancy.
Supported by DS/IZ/2005.
EXPRESSION OF PROENKEPHALIN GENE IN PORCINE THECA
INTERNA AND GRANULOSA CELLS
Jaroslaw Staszkiewicz, Tadeusz Kaminski, Gabriela Siawrys, Bartlomiej E. Krazinski,
Mariusz T. Skowronski, Jadwiga Przala, Stanislaw Okrasa
Department of Animal Physiology, Warmia & Mazury University, 10-719 Olsztyn,
Poland, E-mail: [email protected]
Several studies performed on different species documented the expression of opioid
precursors genes – including that of proenkephalin (PENK) – in the ovary as well as the
presence of their products in various tissues and fluids of the female reproductive tract. The
opioid receptors were found on porcine ovarian cells, and in vitro studies documented
modulatory effect of delta (among others) opioid receptor agonists on steroidogenesis in
porcine granulosa and theca interna cells. The present studies were undertaken: 1/ to establish
the presence of PENK messenger RNA in porcine theca and granulosa cells and 2/ to test the
effect of respective gonadotropins (LH or FSH) on PENK gene expression in these cells.
Theca and granulosa cells were isolated from small (days: 15-16 of the estrous cycle) and
large (days: 19-20), morphologically healthy porcine follicles. Dispersed cells were cultured
in Eagle’s medium under the water saturated atmosphere of 95% air and 5% CO2, in the
presence or absence of respective gonadotropin; theca cells with LH (100 ng/ml) and
granulosa
cells
with
FSH
(100
ng/ml).
Following
24h-incubation, the cells were harvested and the total RNA was isolated. Quantification of
ovarian PENK gene expression was performed through the non-competitive semi-quantitative
RT-PCR technique involving co-amplification of the target mRNA and β-actin control
sequence. To verify specificity of primers in selecting the PENK cDNA, Southern analysis
was performed and the RT-PCR product was sequenced. Different negative controls were
performed.
Proenkephalin mRNA was expressed in both non-stimulated and FSH-stimulated granulosa
cells derived from small and large follicles. In theca interna, only LH-treated cells isolated
from small follicles expressed PENK mRNA.
In our experiment we documented that PENK expression in follicular cells can differ
markedly depending on follicle maturity and gonadotropin stimulation. This suggests that
PENK-derived peptides are locally synthesized in the ovarian follicle cells and might
participate in the regulation of their steroidogenesis as well as the follicle growth and
development.
The project was supported by the State Committee for Scientific Research, Poland (Grant 6
P04C 019 21).
Session II
Central regulations of reproduction
Oral presentation
ROLE OF INTRACELLULAR AND EXTERNAL FACTORS IN THE
OVULATORY RELEASE OF GnRH
Kazimierz Kochman
Institute of Animal Physiology and Nutrition, Polish Academy of Sciences,
05-110 Jablonna near Warsaw, Poland, E-mail: [email protected]
Biosynthesis and release of GnRH are regulated at the several different levels. The most
important regulation is by other neuropeptides and steroids through their specific receptors.
However, the regulation of intracellular reactions is very important and also the impulses
arriving from the outside the nervous system participate in this complex process.
Ribosomal synthesis requires sufficient level of mRNA and the optimal quantity of amino
acids necessary to finish the prohormone formation. Non completed synthesis on ribosome is
recognized as stress by neural cell and signal is transferred to nucleus to react to this situation
by expression of different genes.
Processing of GnRH prohormone is regulated by peptidases, amidating enzyme and the
pyroglutamyl ring formation.
Degrading peptidases play also the important role in the maintenance of GnRH level and may
influence the releasing impulse.
External signals of different character change the gene expression of biological clock proteins
and in this way participate in the function of this regulatory element.
Interplay of all these factors are necessary to the ovulatory impulse releasing GnRH.
EFFECTS OF INTRAVENTRICULAR INFUSION OF OREXIN-A ON GnRHNERVE TERMINALS AND PLASMA GONADOTROPHINS LEVELS IN
IMMATURE FEMALE RATS
Lidia Martyńska1, Ewa Wolińska – Witort1, Magdalena Chmielowska1, Jolanta
Polkowska2, ElŜbieta Wasilewska – Dziubińska1, Wojciech Bik1, Bogusława
Baranowska1
1
Department of Neuroendocrinology, Medical Center of Postgraduate Education,
Marymoncka 99, 01-813 Warsaw, e-mail: [email protected], 2The Kielanowski Institute
of Animal Physiology and Nutrition, 05-110 Jabłonna, Poland,
Orexin-A, recently discovered hypothalamic peptide, except of its strong orexigenic
properties is believed to play a role in the reproduction. The aim of the present study was to
investigate the effect of intraventricular (icv) infusion of orexin-A on GnRH nerve terminals
in the median eminence and on plasma LH and FSH levels in the standard-fed and fasted
immature female rats. Female Wistar rats, 30 days old, were divided into four following
groups: two fed ad libitum with standard diet (n=5 each) and two starved for 36 h, (n=5
each). Orexin-A was administered into the 3rd ventricle of the brain of rats one of standard
and one of starved groups (1 µg/5µl for 5min). Remaining groups were infused with artificial
cerebrospinal fluid (control). One hour after the infusion, blood samples from the heart were
taken, brain’s tissues were removed, fixed and then used for immunohistochemical
localisation of GnRH. Detection of the hormone in the light microscope was followed by the
computer image analysis and expressed as percent area stained and optical density of
immunostaining. Plasma LH and FSH concentrations were determined by radioimmunology.
In both, standard-fed and starved groups of rats, after orexin-A infusions, a striking increase
in the immunoreactive (ir) content of GnRH stored in the nerve terminals of the median
eminence compared to vehicle infused rats was noticed. These observations were supported
by the image analysis. Percent of area stained and optical density of irGnRH were
significantly higher (p<0,05) in orexin-treated, compared to vehicle-treated animals.
Concentrations of LH and FSH were significantly (p<0,01) lower only in fasted, orexininfused group compared to its respective control. The slight but not significant decrease of
both gonadotrophins concentrations was found in standard-fed, orexin-treated group
compared to its respective control. No significant differences were found in the both
gonadotrophins concentrations between vehicle-treated groups. The presented results show
that icv infusions of orexin-A suppress the release of stores of irGnRH from the nerve
terminals and gonadotropins from the pituitary to the circulating blood more evidently in
conditions of undernutrition. It is suggested that orexin-A can suppress the activity of the
hypothalamo- gonadotrophic axis in immature female rats and its effects depend on the level
of nutrition.
Research was supported by 501-2-1-28-02/03 CMKP grant.
REGULATION OF PROLACTIN AND LH SECRETION BY MELATONIN IN
THE SHEEP ON THE LEVEL OF THE PITUITARY GLAND
Tomasz Misztal
Department of Endocrinology, The Kielanowski Institute of Animal Physiology and
Nutrition, Polish Academy of Sciences, 05-110 Jablonna, Poland,
The discovery of melatonin receptors in the pars tuberalis (PT) of sheep pituitary gland and
the examination of the anatomical structure of this adenohypophysial region enabled studying
the mechanisms of the regulatory action of melatonin on the secretion of some pituitary
hormones i.e. prolactin and LH.
Melatonin regulates the seasonal secretion of prolactin in sheep by acting directly on the
pituitary gland. The ovine PT-specific cells secrete tuberalin, the melatonin-related protein,
which induces c-fos gene expression and stimulates prolactin release in pituitary lactotrophs.
The secretory activity of these cells changes throughout the year due to the different 24-h
pattern of expression of the major clock genes. Thus, the endocrine effect of tuberalin is of
importance in the pituitary mechanism of melatonin action in regards to the control of the
seasonal cycle of prolactin. It is suggested that long duration of melatonin signal suppresses
synthesis of tuberalin, induced by an endogenous stimalator(s).
Numerous granular secretory cells of ovine PT secrete LH and are also responsive to the
hypothalamic GnRH. The evidence exists that melatonin attenuated the in vitro GnRHinduced increase in LH secretion from the ovine PT. On the contrary, the short-term infusion
of melatonin into the third brain ventricle increased plasma LH concentration in luteal-phase
ewes. Since the subpopulation of gonadotrophs in the PT is too small to have a noticeable
effect on circulating LH concentration, it is possible that ‘melatonin-sensitive’ LH, secreted
by the PT, may act back on the brain to influence the reproductive neuroendocrine axis via a
short-loop feedback system. Thus, the short-term inhibition of the PT signal by infused
melatonin would enhance the GnRH release from the nerve terminals in the median eminence
and/or the LH release from the pars distalis gonadotrophs.
THE ROLE OF GABAA AND GABAB RECEPTORS IN THE REGULATION
OF GnRH RELEASE IN EWES
Dorota Tomaszewska-Zaremba, Franciszek Przekop
Department of Neuroendocrinology, The Kielanowski Institute of Animal Physiology
and Nutrition, 05-110 Jabłonna, Poland, E-mail: [email protected]
The regulation of hypothalamic gonadotropin-releasing hormone (GnRH) secretion is
provided by both stimulatory and inhibitory neurotransmitters and hormones. Among these
trans-synaptic regulatory systems, γ-aminobutyric acid (GABA), the dominant inhibitory
neurotransmitter in the hypothalamus affects GnRH secretion by two different classes of
membrane receptor molecules: GABAA and GABAB receptors. To examine the role of GABA
in the control of GnRH release from ventromedial-infundibular region of the hypothalamus
(VEN/NI) and medial preoptic area (MPOA) of ewes during different reproduction stages
(seasonal anestrous, follicular phase and luteal phase of the estrous cycle), the extracellular
concentration of GnRH, β-endorphin, noradrenaline, dopamine and their metabolites were
quantified during local stimulation or blockade of GABAA and GABAB receptors in these
structures.
The results show that GABAA and GABAB receptors play important role in the control of
GnRH/LH secretion in ewes. The different neural mechanisms are involved in the suppression
or activation of GnRH/LH release by GABA: directly through the GABAA or GABAB
receptor mechanism on the axon terminals or on perikaria of the GnRH neurons or indirectly
through the GABA receptor mechanism on β-endorphinergic and catecholaminergic neural
systems in MPOA and VEN/NI. The influence of GABA on GnRH release and other studied
neural systems’ activity depends on the physiological state of animals, structure of the
hypothalamus and the type of GABA receptor. Activation of GABAA receptors inhibits and
activation of GABAB receptors stimulates GnRH release in ewes during anestrous and during
follicular phase of the estrous cycle.
THE EFFECTS OF β2 ADRENERGIC AGONIST – FENOTEROL AND
NEUROPEPTIDES VIP AND PACAP38 ON PROGESTERONE RELEASE
FROM CULTURED RAT OVARIAN GRANULOSA CELLS
ElŜbieta Wasilewska – Dziubińska, Magdalena Chmielowska, Ewa Wolińska –
Witort, Lidia Martyńska , Wojciech Bik, Bogusława Baranowska
Neuroendocrinology Department, Medical Centre of Postgraduate Education,
Marymoncka 99, 01-813 Warsaw, Poland, E-mail: [email protected];
[email protected]
Progesterone synthesis in ovarian granulosa and luteal cells may be stimulated by several
factors
including catecholamines and neuropeptides independently of pituitary
gonadotrophins. In this study we compared the effects of fenoterol - β 2 adrenergic agonist
(which is widely used in obstetrical practice) with the effects of neuropeptides VIP and
PACAP38 on progesterone release from cultured ovarian granulosa cells of adult cyclic rats.
We also examined the interaction between β 2 adrenergic and peptidergic stimulation on
progesterone release in short-term and long-term experiments.
There were no differences in progesterone release stimulated by fenoterol, VIP and PACAP38
after 2h incubation. However, after 24 incubation progesterone release stimulated by
fenoterol was strongly higher as compared with progesterone release stimulated by VIP and
PACAP 38. Thus it may suggest that the β 2 adrenergic long-term stimulation of
progesterone release from cultured rat ovarian granulosa cells is more effective than that by
neuropeptides VIP and PACAP38. We also observed that in short-term and long-term
experiments progesterone release stimulated by both fenoterol and VIP and both fenoterol and
PACAP38 was not additive. Progesterone release stimulated simultaneously both by fenoterol
and VIP and both by fenoterol and PACAP38 was diminished in the presence of propranolol
in short-term and long-term experiments. It may suggest that β 2 adrenergic stimulation of
progesterone synthesis in ovarian granulosa cells culture is more important than peptidergic
one.
The exact mechanism (or mechanisms ) underlying adrenergic and peptidergic stimulation of
progesterone synthesis remains unknown. It is conceivable that the β 2 adrenergic receptors of
the granulosa cells are functionally coupled to one or more metabolic events leading to the
production of progesterone. Although the exact nature of the mechanisms underlying this
phenomenon remain unknown, several interpretations may be considered.
Conclusion: The β 2 adrenergic long-term stimulation of progesterone release from cultured
rat ovarian granulosa cells is more important and more effective than that of neuropeptides
VIP and PACAP38.
EFFECTS OF LEPTIN ON THE HYPOTHALAMIC-PITUITARY AXIS IN
DOMESTIC ANIMALS
Dorota A. Zięba 1, Gary L. Williams 2,3
1
Department of Sheep and Goat Breeding, University of Agriculture, Krakow 30-059,
Poland, E-mail: [email protected]; 2Animal Reproduction Laboratory, Texas
A&M University Agricultural Research Station, Beeville, TX, USA; 3 Department of
Animal Science, Texas A&M University, College Station, TX, USA
Leptin, a hormonal peptide synthesized and secreted predominantly from adipocytes, plays a
major role in the central control of energy homeostasis and reproduction in mammals. Leptin
receptor mRNA is localized in the hypothalamus and on gonadotrophs in the anterior
pituitary. Interactions between the ligand and its receptor result in a cascade of signaling
activities that impact gonadotropin and somatotropin secretion. Although the leptin receptor
does not appear to co-localize directly on GnRH neurons, leptin has been shown to stimulate
GnRH secretion in vivo and in vitro. For example, the in vitro release of GnRH from porcine
hypothalamic explants is enhanced in the presence of leptin. Moreover, leptin treatment
increased gonadotropin secretion from porcine pituitary cells in culture and stimulated release
of LH from adenohypophyseal explants from full-fed pigs. In contrast to these observations in
monogastric species, stimulation of the hypothalamic-pituitary axis by leptin in ruminants is
observed mainly in animals and/or tissues that have been pre-exposed to negative energy
balance. In fact, it is now believed that the effects of leptin are greatest during acute
undernutrition in all animals. Leptin prevented fasting-induced reductions in LH pulse
frequency in peripubertal heifers and castrated, estradiol-treated rams, increased the
magnitude of GnRH and LH pulses in fasted beef cows, and enhanced basal secretion of LH
in vivo in undernourished lambs and long-term food-restricted sheep, and from pituitary
explants of fasted cows. However, leptin was unable to accelerate the frequency of LH pulses
in sexually-immature heifers and male lambs, regardless of nutrient status, and had no effect
on the secretion of GnRH and LH in full-fed cattle or from hypothalamic and anterior
pituitary explants derived thereof. Leptin also affects the somatotropic axis, causing release of
GH and this effect is also dependent upon nutritional history of animals. Continuous central
infusion of leptin had no effect on circulating concentrations of GH in ovariectomized, fullfed sheep, but increased circulating GH in chronically undernourished sheep and in fasted
heifers. The ability to detect such effects is related to species, as well as the timing, duration,
and dose of leptin. Recently, leptin has been shown to exert direct effects on GH gene
expression and this activity is modulated by nitric oxide. Collectively, leptin serves as a
metabolic signal that is translated via several neuropeptide systems within the brain and
anterior pituitary. These include pathways that regulate both the reproductive neuroendocrine
and somatotropin axes, but both are linked to leptin’s innate involvement in food intake,
satiety and overall energy balance.
Poster presentation
EFFECT OF STRESS ON THE EXPRESSION OF GnRH GENE AND
GnRH RECEPTOR (GnRH-R) GENE IN THE PREOPTIC AREAHYPOTHALAMUS, AND ON THE GnRH-R GENE IN THE
SYALK/MEDIAN EMINENCE AND ANTERIOR PITUITARY GLAND
IN THE EWES DURING THE FOLLICULAR PHASE OF THE
ESTROUS CYCLE
Magdalena Ciechanowska1, Magdalena Łapot1, Tadeusz Malewski2, Krystyna
Mateusiak1, Franciszek Przekop1
1
The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy
of Sciences, 05-110 Jabłonna, 2Institute of Genetics and Animal Breeding,
Polish Academy of Sciences, Jastrzębiec, 05-552 Wólka Kosowska, Poland,
The RT-PCR technique was used to analyze GnRH mRNA and its receptor (GnRH-R mRNA)
in the preoptic area, anterior and ventromedial hypothalamus, and GnRH-R mRNA in the
stalk/median eminence and anterior pituitary gland of ewes subjected to short or prolonged
footshock stimulation during the follicular phase of the estrous cycle. The state of stress was
induced by mild alternative current pulses (i = 3 mA) on the feet of ewes for 3 h on day 16 of
the estrous cycle (for short-stressed ewes) and for 5 h daily during 4 consecutive days (from
days 13 to 16 of the estrous cycle) for prolonged-stressed animals. To establish the influence
of stress on LH secretion, two series of blood samples were collected from each animal in 10
min intervals for 5 hours on day 16 of the estrous cycle in unstressed and stressed ewes. In
control ewes the GnRH mRNA was found in structures throughout the preoptic area, anterior
and ventromedial hypothalamus; the lowest levels of GnRH mRNA was found in the anterior
hypothalamus. No mRNA was detected in the stalk/median eminence. The concentration of
GnRH mRNA increased significantly in the preoptic area, anterior and ventromedial
hypothalamus of ewes subjected to short stress. The prolonged stressful stimuli significantly
decreased GnRH mRNA levels in all analyzed structures. In control ewes, GnRH-R mRNA
was found in different levels in the analyzed tissues: the highest concentrations were found in
the anterior pituitary gland and the stalk/ median eminence. In short stressed ewes the
significant augmentation of mRNA encoding GnRH-R was detected in the preoptic area,
entire ventromedial hypothalamus, stalk/median eminence and anterior pituitary gland. With
the exception of the preoptic area, GnRH-R mRNA was significantly reduced in all structures
of animals subjected to prolonged footshocking; GnRH-R mRNA in the preoptic area did not
differ from control values. The above mentioned changes in GnRH mRNA and GnRH-R
mRNA levels under short or prolonged stress were associated with an increase or decrease of
LH concentration in blood plasma thus suggesting, at least in some extent, the existence of a
direct relationship between GnRH mRNA and GnRH-R mRNA expression with LH secretion.
Further studies are necessary to determine the relationship that exists between GnRH mRNA
and GnRH-R mRNA concentrations with the biosynthesis of GnRH and GnRH-R.
EXPRESSION OF GnRH GENE AND GnRH RECEPTOR (GnRH-R)
GENE IN THE PREOPTIC AREA HYPOTHALAMUS AND GnRH-R
GENE IN THE STALK/MEDIAN EMINENCE, AND ANTERIOR
PITUITARY GLAND OF ANESTROUS EWES
Magdalena Ciechanowska1, Magdalena Łapot1, Tadeusz Malewski2, Franciszek
Przekop1
1
There is a general agreement that annual cycles in the reproduction of sheep and other
seasonal breeders are primarily due to changes in the environmental factors which activate
or inhibit the specific GnRH afferents and gonadotropin secretion. Despite the number
studies that have been concerned with the mechanism of GnRH/LH release during the
breeding and non-breeding season there is no data whether the photoperiod may affect
GnRH gene and GnRH-R gene expression during annual cycles in reproduction. Given the
observation that seasonal change in the innervation of GnRH neurons is associated with
the GnRH release in the ewe, we put a question how the photoperiod may also affect the
expression of GnRH gene as well as GnRH-R gene. In the first study to clarify the
anestrous on the molecular basis of GnRH biosynthesis it was examined the levels of
GnRH mRNA and the GnRH-R mRNA in the preoptic area, anterior and ventromedial
hypothalamus, and the GnRH-R mRNA in the stalk/median eminence and anterior
pituitary gland. Their values were compared with these obtained from ewes during luteal
phase of the estrous cycle. The reason for such selection is the fact, that in both states the
GnRH secretion is suppressed: by dopaminergic system in the hypothalamus in anestrous
ewes and by progesterone during luteal phase of the estrous cycle of sheep. Results: In
anestrous ewes the comparable levels of GnRH mRNA were found in the preoptic area,
anterior hypothalamus and ventromedial hypothalamus; the GnRH-R mRNA at different
levels were detected throughout preoptic area, anterior and ventromedial hypothalamus,
stalk/median eminence and anterior pituitary gland. The highest concentrations of GnRHR mRNA were detected in the stalk/median eminence and anterior pituitary gland. In ewes
during luteal phase of the estrous cycle the concentration of both GnRH mRNA and
GnRH-R mRNA were significantly higher in all structures than in the anestrous ewes. The
obtained results indicate for the first time that in non-breeding season the expression of
GnRH gene and GnRH-R gene is suppressed as compared to ewes during luteal phase of
the estrous cycle. In conclusion it is suggested that the decrease of the expression of GnRH
gene and GnRH-R gene in the preoptic area-hypothalamus and GnRH-R gene in the
stalk/median eminence and the anterior pituitary gland among others leads to anovulatory
state in sheep during long photoperiod.
EFFECT OF INTRACEREBROVENTRICULAR INFUSION OF GENISTEIN
OR ESTRADIOL ON FSH AND GH SECRETION IN OVARIECTOMIZED
EWES
Konrad Górski, Tomasz Misztal, Katarzyna Romanowicz
Nutrition, 05-110 Jabłonna, Poland; E-mail: [email protected]
Genistein belongs to a family of phytoestrogens, which are found in many plants that are used
in ruminant feeds. Through binding with both isoforms of estrogen receptor α and β,
phytoestrogens are capable of evoking typical estrogenic responses and/or have the potential
to exert antagonistic effects. The aim of this study was to compare the effects of a brief
intracerebroventricular (icv) infusion of genistein and 17β-estradiol (E2) on the secretion of
FSH and growth hormone (GH) in ovariectomized ewes, during the breeding season. The
ewes (n=15) received the following treatment: 1) icv infusion of vehicle (control, n=5), icv
infusion of genistein (n=5) and icv infusion of E2 (n=5). The estrogenic compounds were
infused in the concentration of 10 µg/100 µl/h, from 10.00 to 14.00 h and blood samples were
collected from 8.00 to 20.00 h, at 10 minutes intervals. Plasma FSH and GH concentrations
were assayed by the RIA methods and the data are shown as the means (± SEM) of 2-hour
periods.
No significant differences in FSH concentrations were found in each group of ewes and
among them throughout the experimental period.
In the control group, a significant increase in GH concentration was observed after the
infusion compared to the values recorded before and during it (P<0.05 – P<0.001). In
genistein-infused ewes, the GH concentration increased significantly during the last two hours
of the infusion, compared to the pre-infusion period (P<0.01) and reached the maximum value
after the infusion (P<0.001). In E2-infused ewes, plasma GH concentration increased
significantly during the first two hours of the infusion compared to the pre-infusion period
(P<0.05). The second increase in the GH concentration was recorded in this group during the
evening hours (P<0.001) and did not differ significantly from the concomitant concentrations
in controls and in genistein-infused ewes
These results demonstrated that in ovariectomized ewes, the FSH secretion, in contrast to GH,
was stable and did not undergo influences of the estrogenic compounds. Both, genistein and
E2 stimulated daily GH secretion but a response of this hormone was weaker for genistein.
These results indicate that the estrogenic compounds may affect the regulation of GH
secretion at the level of the central nervous system.
MECHANISM OF SEXUAL SELECTION IN BANK VOLES,
CLETHRIONOMYS GLAREOLUS
Małgorzata Kruczek
Department of Mammalian Reproduction, Institute of Environmental Sciences,
Jagiellonian University, 30-387 Krakow, Poland, E-mail: [email protected]
To the extent that most female rodents make a greater parental investment than do males, they
should be particulary adapted to discriminate among potential partners. The studies presented
here demonstrate that bank vole females used males olfactory cues for mate selection. They
were able to distinguish between hormonally active and inactive males and between dominant
and subordinate males, strongly preferring the odour of dominant males. In two-choice
preference test, females spent more time investigating dominant males odour (35.0±7.4 sec.)
than that from subordinate ones (10.2±3.4 sec.).
To establish which olfactory system, main or vomeronasal, is involved in mate selection the
VNX and OBX females were tested for their ability to distinguish between intact and
castrated males. It has been shown that vomeronasalectomy eliminate the ability of females to
recognize the hormonal state of the potential, sexual partner. The deficit in time spent sniffing
tested males exhibited by olfactory bulbectomized female (6.5±2.8 sec.) was much more
severe than those observed in VNX females (26.9±5.2 sec.).
It can be concluded that both olfactory systems are involved in females sexual selection.
Males olfactory cues are also used by bank vole females for kin recognition. In two-choice
preference test adult females spent more time with the odour of non-sibling males (61.7±sec.)
in comparison with time spent with the olfactory cues from sibling males (10.4±sec.) or social
brothers (9.4±2.8 sec.). If the females had a choice between brothers and social brothers they
always choose non-sibling males (11.0±1.2 sec. for brothers and 42.5±4.8 sec. for social
brothers).
These findings indicate that bank vole females use a genetically based mechanism to
recognize their kin, however social association between males and females early in life cannot
be eliminated.
EFFECT OF STRESS ON THE EXPRESSION OF GnRH GENE AND
GnRH RECEPTOR (GnRH-R) GENE IN THE PREOPTIC AREAHYPOTHALAMUS, AND OF THE GnRH-R GENE IN THE
STALK/MEDIAN EMINENCE AND THE ANTERIOR PITUITARY
GLAND OF ANESTROUS EWES
Magdalena Łapot1, Magdalena Ciechanowska1, Tadeusz Malewski2, Krystyna
Mateusiak1, Franciszek Przekop1
1
Using RT-PCR technique it was analysed GnRH mRNA and GnRH-R mRNA in the
preoptic area, anterior and ventromedial hypothalamus, and GnRH-R mRNA in the
stalk/median eminence and in the anterior pituitary gland of anestrous ewes subjected
to intermittent short or prolonged footshock stimulation. The state of stress was induced
by mild alternative current pulses (i = 3 mA) o the feet of ewes for 3 h (short stress) or
for 5 h daily during 4 consecutive days (prolonged stress). To establish the effect of
stress on LH secretion two series of blood samples were collected from each animal in
10 min intervals for 5 h: first on the day prior stressing and second on the first day, or
the fourth day of stimulation in short or prolonged stressed animals, respectively. In
control ewes the comparable levels of GnRH mRNA were found in the structure
throughout the preoptic area, anterior and ventromedial hypothalamus. In non-stressed
ewes the overall distribution of GnRH-R mRNA at different levels was detected in
tissue continuum throughout the preoptic area, anterior and ventromedial
hypothalamus, stalk/median eminence and the anterior pituitary gland: the highest
concentration was in the stalk/median eminence and in the anterior pituitary gland. The
short and prolonged footshock stimulation significantly increased GnRH mRNA in all
analysed tissue: the highest responses in GnRH mRNA concentration were in the
preoptic area and ventromedial hypothalamus. Both kinds of stress increased
significantly concentration of GnRH-R mRNA in the preoptic area, anterior and
ventromedial hypothalamus, stalk/median eminence and anterior pituitary gland: the
highest responses to prolonged stress were found in the preoptic area, anterior and
ventromedial hypothalamus. In spite of profound up-regulation of GnRH mRNA and
GnRH-R mRNA under short and prolonged stress condition, the increase of LH
secretion followed only in shortly stressed sheep. Presented results suggest that the
increase of transcriptional activity of GnRH gene and GnRH-R gene is not directly
related with an increase of GnRH release and GnRH-R activity. It remains to determine
the relationship that exists between GnRH mRNA and GnRH-R mRNA expression
with the mechanism controlling GnRH and GnRH-R biosynthesis and LH secretion.
EFFECT OF MELATONIN ON LH SECRETION IN PREPUBERTAL SHEEP
Edyta Molik1, Tomasz Misztal2, Katarzyna Romanowicz2, Edward Wierzchoś1,
Maciej Murawski1 Kinga Szwiertnia1
1
Department of Sheep and Goat Breeding, Agricultural Academy of Cracow, Poland,
2
Nutrition, Jablonna, Poland.
Chronic administration of exogenous melatonin in adult anestrous ewes leads to an increase in
the secretion of LH during treatment. Exactly how melatonin exerts this effect is, however,
not well understood. Its action on the hypothalamo-pituitary-gonadotropic system in
prepubertal animals is also not fully examined. Thus, the aim of this study was to investigate
the long-term effect of melatonin on the LH secretion in prepubertal female ewes.
Twenty female lambs, born in February, were used in the experiment after weaning, from
May to the end of November. All lambs were maintained under the natural lighting conditions
and half of them (n = 10) obtained the long-term acting melatonin implants (MelovineTM,
France). Eight series of blood collection were performed in all animals at 4-week intervals
and the first started before melatonin treatment. Blood samples were collected from 12.00 to
18.00 h at 20-min. intervals and the LH concentration was assayed by the RIA method. The
results are shown as the means ± SEM.
The mean plasma LH concentrations recorded in May, on the beginning of the experiment,
did not differ significantly (6.47 ± 0.64 ng/ml, respectively). The first significant difference
(P<0.01) in LH concentration between groups was found in the third months (August) of the
experiment (5.45 ± 0.49 ng/ml for control vs 6.04 ± 0.83 ng/ml for melatonin treated ewes).
The LH concentration increased in both groups of ewes during the next months, being
persistently higher in ewes treated with melatonin (P<0.01). The highest plasma LH
concentration in melatonin treated ewes occurred in November (9.35 ± 3.41 ng/ml, for
controls vs. 6,48 ± 2,01 ng/ml). These results show that the secretion of LH is markedly
elevated in ewes bearing the implants of melatonin, compared to these naturally matured and
indicate that melatonin may play a stimulatory role in the maturation of the gonadotropic
system in the sheep, which experienced the long-day photoperiod before the treatment.
MODULATION OF HYPOTHALAMO-PITUITARY-GONADAL AXIS BY
OPIOIDS AND GLUCOCORTICOIDS
Krystyna Pierzchała-Koziec
Department of Animal Physiology, University of Agriculture, 30-059 Cracow,
Interest in the role of the opiate system in the mediation of reproduction remained high in the
last decade, although the emphasis changed somewhat from previous years. Much research
focused on the differential roles of the receptor types and subtypes. There was also significant
concern about the interaction of the opioid system with other systems, both neurotransmitter
system and those involved with specific functions. The opiates modulate the reproduction in
male and female of many species of mammals and birds mostly by inhibition of the activity of
hypothalamo-pituitary-gonadal axis. However, the mechanism by which negative feedback
control systems act to regulate opioids synthesis and secretion in sheep is still uncertain.
A reduction in inhibition of gonadotropin releasing hormone secretion by endogenous opioid
system in the hypothalamus is thought to be permissive of the preovulatory surge of GnRH
and luteinising hormone.
The opioid peptides seem to be involved in the regulation of the estrus cycle, pregnancy and
developmental processes. Chronic morphine produced more distinct expression of
proenkephalin mRNA patches of neostriatum than in the striatum. However ,the amounts of
adrenal preproenkephalin mRNA were lower what may indicate that the changes in the gene
expression were dependent on glucocorticoids. Cortisol may play an important role in the
regulation of adrenal proenkephalin mRNA and synthesis of enkephalin, because there was
a drop in proenkephalin mRNA before birth at the time of increased cortisol.
Opioid effects depend on the stage of estrus cycle and the gonadal steroid levels. It was
previously showed that prolonged treatment with progesterone changed the concentration and
synthesis of endogenous opioid peptides in the sheep brain. As a part of study dealing with the
interaction of opioids and gonadal steroids the study was carried out to assess the effects of
prolonged administration of progesterone and opioid receptor antagonist or agonist on the
activity of the hypothalamo-pituitary-gonadal (HPA) axis in ewes.
Long term treatment with progesterone decreased the plasma levels of Met-enkephalin and
cortisol and their concentrations in the adrenals (opioid and cortisol) and the HPA axis
(opioid). Injections of naltrexone and Met-enkephalin significantly changed the effect of
progesterone on the synthesis, release and receptor binding of endogenous opioid and cortisol.
The results showed that synthesis of proenkephalin occurred also in the ovary what may
suggests that the interaction of opioids and steroids during steroidogenesis is controlled by
locally produced opioids. It may be assumed that opioids and glucocorticoids are the
regulators of the HPA axis activity in sheep during estrus cycle.
Supported by the State Committee for Scientific Research as a Solicited Project PBZ-KBN084/PO6/2003/3.10
THE ROLE OF STEROID HORMONES IN THE MODULATION OF OPIOID
PEPTIDES RELEASE FROM HYPOTHALAMO-PITUITARY AXIS IN
SHEEP
Krystyna Pierzchała-Koziec¹, Barbara Kępys², Joanna Zubel¹, Marcin Śmigielski¹
¹Department of Animal Physiology, University of Agriculture, 30-059 Cracow,
E-mail: [email protected],²Institute of Molecular Biology, Jagiellonian
University, Cracow, Poland
Opioid peptides inhibit the activity of hypothalamo-pituitary-gonadal axis in many species of
mammals and birds. However, their effects depend on the stage of estrus cycle and the
gonadal steroid levels. It was previously showed that prolonged treatment with progesterone
changed the concentration and synthesis of endogenous opioid peptides in the sheep brain. As
a part of study dealing with the interaction of opioids and gonadal steroids the study was
carried out to assess the effects of prolonged administration of progesterone on the in vitro
release of Met-enkephalin from the hypothalamus and pituitary of sheep.
The experiment was carried out on fifteen 2 years old ewes divided into control and two
experimental groups received progesterone (intravaginal sponges for 12 days) and injected
with saline (P) and naltrexone (P+NAL, 3mg/kg i.v.) on days: 1, 2, 5, 12 and 15. Sheep from
all experimental groups received an injection of 600 I.U. of PMSG after removing sponges.
Two days later, hypothalamus and pituitary were dissected out and directed to in vitro release
of Met-enkephalin. Fragments of tissues were treated for 20 min with naltrexone or estradiol
and the 2 ml fractions were collected and directed to the estimation of Met-enkephalin
concentration. The opioid level was measured by radioimmunoassay method according to
Pierzchała and Van Loon(1990).
The basal level of Met-enkephalin release from hypothalamus was decreased by naltrexone
and estradiol from 0.49±0.01 to 0.07±0.01 and to 0.29±0.01 pmol/g/20min, respectively
(P<0.001). Long term treatment with progesterone potentiated the effect of in vitro naltrexone
and estradiol but did not change the basal level of the opioid release. In contrast, injection of
naltrexone increased the basal level of Met-enkephalin release from 0.53±0.01 observed in
progesterone treated ewes to 0.81±0.01 pmol/g/20min in opioid receptor antagonist injected
sheep (P<0.05).Combination of exogenous and endogenous naltrexone resulted in significant
release of opioid from hypothalamus - to 4.34±0.40 pmol/g/20min (P<0.001). It seems
probable that naltrexone inhibited the in vivo release of an opioid peptide during the
progesterone treatment and/or stimulated the synthesis of enkephalin precursor. On the other
hand, in vitro estradiol almost completely inhibited the release of an opioid from the
hypothalamus of ewes treated with progesterone and naltrexone.
The Met-enkephalin release from the pituitary of control sheep was decreased by naltrexone
and estradiol in a similar manner – from 15.0±1.2 to 3.1±0.3 and to 2.4±0.1 pmol/g/20min,
respectively (P<0.001). Progesterone decreased the release of Met-enkephalin to 4.1±0.3
pmol/g/20min (P<0.3) and this effect was potentiated by in vitro naltrexone and estradiol.
Naltrexone given with progesterone partially reversed the inhibition caused by progesterone
and modulated the in vitro effect of estradiol.
The results clearly showed that progesterone given for 12 days and in vitro estradiol
significantly affected the release of an endogenous opioid peptides –Met-enkephalin from
hypothalamus and pituitary what may suggest the involvement of opioid peptides into central
regulation of estrus cycle in sheep.
Supported by the State Committee for Scientific Research as a Solicited Project PBZ-KBN084/PO6/2003/3.10.
THE EFFECT OF PROLONGED PROGESTERONE TREATMENT ON THE
OPIOID-LIKE PEPTIDES IN THE SHEEP BRAIN
Krystyna Pierzchała-Koziec¹, Joanna Zubel¹, Olaf Katarzyński¹, Janusz Rząsa¹,
Andrzej DroŜdŜ²
¹Department of Animal Physiology, Agricultural University, 30-059 Cracow,
E-mail: [email protected], ²National Research Institute of Animal Production,
Balice, Poland
The regulation of reproduction process is very complicated and depends on many different
hormones at every level of hypothalamo-pituitary-gonadal axis. The effect of opioids on the
concentration of steroid hormones was documented in several experiments but the data
concerning the effect of progesterone or estradiol on the synthesis and concentration of
endogenous opioids are very scarse. Thus, the aim of the study was to assess the effects of
long term progesterone treatment on the Met-enkephalin concentration in sheep hypothalamus
and pituitary.
The experiment was carried out on 20 two years old ewes divided into control and three
experimental groups received progesterone (intravaginal sponges for 12 days) and injected
with saline (P), Met-enkephalin (P+MET, 1mg/kg i.v.) and naltrexone (P+NAL, 3mg/kg i.v.)
on days: 1, 2, 5, 12 and 15. Sheep from all experimental groups received an injection of 600
I.U. of PMSG after removing sponges. Two days later, hypothalamus and pituitary were
dissected out and directed to the estimation of Met-enkephalin concentration. The opioid level
was measured by radioimmunoassay method according to Pierzchała and Van Loon (1990).
Data was statistically analyzed by Fisher’s test followed by Duncan’s test.
Long term treatment with progesterone significantly decreased the concentration of Metenkephalin in the hypothalamus from 10.4±0.8 to 5.5±0.4 pmol/mg (P<0.001). Injections of
exogenous Met-enkephalin almost completely reversed the inhibiting effect of progesterone
what resulted in increased concentration of this peptide (8.9±0.6 pmol/mg, P<0.05 compare to
group received progesterone alone). Unexpectedly, injections of naltrexone, an opioid
receptor antagonist, did not change the inhibition of Met-enkephalin level caused by
progesterone (6.5±0.4 pmol/mg). In contrast, the concentration of opioid peptide in pituitary
was not changed by prolonged treatment with progesterone (11.9±1.2 compare to 9.7±0.7
pmol/mg observed in control sheep). Injections of Met-enkephalin increased the concentration
of endogenous peptide to 19.1±1.9 pmol/mg (P<0.01). Treatment with naltrexone did not
affect the concentration of Met-enkephalin in the pituitary (10.6±1.1 pmol/mg).
The results showed that progesterone affected the Met-enkephalin concentration mainly in
hypothalamus what may suggest that opioid interaction with GnRH is dominant during the
estrus cycle. It is interesting that progesterone did not changed the opioid concentration in
pituitary, probably Met-enkephalin released from hypothalamus was transported to pituitary
and stored there. Taking together, the results clearly showed the interaction of progesterone
and endogenous opioid peptides in the central nervous system.
Supported by the State Committee for Scientific Research as a Solicited Project PBZ-KBN084/PO6/2003/3.10
LONG FORM LEPTIN RECEPTOR IN THE HYPOTHALAMUS AND
PITUITARY OF PREGNANT PIGS
Nina Smolińska1, Gabriela Siawrys1, Tadeusz Kamiński1, Jadwiga Przała1,3, Alina
Gajewska2, Kazimierz Kochman2, Stanisław Okrasa1
1
10-718 Olsztyn-Kortowo 5, 2The Kielanowski Institute of Animal Physiology and
Nutrition, Polish Academy of Sciences, 05-110 Jablonna n. Warsaw, Poland,
3
Leptin is a hormone that plays an important role in the regulation of neuroendocrine and
reproductive functions through its effect on the hypothalamic-pituitary-gonadal axis after
binding to its receptors, of which a long form (OB-Rb) seems to be especially important. To
date, the leptin receptor has been identified in many organs including the hypothalamus and
pituitary, however, a period critical for successful embryo implantation has not been
previously examined.
The aim of the present study was to characterize the expression levels of porcine long form
leptin receptor in the different areas of the hypothalamus and pituitary on days 10-12 of the
oestrous cycle and 14-16 of pregnancy in pigs.
Total RNA was isolated from medial basal hypothalamus (MBH), preoptic area (POA), stalkmedian eminence (SME), anterior (AP) and posterior (NP) pituitary. The semiquantitative
PCR profiles consisted of an initial denaturing step at 95°C for 15 min, 38 cycles for OB-Rb
primers and 28 cycles for GAPDH primers (as an internal control) of denaturing at 94°C for 1
min, annealing at 58°C for 1 min, extension at 72°C for 1 min, and a final extension at 72°C
for 10 min. Protein (150 µg) extracted from all samples, was loaded onto a 5% gel and
subjected to SDS-PAGE. The gel was electroblotted onto a nitrocellulose membrane, which
then was blocked in TBST containing 5% skimmed milk powder for 5 h. Next, the membrane
was incubated with goat polyclonal OB-Rb antibodies for 12 h. The nitrocellulose was then
washed and incubated with rabbit anti-goat IgG for 1,5 h.
On days 10-12 of the oestrous cycle and 14-16 of pregnancy, the OB-Rb mRNA expression
in MBH and SME was at the same level. During early pregnancy, long form leptin receptor
gene expression in POA was suppressed in comparison with days 10-12 of the oestrous cycle
(p<0,05), whereas in AP and NP expression of the OB-Rb mRNA was greater during early
pregnancy (p<0,001). The sequence of the 382 bp PCR products showed a 100% homology
with the known gene sequence for pig OB-Rb. During early pregnancy Western blot analysis
revealed that level of OB-Rb protein in POA was greater in comparison with days 10-12 of
the oestrous cycle (p<0,05), whereas in AP expression of the protein was suppressed
(p<0,05). There were no significant differences in the OB-Rb protein levels between these
periods in MBH, SME and NP. Different pattern of OB-Rb gene and protein expression in
some of examined tissues suggests existence of tissue-specific posttranscriptional processing
of OB-Rb gene product.
In conclusion, the finding of changes in leptin receptor expression in the hypothalamus and
pituitary of pregnant pigs in relation to cyclic animals supports the idea that leptin may be
indirectly involved in the regulation of implantation-critical period of pregnancy.
This research was supported by the State Committee for Scientific Research (project: No PBZ
KBN-084/P06/2002 and No 0206.0805).
PATTERNS OF STORAGE AND SYNTHESIS OF GONADOTROPHIC
HORMONES IN THE PITUITARY CELLS DURING EARLY ADULTHOOD
OF FEMALE SHEEP
Marta Wańkowska, Anna Wójcik-Gładysz, Jolanta Polkowska
Nutrition, Polish Academy of Sciences, 05-110 Jabłonna, Poland,
The aim of this study was to describe the synthesis and storage patterns of luteinizing
hormone (LH) and follicle-stimulating hormone (FSH) in the population of the pituitary
gonadotrophic cells during early adulthood when ovarian cyclicity including ovulation
became manifested. An examination of the histomorphological changes has been made in the
ovary-intact sheep during two phases of the oestrous cycle, the late follicular phase (Day 15;
n=3) and the preovulatory phase (Day 17; n=3). Serum progesterone profiles attested, that it
was the second oestrous cycle after the first preovulatory surge that resulted in a full luteal
phase of 14-16-days’ duration. The immunoreaction was developed in the adenohypophyseal
cells by immunohistochemistry using polyclonal antibodies anti-oLHβ and anti-oFSHβ.
Staining of mRNAs in gonadotrophs was performed by hybridohistochemistry using
riboprobes made on homologous sheep cDNAs for LHβ or FSHβ subunits. The populations
of immunostained or hybridohistochemically stained gonadotrophs were optically detected
and digitally analysed. The percentage of the adenohypophyseal area occupied by
gonadotrophs containing in situ hybridised LHβ-mRNA or FSHβ-mRNA was higher (P<0.05)
at the preovulatory phase in comparison with the follicular phase. The percentage of
adenohypophyseal area occupied by both the immunoreactive (ir)-LHβ and ir-FSHβ cells was
lower (P<0.05) at the preovulatory phase compared with the earlier stage. In conclusion the
similar patterns of the pretranslational synthesis of the LHβ and FSHβ subunits and the
storage of the both gonadotrophins were observed. These findings may imply the similar
intrapituitary regulation of LH and FSH posttranscriptional processing during the pubertal
transition to the adulthood in the female sheep.
DIRECT EFFECT OF BOAR PHEROMONE 5α
α-ANDROSTENOL ON Gn-RH
AND OXYTOCIN RELEASE FROM PORCINE HYPOPHYSIS
Barbara Wąsowska, Jarosław Całka1, Jolanta Chłopek, Stanisława StefańczykKrzymowska
Department of Local Physiological Regulations, Institute of Animal Reproduction and
Food Research of The Polish Academy of Sciences, Tuwima 10, 10-747 Olsztyn,
Poland, E-mail: [email protected]; 1Department of Functional Morphology,
Division of Animal Anatomy, University of Warmia and Mazury in Olsztyn,
Oczapowskiego 13, 10-718 Olsztyn, Poland
The existence of a humoral pathway for priming action of boar pheromone 5α-androstenol, in
addition to the standard olfactory pathway typical for signaling pheromone, has been earlier
demonstrated in the gilts. Results of our study performed on gilts during sexual maturation
indicated the possibility of the action of 5α-androstenol as a priming pheromone in the central
neural system with omitting the olfactory system. This study were designed to test in in vitro
condition the direct effect 5α-androstenol on GnRH and oxytocin release from chosen
structures of the hypothalamus during sexual maturation in gilts.
The gilts at the age of 192-195 days (n=15) were killed and the following structures were
collected: preoptic area (POA), supraoptic nucleus (SOA), paraventricular nucleus (PV) and
mediobasal hypothalamus (MBH). The tissues were incubated with 5α-androst-16-en-3-ol in
doses 4x10-9 M and 2x10-8 M or with buffer solution (control). The incubation medium was
assayed for GnRH and oxytocin concentration by RIA. The effect of 5α-androstenol on
GnRH release from the hypothalamus structures was dependent on the dose. Lower dose of
5α-androstenol was effective only in MBH where its increased GnRH release by 255% in
comparison to control group. In contrary, this dose decreased GnRH release from PV by
182%. Higher dose of 5α-androstenol caused considerable increase of GnRH release from
SOA, POA and MBH by 347%, 194% and 86%, respectively. 5α-androstenol had weaker
effect on oxytocin release from the hypothalamus structures. The lower dose produced gentle
decrease of oxytocin release from SOA and POA by 16% and 20%, respectively, but the
increase of oxytocin release from PV by 11% in comparison to control group. Higher dose of
5α-androstenol increased oxytocin concentration in medium only after incubation of MBH
(by 43%).
The results confirmed the possibility of the direct effect of boar pheromone 5α-androstenol as
a priming pheromone on the release of GnRH and oxytocin from the hypothalamus structures
during attainment of the puberty in gilts. It is interesting, that in gilts during sexual maturation
more effective was higher dose of 5α-androstenol, whereas in pubertal gilts in similar
experiment condition – lower dose.
SEASONAL AND DOSE-DEPENDENT EFFECTS OF LEPTIN ON LH
SECRETION BY OVINE ADENOHYPOPHYSEAL CELLS
Dorota A. Zięba1, Beata Klocek1, Izabela Derecka1, Maciej Murawski, Katarzyna
Romanowicz2
1
Poland, E-mail: [email protected]; 2Department of Endocrinology, The
Kielanowski Institute of Animal Physiology and Nutrition, Jablonna, Poland.
Leptin regulates the secretion of several pituitary hormones (e.g. in pigs, cattle and sheep). In
the ovine adenohypophysis, while leptin receptor is expressed in almost 90% of the
gonadotropes in the pars tuberalis, it is observed in only about 30% of the cells in the pars
distalis. However, leptin plays an important role in the control of LH release and, as
demonstrated in cattle, mediates part of this effect directly at the adenohypophyseal level.
Despite the provision of unlimited food, sheep exhibit increased appetite and body weight as
well as high concentrations of leptin during long days (LD), and decreased appetite and body
weight in response to short days (SD). Current studies were performed on seasonal breeding
sheep with a focus on the putative relationships between photoperiod (melatonin) and leptin.
Melatonin receptors are localized at suprachiasmatic and dorsomedial nuclei, and leptin
receptors are localized in the accurate, ventromedial nuclei, and lateral hypothalamic area.
Connections exist between these hypothalamic nuclei and it is speculated that melatonin and
leptin feedback may be integrated ultimately within the paraventricular nucleus, a critical
regulatory center for appetite control. The hypothesis underlying the proposed studies is that
photoperiod affects leptin secretion specifically in SD, when endogenous leptin
concentrations are low and animals seem to be sensitive to leptin. Adenohypophyses were
collected from ewes during short and long photoperiods and cells were dispersed and cultured
for 3 days. On day 4, adenohypophyseal cells were treated with media alone (control) or
media containing 50 and 100 ng/ml recombinant ovine leptin (oleptin). Three independent
replications were performed. None of the doses of oleptin stimulated (P > 0.05) the release of
LH during the long day photoperiod and the amount of hormone which was release into media
was much lower (P < 0.05) compared to that in media from SD cells culture. During the
breeding season, (SD) oleptin at a dose of 50 ng/ml greatly increased (P < 0.01) LH release
(160%) compared to control-treated cells, which was confirmed in three replicates.
Furthermore, the dose of 100 ng/ml oleptin also lightly increased (P < 0.05) LH secretion
(122%). Results support previously demonstrated reports in cattle that leptin is able to act
directly at the anterior pituitary level to modulate LH release, and this effect is dependent
upon dose, with lower doses more effective. Moreover, results confirm findings that sheep
have a unique potential to be insensitive to leptin during LD and can serve as models for
reversible leptin insensitivity, a phenomenon observed in another seasonal species, the
Siberian hamster (Phodopus sungorus).
Session III
Regulation of corpus luteum function
Oral presentation
IN VIVO STUDIES ON THE VASCULAR FUNCTION IN THE BOVINE
OVARY: DETERMINATION OF BLOOD FLOW AND HORMONAL
SECRETION IN THE FOLLICLE AND CORPUS LUTEUM
Tomas J. Acosta
Laboratory of Reproductive Endocrinology, Faculty of Agriculture, Okayama
University, Okayama 700-8530, Japan, E-mail: [email protected]
Changes in ovarian blood flow are thought to be involved in the cyclic remodeling of the
ovary that occurs during follicular growth, ovulation, and development of the new corpus
luteum (CL). Therefore we conducted two in vivo experiments to assess ovarian blood flow
and factors that may induce these changes in cattle. In the first study, cycling cows were
examined by transrectal color and pulsed Doppler ultrasonography to determine the blood
flow within the wall of the preovulatory follicle and in the early CL. Ultrasonographic
examinations began 48 h after a luteolytic injection of prostaglandin (PG) F2 given at the
mid luteal stage. Cows showed LH-surge (Day 0) followed by ovulation. In the color Doppler
image of the preovulatory follicle, blood flow before the LH-surge was detectable in a small
area at the base of the follicle. However, clear increase in follicular blood flow was detected
synchronously with the initiation of the LH-surge. In the developing CL, the blood flow
gradually increased in parallel with the increase in CL volume and plasma concentration of
progesterone from Day 2 to Day 5. In the second study, the local release of vasoactive
substances, such as angiotensin (Ang) II, endothelin-1 and PG were determined by surgical
implantation of the capillary membranes of a microdialysis system (MDS) within the bovine
follicle wall and the developing CL along with ovarian venous and jugular catheters to collect
simultaneous, real-time information on their ovarian and systemic profiles. PGF2
concentrations in ovarian venous plasma (OVP) were higher during the pre-LH surge period
and dropped towards the onset of the LH surge. Acute increases in PGF2 concentrations in
the OVP and in the MDS perfusates were observed around the time of ovulation. The
structural and functional changes that take place in the ovary during ovulation and CL
development were closely associated with a local change in the blood flow. The overall
results suggest that a physiological relevant “cross-talk” between vascular components
(endothelial cells) and vasoactive substances exists in the bovine ovary.
NITRIC OXIDE AS A REGULATOR OF THE BLOOD FLOW AND
SECRETORY FUNCTION OF THE OVARY
Jerzy Jaroszewski
Department of Pathology and Pharmacology, Faculty of Veterinary Medicine,
University of Warmia and Mazury in Olsztyn, Oczapowskiego 13, 10-718 Olsztyn,
The ovary is a complex endocrine organ that undergoes profound structural and functional
changes during the oestrous cycle. The mechanism controlling these changes is complex and
is depending on the innervation, endocrine regulation and blood flow. Haemodynamic
changes are observed in all phases of the ovarian tissue remodelling, and corpus luteum is one
of the most highly vascularized organ, receiving the greatest rate of blood flow per unit of
tissue of any organ in the body. Recently, it has been documented that nitric oxide may play
an important role in the regulation of blood vessel tension. In blood vessels of the
reproductive tract, nitric oxide is released from endothelial cells, nerve fibres and vascular
smooth muscle cells. Histochemical study have demonstrated a higher nitric oxide synthaserelated NADPH-d activity in the bovine ovarian artery during the middle-luteal phase, as
compared to the follicular phase. Moreover, nitric oxide donor caused higher relaxation of the
porcine ovarian artery segments collected from luteal phase, than in the vessels from the
follicular one. Similarly, intensification of vasocontractile action of norepinephrine in the
porcine ovarian artery pretreated with nitric oxide synthase inhibitor was higher in the luteal,
than in the follicular phase.
Color Doppler ultrasonography study showed that after prostaglandin F2α treatment blood
flow within the bovine corpus luteum initially increased within first two hours, decreased to
the level before drug administration at 4 hour and then decreased to a lower level beginning at
8th hour. Another study showed that plasma concentration of nitrite/nitrate (stable metabolites
of nitric oxide production) was also elevated during the first two hours after prostaglandin F2α
treatment and thereafter slowly decreased. These data indicate that nitric oxide can be a factor
responsible for the increase in the blood flow after prostaglandin F2α treatment. It has been
documented that nitric oxide donor strongly stimulates production of prostaglandin F2α and
leukoriene C4 (two luteolytic factors), as well as prostaglandin E2, a luteotrophic factor in the
corpus luteum, while nitric oxide synthase inhibitor has an opposite action. Both nitric oxide
and prostaglandin E2 are potent vasodilators. Therefore, during first hours after prostaglandin
F2α treatment they relax vascular smooth muscle cells and maintain the luteal blood flow
required for invasion of immune cells into the corpus luteum during luteolysis.
In conclusion, nitric oxide can affect ovary function by direct influence on its secretion as
well as by the changes in the blood flow through this gland.
AUTOREGULATION OF LUTEAL FUNCTION IN CATTLE
Jan Kotwica
Institute of Animal Reproduction and Food Research, Polish Academy of Sciences,
10-718 Olsztyn-Kortowo, Poland, E-mail: [email protected]
In further studies it has been found that P4 can affect gene expression of enzymes involved in
the luteal steroidogenesis. Concentrations of mRNA for steroid acute regulatory protein
(StAR), 3β-HSD, cytochrome P450scc and concentration of their protein products were
increased in luteal cells stimulated with LH, PGE2 and P4. While NO donor (S-NAP)
inhibited both concentrations of mRNA and protein for StAR, 3β-HSD, cytochrome P450scc.
The corpus luteum (CL) is a transient reproductive gland that produces progesterone (P4), a
hormone required for the establishment and maintenance of pregnancy. Hormonal and neural
signals from brain centres are critical for normal luteal function in domestic animals,
including cow. However, CL is also able to synthesize itself a lot of active substances which
regulate P4 synthesis/secretion independently on central regulation.
Bovine CL can to synthesize noradrenaline (NA) from dopamine as a precursor. Further, NA
increases the activity of 3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P-450scc
and PGA the crucial enzymes in the synthesis of P4 and OT. Thus NA stimulates directly P4
synthesis and, by the post-translational processing of ovarian OT synthesis which, in turn,
affects P4 synthesis. By contrast, P4 reduces the rate of NA degradation by inhibiting activity
of MAO and COMT, the enzyme primarily responsible for the intracellular degradation of
catecholamines, and so increasing the half-life of the neurotransmitter. Thus it is assumed that
NA is involved in basal P4 synthesis/secretion and, furthermore NA protects and supports the
CL in situations which could impair CL function.
P4 itself, with an intensity comparable to LH, is able to increase activity of 3β-HSD and
cytochrome P-450scc in luteal cells, while P4 antagonist diminishes influence of P4. These
effects were seen only in CL from days 5-10 of the cycle. Bovine CL exhibited specific
nuclear staining for P4 receptor (PR) more evident on days 5-10 of the oestrous cycle than on
any other stage of the estrous cycle. Moreover, Western blot analysis revealed that on days 516 of the cycle, CL contained 3-times more of isoform B than A of PR, in contrast to the
endometrium which had more PR-A. We conclude that P4 supports its own synthesis and the
specific PR within luteal cells can be involved in this process. But non-genomic mechanism
of P4 effect on luteal cells cannot be excluded.
Next we found that P4 and PGE2 increased bcl-2 expression (protects cells from apoptosis)
and decreased level of active caspase-3 (which is pivotal executor of apoptosis) in luteal cells.
Expression of bcl-2 was inhibited by staurosporine 3,3’,4,4’-tertrachlorobiphenyl (PCB)-77.
Moreover, aminoglutethimide (blocker of cytochrome P450scc), NO donor (NONOate),
PCB-77 and staurosporine increased in cells bax expression (associated with cell death). The
same factors increased caspase-3 activity in the cells. The ratio of bax/bcl-2 was decreased by
P4 and increased by staurosporine, NONOate, aminoglutethimide, and PCB. Thus, high
concentration of P4 in CL protects luteal cells from apoptosis while disruption of
steroidogenesis can induce cell death program. The data suggest that CL has a wide area of
autonomy and it can itself regulate own function.
REGULATION OF FUNCTIONAL LUTEOLYSIS IN RODENTS
Masugi Nishihara
Department of Veterinary Physiology, Veterinary Medical Science, The University of
Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan,
In the corpus luteum of rats and mice, 20α-hydroxysteroid dehydrogenase (20α-HSD)
catalyzes the conversion of progesterone to a biologically inactive metabolite, 20αdihydroprogesterone (20α-OHP). The reduction of progesterone by 20α-HSD is recognized
to play an important role in the functional luteolysis in these rodent species. In these rodents,
newly formed corpora lutea begin to express 20α-HSD within 2-3 days after ovulation. The
decreased progesterone levels allow the subsequent ovulations every 4-5 days unless the
cervix of the uterus is stimulated by copulation. Cervical stimulation induces the prolactin
surge from the anterior pituitary as a neuroendocrine reflex, which exerts luteotrophic actions
by suppressing the gene expression of 20α-HSD thereby maintains the luteal function during
pseudopregnancy or pregnancy. At the end of pseudopregnancy or pregnancy, the expression
of 20α-HSD is markedly elevated by prostaglandin F2α signaling with a resultant decrease in
progesterone secretion. We have cloned the mouse 20α-HSD gene and found that mouse
20α-HSD genomic structure spanned approximately 18 kb from exon 1 to exon 9. The
similarity in the arrangements of exons between mouse and human 20α-HSD genes indicates
their close relationship in the evolutional process. A reporter assay using reporter constructs
of various lengths of the 5'-flanking region revealed that the region between -83 and -60
upstream of the transcription start site was essential for transcriptional activity. Furthermore,
mutational analysis demonstrated that a putative Sp1 site in this region was critical in the
expression of the reporter gene. To determine the actual contribution of 20α-HSD to the
functional luteolysis, we generated a strain of mice with targeted disruption of 20α-HSD
gene. In the 20α-HSD-/- mice that were deficient with the genomic region essential for the
catalytic reaction (exons 2-4), neither 20α-HSD activity in the corpus luteum nor an increase
in serum concentrations of 20α-OHP during pseudopregnancy and pregnancy was detected.
The durations of estrous cycle and pseudopregnancy were extended for a few days. However,
the functional luteolysis was achieved at the term of pregnancy, and the duration of pregnancy
was not extended even in 20α-HSD-/- mice. These observations support the notion that 20αHSD is indeed involved in functional luteolysis at the term of pseudopregnancy as well as
during the estrous cycle, but at the same time suggest that another mechanism(s) yet
unclarified is also prerequisite for the functional luteolysis in rodents.
REGULATION OF APOPTOSIS IN THE HUMAN CORPUS LUTEUM
Norihiro Sugino
Division of Obstetrics and Gynecology, Department of Reproductive, Pediatric and
Infectious Science, Yamaguchi University School of Medicine, Minamikogushi 1-1-1,
Ube, 755-8505 Japan, E-mail: [email protected]
Ephemerality and prolongation of corpus luteum (CL) function have been a matter of concern
for many years. In humans, the life span of the CL is 14 days after ovulation if pregnancy
does not occur, but the CL can be further maintained if pregnancy occurs. The regression of
the CL is necessary for follicular development in the next reproductive cycle whereas the
rescue of the CL is essential for the maintenance of pregnancy. Accumulating data have
shown that apoptosis plays important roles in the regulation of the life span of the CL in
several species. The present study was, therefore, undertaken to investigate the regulation of
apoptosis in the CL in humans. For this purpose, the frequency of apoptosis and expression
of Bcl-2 (inhibitor of apoptosis) and Bax (inducer of apoptosis) were examined in the CL
during the menstrual cycle and in early pregnancy. The number of apoptotic cells, determined
by TUNEL method, was much greater in the regressing CL than that in the midluteal phase
CL whereas there were no apoptotic cells in the CL of pregnancy. Immunohistochemistry
revealed that Bcl-2 expression was observed in luteal cells in the midluteal phase and early
pregnancy, but not in the regressing CL, whereas Bax expression was observed in the
regressing CL, but not in the midluteal phase and early pregnancy. bcl-2 mRNA levels in the
CL during the menstrual cycle were highest in the midluteal phase and lowest in the
regressing CL. In the CL of early pregnancy, bcl-2 mRNA levels were higher than those in
the midluteal phase. In contrast, bax mRNA levels were highest in the regressing CL and
remarkably low in the CL of early pregnancy. Western blot analyses also showed the result
similar to changes in mRNA expression of Bcl-2 and Bax. When corpora lutea of the
midluteal phase were incubated with hCG, hCG increased the mRNA and protein levels of
Bcl-2 and decreased those of Bax. In conclusion, apoptosis play important roles in the
regulation of the life span of the human CL and is regulated by Bcl-2-Bax system.
Poster presentation
EFFECT OF PROSTAGLANDIN F2αα ON BOVINE CORPUS LUTEUM
DEPENDS ON CELL COMPOSITION AND CONTACT
Anna Korzekwa1, Izabela Wocławek-Potocka1, Jerzy J. Jaroszewski2, Mamadou M.
Bah1, Beata Barszczewska2, Dariusz J. SkarŜyński1
1
Department of Reproductive Immunology, Institute of Animal Reproduction and Food
Research, PAS, 10-747 Olsztyn, Poland; 2Department of Pathology and
Pharmacology, Faculty of Veterinary Medicine, University of Warmia and Mazury,
10-718 Olsztyn, Poland.
It is well known that prostaglandin (PG)F2α is main luteolytic factor in cattle. However, its
direct luteolytic effect on steroidogenic cells of the bovine corpus luteum (CL) is
controversial. Therefore, we suggest that the contact between different CL cell types is
necessary for induction of PGF2α-dependent luteolytic mechanisms in bovine CL. The aim of
this study was to compare PGF2α influence on the bovine CL using different in vivo and in
vitro methods and to show whether PGF2α action on the CL depends on the cell to cell contact
and cell composition. In Exp. 1, the heifers (n=4; 15 Day of the estrous cycle) were injected
with PGF2α analogue (aPGF2α; 25 mg) into aorta abdominalis. PGF2α induced temporary (in
minutes) increase of P4 level followed by the CL regression after 24 h (the decrease of P4
level; P<0.05). In Exp. 2, PGF2α in three doses (2,5; 5 or 10 mg; each n=3; 15 Day of the
estrous cycle) was perused into CL via microdialysis system in vivo. P4 concentrations in the
perfusate samples from CL treated with 5 mg of aPGF2α increased 0.5 h after drug perfusion
in comparison to the period before treatment. Concentrations of P4 in the jugular plasma
samples after first hour from the drug perfusion started to decrease and at 24 h were lower
compared to the period before treatment. In Exp. 3, the direct effect of PGF2α (10-6M) on the
enzymatically isolated CL steroidogenic cells (Day 15-17 of the estrous cycle) was examined.
Prostaglandin F2α increased P4 production in steroidogenic CL cells (P<0.05). In Exp. 4, the
role of paracrine regulations in PGF2α-induced regression of the CL was studied. The cocultures of steroidogenic, endothelial and/or immune cells were incubated 18 h in glass tubes
with PGF2α (10-6M). Although, PGF2α increased P4 secretion (135 % of control) in the
homogeneous steroidogenic cell culture, we observed the decrease of P4 secretion (88 % of
control) in co-cultures of CL cells. In summary, the contact between three main types of CL
cells is necessary to appear the luteolytic effect of PGF2α on the steroidogenic bovine CL
cells. Moreover, some of endothelial and immune cell products participate in PGF2α−induced
luteolysis.
Supported by the basic fund of PAS.
ANALOGUES OF PROSTAGLANDIN (PG)F2αα DIFFERENTLY REGULATE
THE FUNCTION OF BOVINE CORPUS LUTEUM (CL)
Wojciech Pilawski, Katarzyna M. Deptuła, Katarzyna K. Piotrowska, Anna Korzekwa
& Dariusz J. SkarŜyński1
Research, PAS, 10-747 Olsztyn, Poland
Analogues of PGF2α (aPGF2α) may have negative influence on reproductive functions of
cows. Pharmacological manipulation of the estrous cycle may cause various ovarian
dysfunctions including abnormal progesterone (P4) production. Therefore, in the present study
we examined the direct effects of different aPGF2α on the secretory function of steroidogenic
CL cells and on the motor function of ovarian artery in vitro. In Exp. 1 enzymatically isolated
steroidogenic CL cells (5-8 day of estrous cycle), were incubated natural PGF2α (Sigma) and
three different aPGF2α: dinoprost (Dinolityc, Upjohn-Pharmacia), cloprostenol (Bioestrovet,
Biowet) or luprositiol (Prosolvin, Intervet), all substances were at doses 10-7-10-5M. In the
medium concentration of P4 and PGE2 were measured by EIA. Natural PGF2α and dinoprost
slightly stimulated P4 and PGE2 secretion in the cells (P<0.05). There were no significant
differences in the action of natural PGF2α and dinoprost in respect to P4 and PGE2 (P>0.05).
Luprositiol strongly stimulated P4 (P<0.001). In contrast, cloprostenol inhibited P4 secretion
(P<0.05). In Exp. 2. we examined possible influence of aPGF2α on the ovarian artery
contraction. Ovarian arteries were collected from the broad ligament of the uterus (5-8 days of
the cycle). The contractions of circles of ovarian arteries (3-5 mm) were measured in Hugo
Sachs Electronic using transducer F-30. Arteries were stimulated with natural PGF2 α and with
three analogues: dinoprost, cloprostenol or luprositiol (10-7-10-5M). The strongest effect on the
ovarian artery contraction was observed after stimulation with luprositiol (P<0.001). The
stimulatory effect on arterial motor function was also observed after cloprostenol treatment
(P<0.05). Dinoprost and natural PGF2α did not affect contraction of the artery (P>0.05).
During termination of CL function not all analogues could reflect physiological effects of
endogenous PGF2α. The only effects of dinoprost on CL function in vitro were conferrable
and similar to the action of PGF2α. In terms of luteal P4 secretion, cloprostenol has opposite
effect to natural PGF2α action. Regarding the regulation of ovarian artery contraction, the
effects of cloprostenol and luprositol were much stronger than natural PGF2α. The highest
contractility of ovarian artery may result in disorders of the ovarian blood flow during
luteolysis, at follicular development and maturation. Our recent in vivo studies revealed that
synchronization of the estrus using analogues of PGF2α decreases sensitivity of the CL on
luteotropic factors. Thus, lower P4 production and reduction in CL sensitivity on luteotropic
factors might be the reason of the early embryo mortality in embryo-recipient cows.
Supported by the Grant KBN-MNiI 3P06K 047 25.
REGULATION OF APOPTOSIS IN BOVINE LUTEAL CELLS
Robert Rękawiecki, Ewa Liszewska, Jan Kotwica
Institute of Animal Reproduction and Food Research; Polish Academy of Sciences,
The corpus luteum (CL) is a transient reproductive gland that produces P4 the steroid
hormone responsible for the establishment and maintenance of early pregnancy. In the
absence of pregnancy the CL decreases the production of P4 followed by the regression of CL
structure, which includes apoptosis of cells. The aim of this study was to investigate the effect
of hormones and factors, that either support or impair P4 synthesis, on apoptosis in luteal
cells.
Bovine luteal cells from days 6-10 and 11-15 of the estrous cycle were incubated (6h) with
factors which support: LH (100ng/ml); PGE2 (10-6M); or disrupt steroidogenesis:
aminoglutethimide (1.5x10-4M); NO donor-NONOate (10-4M); polychlorinated biphenyl
(PCB)-77 (100 ng/ml), and staurosporine (1 µg/ml) which was used as negative control.
Moreover, cells were treated with P4 (10-5M). Concentrations of P4 in medium after treatment
was determined. In Experiment 1, RNA from luteal cells, was isolated and subjected for RTPCR. Next cDNA was amplified by PCR using primers to determine bax expression
(associated with cell death), bcl-2 expression (protects cells from apoptosis). Products of PCR
separated on gel were analyzed using Kodak EDAS290 system. In Experiment 2 luteal cells,
treated as above, after 24h of incubation were lysed. Thereafter, activity of caspase-3 was
analyzed by means of Caspase 3 Assay Kit.
Progesterone increased (P<0.001) whereas staurosporine decreased (P<0.01–0.001) bcl-2
expression in both stages of the estrous cycle. The expression of bcl-2 in luteal cells from day
11-15 of the estrous cycle was stimulated by PGE2 (P<0.01) and inhibited by PCB77
(P<0.01). In luteal cells from both stages of the cycle treated with aminoglutethimide,
NONOate and staurosporine bax mRNA was increased, but this effect was more evident in
cells from day 11-15 (P<0.001) than from day 6-10 (P<0.05 – 0.01) of the estrous cycle. PCB77 stimulated the expression of bax in cells from 11-15 days of cycle (P<0.01) only.
Treatment of luteal cells with P4 (P<0.05) and PGE2 (P<0.01) for 24h decreased level of
active caspase-3 while aminoglutethimide (P<0.05), NONOate (P<0.05) and staurosporine
(P<0.001) increased activity of caspase-3 in the cells.
The overall results suggest indirectly that high intra-luteal concentrations of P4 suppresses
apoptosis in bovine luteal cells through the stimulation of bcl-2 mRNA expression and
inhibition of caspase-3 activity. Whereas impairment of steroidogenesis, followed by the
decline of P4 synthesis, increases both bax mRNA expression and caspase-3 activity and
concomitantly it decreases bcl-2 mRNA expression. This indicates that stimulation of P4
synthesis protects luteal cells from death, whereas the decrease of luteal cells ability to
produce P4 turns on program of apoptosis.
Supported by grant (PBZ-KBN-084/P06/2002).
POSSIBLE ROLES OF INTERLEUKIN-4 AND INTERLEUKIN-6 IN
REGULATING LUTEOLYSIS IN PIGS
Ryosuke Sakumoto1, Kiyoshi Okuda2
1
Department of Physiology and Genetic Regulation, National Institute of
Agrobiological Sciences, 305-0901 Tsukuba, Japan, 2Laboratory of Reproductive
Endocrinology, Okayama University, 700-8530 Okayama, Japan,
Inflammatory cytokines are known to regulate ovarian function in many species, and to have
luteolytic actions in corpus luteum (CL). The objective of the present study was to investigate
whether interleukin (IL)-4 and IL-6 play roles in the mechanism of luteolysis in porcine CL.
The gene expressions of IL-4, IL-6 and their specific receptors (IL-4R and IL-6R) were
determined in the porcine CL obtained from early (days 2-5 after ovulation), mid- (days 812), late (days 14-17), and regressed (days 19-21) stages of the estrous cycle (4 pigs per each
stage). Protein expressions of IL-4 and IL-6 were also examined in the CL of each stage.
Moreover, the effects of these cytokines on progesterone (P4), estradiol-17β (E2) and
prostaglandin (PG) F2α secretion by cultured luteal cells were studied. Proteins as well as
mRNAs of IL-4 and IL-6 were detected in the CL at all luteal stages. The protein
concentrations of IL-4 and IL-6 in the CL did not significantly alter during the estrous cycle.
Furthermore, mRNAs of the IL-4R and IL-6R were clearly expressed in the CL throughout
the estrous cycle. Real-time PCR analysis revealed that IL-6R mRNA expression was higher
in the regressed CL than in the CL at other stages (P<0.01). The abundance of IL-4R mRNA
was constant in the CL during the estrous cycle. When cultured luteal cells obtained from
mid-stage CL were exposed to IL-6 (1-100 ng/ml), it inhibited P4 and E2 secretion by the
cells (n=6; P<0.05). Although IL-4 (1-100 ng/ml) did not significantly alter P4 secretion, it
inhibited E2 secretion by the cells in a dose-dependent fashion (n=6; P<0.05). In addition, IL4 and IL-6 slightly, but not significantly, inhibited PGF2α secretion by the cells. These results
suggest that IL-4 and IL-6 are locally produced in the porcine CL during the estrous cycle,
and that they inhibit steroid production from luteal cells via their specific receptors.
Collectively, we suggest that both IL-4 and IL-6 act as intraluteal regulators to establish
luteolysis in the porcine CL.
This Research was supported by Grants-in-Aid for Scientific Research (No. 14760183, No.
14360168) from the Japan Society for the Promotion of Science (JSPS).
SYNCHRONIZATION OF OVARIAN CYCLE IN CYNOMOLGUS (Macaca
fuscicularis) AND JAPANESE (Macaca fuscata) MONKEYS
Ryuzo Torii, Hideaki Tsuchiya, Norio Okahara, Junko Narita, Takahiro Nakagawa,
Tatsuyuki Takada
Research Center for Animal Life Science, Shiga University of Medical Science
Seta Tsukinowa-cho, Otsu, Shiga 520-2192, Japan,
Purpose: The Center continues to advance AI, IVF-ET, and ICSI-ET technique in
cynomologus and Japanese monkeys, but availability of ET recipients remains difficult. Here
we report results of efforts to schedule recipient availability in accordance with investigation
timetables via ovulation synchronization.
Materials and methods: Subjects comprised 10 cynomolgus (menstruation invisible in 6,
menstruation periodically visible in 4) and 5 Japanese monkeys (non-mating season).
Progesterone (P) administered to all subjects via ventral SC implantation of silicone tubes
containing P powder. Tubes removed after a set time period. Cephalic vein blood draws
conducted twice-weekly, results measured by EIA.
Results: In all subjects serum P increased following P tube implantation and rapidly decreased
upon removal. Menstruation occurred 3 to 4 days after removal in 9 of 10 cynomolgus and 3
of 5 Japanese monkeys. Menstruation occurred in remaining cynomolgus and an additional
Japanese monkey 8 to 10 days after removal. Ovulation confirmed by laparoscopy in 3 of 10
cynomologus and 3 of 4 Japanese monkeys after first menses. Ovulation confirmed after
second menses after removal in 3 of 7 cynomologus.
Discussion: P tube implantation/removal induced menstruation at high rates in all subjects.
Ovulation occurred in about 70% of induced subjects. Results indicate feasibility of
scheduling IVF-ET/ICSI-ET recipients in accordance with investigation timetables via
ovulation synchronization.
THE EFFECT OF STEROID HORMONE INFUSION INTO THE OVARIAN
ARTERY ON INHIBIN CONCENTRATION IN PORCINE FOLLICULAR
FLUID
Barbara Wąsowska, Jolanta Chłopek
Food Research of The Polish Academy of Sciences, Tuwima 10, 10-747 Olsztyn,
Our earlier studies have shown that retrograde transfer of steroid hormones in the periovarian
vascular complex increased their concentration in blood supplying the ovary. The purpose of
the study was to establish whether elevated supply of steroid hormones: progesterone (P4),
estradiol (E2) and testosterone (T) into the ovary during luteal phase of the estrous cycle may
regulate the ovarian function through feedback mechanism based on inhibin secretion.
Sexually matured gilts (n=9) after two controlled estrous cycle were used in this study. On
day 9 of the estrous cycle, the gilts were anaesthetized and the catheters were inserted into
branch of uterine artery connected by anastomoses with ovarian artery. The cannulated uterine
artery branch was ligatured below anastomoses. This way of cannulating the uterine artery
branch makes it possible to infuse hormones to the ovary without the necessity of surgical
intervention in the area of the periovarian vascular complex. During next three days, P4, E2
and T were infused into the right ovarian artery (experimental ovary – EO) and saline was
infused into the left ovarian artery (control ovary – CO). The doses of hormones were adopted
on the base of the rate of the retrograde transfer of these hormones in luteal phase. The
progesterone infusion rate was 0.62 µg/min (a physiological dose), 1.25 µg/min and 1.87
µg/min on the first, second and third day, respectively. The estradiol infusion rate was 156
pg/min (a physiological dose), 312 pg/min and 486 pg/min on the first, second and third day,
respectively. The testosterone infusion rate was 249.6 pg/min (a physiological dose), 499.2
pg/min and 748.8 pg/min on the first, second and third day, respectively. The infusion was
performed for 6 hours per day. Seven days after infusions were stopped, the gilts were
humanely killed and follicular fluid was collected from EO and CO. In follicular fluid
samples concentration of inhibin was determined by RIA method. Mean hormone
concentration from EO and CO were compared by one-way analysis of variance followed by
unpaired Student’s t test. The infusions of progesterone decreased significantly the
concentration of inhibin in EO: 4454±520 pg/ml in comparison to the CO: 7810±453 pg/ml,
P<0.05 in progesterone-treated group. The hormone concentration did not differ significantly
between: EO: 7219±1023 pg/ml and CO: 7592±931 pg/ml in estradiol-treated group and
testosterone-treated group (EO: 6953±823 pg/ml and CO: 6999±1300 pg/ml).
In conclusion, it was demonstrated that experimental elevation of progesterone concentration
in blood supplying the ovary changed the secretion of inhibin which modulate ovarian
steroidogenesis during luteal phase of porcine estrous cycle.
Supported by State Committee for Scientific Research (3P06D 02323).
RETROGRADE TRANSFER OF TESTOSTERONE TO THE PORCINE
OVARY IN LUTEAL AND FOLLICULAR PHASES OF THE ESTROUS
CYCLE IN VIVO
Barbara Wąsowska, Stanisława Stefańczyk-Krzymowska and Jolanta Chłopek
Division of Reproductive Endocrinology and Patophysiology, Institute of Animal
Reproduction and Food Research of The Polish Academy of Sciences, Tuwima 10,
The present study is the first in which the efficiency of the retrograde transfer of testosterone
in periovarian vascular complex has been calculated in pigs in in vivo conditions. Crossbred,
sexually mature gilts, after two controlled estrous cycle were used in this study. On days 1719 (follicular phase, n=5) or days 10-12 (luteal phase, n=12) of the estrous cycle, the gilts
were anaesthetized. Catheter was inserted into a branch of the uterine artery connected by
anastomoses with the ovarian artery. Cannulated uterine artery branch was ligatured below
anastomoses. This way of cannulating the uterine artery branch makes it possible to collect
arterial blood supplying the ovary. Additional catheters were inserted into ovarian and jugular
veins. The reproductive tract was replaced in the abdominal cavity in its physiological
position. Simultaneous venous blood samples were collected every 5 min during a 30-min
period from the jugular vein and ovarian vein, and arterial blood supplying the ovary. The
blood flow recorded in the ovarian artery was in follicular phase 19.9±0.3 ml/min and in
luteal phase 25.9±0.6 ml/min. The plasma samples were assayed for testosterone
concentration by RIA method. Mean concentration of testosterone in systemic blood reaching
the initial part of the ovarian artery was 13.8±0.5 pg/ml in luteal phase and 12.1±0.3 pg/ml in
follicular phase. The concentration of testosterone in blood supplying the ovary averaged in
luteal and follicular phase of the estrous cycle 15.9±0.9 pg/ml and 14.2±0.5 pg/ml,
respectively and in both phases of the estrous cycle was higher than in systemic blood. The
concentration of testosterone in ovarian vein of gilts was in luteal phase and follicular phase
178.6±9.1 pg/ml and 44.5±0.8 pg/ml, respectively and the values differed significantly
(P<0.01). The efficiency (E) and rate (R) of the retrograde transfer were calculated according
the formulas: E=(a-b)x100/c and R=(a-b)xV, where a - hormone concentration in blood
supplying the ovary, b – hormone concentration in systemic blood reaching the ovarian artery,
c - hormone concentration in the ovarian venous effluent and V – volume of blood plasma
flowing through the ovarian artery (in ml/min). The efficiency of retrograde transfer of
testosterone to the ovary was in luteal phase: 1.11±0.6% and follicular phase: 4.72±0.8% of
testosterone concentration the ovarian effluent. The efficiency was not dependent on the
concentration of hormone in the ovarian venous blood. The rate of testosterone retrograde
transfer was 32.6±0.6 pg/min and 25±0.7 pg/min in luteal and follicular phase, respectively.
We concluded that retrograde transfer of testosterone in periovarian vascular complex,
increasing their concentration in blood supplying the ovary may modify the secretion of the
ovarian hormones in follicles and corpus luteum.
Supported by State Committee for Scientific Research (3P06D 02323).
Session IV
Andrology I
Oral presentation
NANOS1 PROTEIN INTERACTS WITH TRANSLATION FACTORS IN
HUMAN GERM CELLS
Barbara Ginter-Matuszewska, Kamila Kusz, Anna Spik, Katarzyna Matylla, Jadwiga
Jaruzelska
Institute of Human Genetics, Polish Academy of Science, ul. Strzeszyńska 32, 60-79
Poznań, Poland, e-mail: [email protected]
Nanos is translational repressor in body patterning and germ cells development in Drosophila.
We have previously identified human homologous protein (NANOS1) which is expressed in
the germ line of man, and is crucial for self-renewal of spermatogonia stem cells. The role of
NANOS1 in translation would potentially involve interaction with translation factors. To test
this possibility we performed a yeast two-hybrid search for NANOS-binding proteins. For that
purpose, we cloned NANOS1 cDNA in pAS2.1 vector in fusion with GAL4 DNA-binding
domain. This construct was subsequently used as the “bait” to screen human testis cDNA
library in yeast. We identified three candidate NANOS1-binding proteins, all of them being
expressed in the male germ-line: 1/ PABPC3, is a poly(A)-binding protein specifically
expressed in round spermatids. Interaction with PABPC3 indicates that besides spermatogonia
self-renewal, NANOS1 might also play a role in completion of meiosis. Involvement of
PABPC3 at that stage of spermatogenesis was further supported by our finding that it binds
VASA, a meiotic DEAD-box helicase. 2/ HSPC021 protein is tightly associated with
mammalian translation initiation factor eIF3 in various cell types. 3/ DEAD-box RNA
helicase DP103 has potential role in translation initiation and RNA splicing. We found that
DP103 also interacts with PUMILIO2, a homologue of Drosophila Pumilio, which together
with Nanos acts as co-respressor of mRNA translation. These initial results indicate that
NANOS1 may be involved in translational machinery in human germ cells. Further
experimental approaches aiming at validation of interactions identified in yeast are underway.
This study was supported by State Committee for Scientific Research (6PO5E 001 21 and 3
PO5E 013 25).
THE ROLE OF OCT-4 TRANSCRIPTION FACTOR IN DEVELOPMENT OF
THE HUMAN MALE GERM CELLS
Charles Koyias, Anna Spik, Jadwiga Jaruzelska
Institute of Human Genetics Polish Academy of Sciences, 32 Strzeszyńska, 60-479
Poznań, Poland, [email protected]
OCT4 transcription factor is expressed in embryonic stem cells in mammals, but disappears at
the onset of embryonic differentiation. However, it remains persistent in primordial germ
cells. In mice, OCT4 is expressed only in spermatogonia stem cells, and ceases once meiosis
is initiated. The germ-line expression profile of OCT4 prompts us to hypothesize that it is a
key factor in genetic control of the fate of spermatogonia stem cells (self-renewal versus
differentiation). To dissect this genetic control we attempted to identify proteins interacting
with OCT4 in the male germ cells by yeast two-hybrid screening of the human testis cDNA
library. The “bait” was generated by OCT4 amplification from testis cDNA and cloned in
fusion with DNA-binding domain of the yeast GAL4 transcription factor. This construct was
used for yeast transformation together with testis cDNA cloned in fusion with the GAL4
trans-activation domain. Clones were isolated, analyzed, and aligned with GeneBank
sequences. Interestingly, our study revealed that in the human testis tissue OCT4 is
transcribed from retroposon, while the ancestral OCT4 gene which is characterized by
exon/intron structure is expressed in embryonic stem cells. This differential expression may
indicate that retroposition resulted in a new function of OCT4 gene, and namely in germ cell
development. We found that OCT4 interacts with Snapin, a component of SNARE complex
of proteins which is expressed in brain, and is required for synaptic vesicle docking and
fusion. Although Snapin mRNA is highly expressed in human testis tissue, its role in
reproduction was not reported so far. Further studies are underway to elucidate the role of
OCT4-Snapin interaction in germ cell development.
This study was supported by the State Committee for Scientific Research 3PO5A 005 25.
FACTORS AFFECTING MALE FERTILITY
Mariola Marchlewicz
Department of Histology and Embryology, Pomeranian Medical University,
70-111 Szczecin, Al. Powstańców Wielkopolskich 72, Poland,
Number of spermatozoa in ejaculate has significantly decreased during the past 60 years.
Reasons of impaired sperm function are thought to be complex and unpredictable because
influence of genetic, environmental, occupational factors is added up. The substances present
in the environment which imitiate estrogen action are also responsible. They are widespread,
resistant to biodegradation, lipophylic and bioavailable. Chemical compounds containing
phenolic ring like: chlorinated pesticides, polychlorinated biphenyls (PCBs), dioxins (TCDD)
and phytoestrogens display estrogenic activity.
Action of these factors on the gonad may begin already in fetal stage i.e. many years before
sexual maturity and their effects may be reflected in the functional state of the mature testis.
About 8. weeks following fertilization Leydig cells begin to synthesize androgens required for
further development of the reproductive system. In Sertoli cells testosterone is converted by
means of 5 alpha-reductase to DHT, which is necessary for testis descent and external sexual
organ differentiation. Sertoli cells also contain P450 cytochrome aromatase converting
androgens to estrogens. The synthesis of insulin-like factor 3 (Insl-3) in Leydig cells may be
inhibited during early stages of development by exposition to exogenous estrogens. Insl-3
plays an important role in transabdominal testis descent. Chemical substances acting as
estrogens may also cause androgens deficiency by inhibiting expression of steroidogenic
enzymes pathway. Hypospadiasis or incomplete testicular descent (cryptorchidism) may be
the consequence. The number of Sertoli cells is of particular importance for the number of
spermatozoa produced by the mature gonad. During the spermatogenesis only a certain
number of germinal cells may be bond to Sertoli cell with appropriate junctions and undergo
its protective and regulatory influence. Sertoli cells proliferation takes place primarily at fetal
and neonatal life and is influenced by FSH. Chemicals with estrogenic activity could lead to
permanent decrease in Sertoli cells number, testis size and sperm production. Exogenous
estrogens act through ERβ in Sertoli cells and inhibition of FSH release from pituitary gland.
Gonocytes (fetal germ cells) also appear to be affected adversely by the abnormal testicular
environment.
During adulthood environmental factors (pesticides, solvents) may act upon germinal cells
and Sertoli cells and other organs of the reproductive system (including epididymis) causing
dramatic changes in semen. Some habits and social drugs are also detrimental for man
fertility.
The research project Nr PBZ-KBN-084/P06/2002 supported by Polish Scientific Committee
was performed between 2003 and 2005.
SIAH1 IS CANDIDATE mRNA REGULATED BY NANOS1—PUMILIO2
PROTEIN COMPLEX IN THE HUMAN GERM CELLS
Anna Spik1, Sławomir Oczkowski2, Maciej Kotecki1, Piotr Formanowicz2,3, Jacek
BłaŜewicz2,3, Jadwiga Jaruzelska1
1
Institute of Human Genetics, Polish Academy of Science, Poznań; 2Institute of
Computing Science, Poznań University of Technology; 3Institute of Bioorganic
Chemistry Polish Academy of Science, Poznań, Poland,
The nanos-pumilio protein complex is translational repressor of specific mRNAs during
morphogenesis and development of Drosophila melanogaster germ cells. Both proteins bind
specific GUUGU (A) and AUUGUA (B) elements situated in 3’UTR of target mRNAs. We
have identified homologous complex (NANOS1 and PUMILIO2 respectively) in the germline of the man and we currently search for potential human mRNAs targets.
Here, we describe candidate mRNA SIAH1 which is expressed in human testis tissue.
This mRNA contains GUUGU (A) and AUUGUA (B) elements in 3’UTR, as mRNA targets
of nanos and pumilio in Drosophila. The SIAH1 is homolog of Sina (seven in absentia) gene
which was shown to be important for fertility of the fly and mouse. Interaction between
NANOS1-PUMILIO2 complex and SIAH1 mRNA was tested using the electrophoresis
mobility shift assay. For that purpose, mRNA SIAH1 was in vitro transcribed at the presence
of radioactive precursor, while NANOS1 and PUMILIO2 proteins were overexpressed in
bacteria and then purified by affinity chromatography. We have shown that NANOS1 and
PUMILIO2 bind 3’UTR of mRNA SIAH1 with high affinity and specifically. Binding
specificity was estimated by 1/ competition assay using appropriate RNA competitors, 2/
testing mutated SIAH1 mRNAs lacking GUUGU (A) or AUUGUA (B) motifs for PUMILIO2
binding. Our results support the hypothesis that SIAH1 mRNA is regulated by NANOS1PUMILIO2 complex in the human male germ cells. In vivo experiments are carried out to
confirm these findings.
This study was supported by State Committee for Scientific Research PBZ-KBN084/PO6/2002.
CHANGES OF SURFACE OF THE EPIDIDYMAL EPITHELIAL CELLS
STIMULATED WITH LH/hCG IN VITRO
Małgorzata Świder−Al−Amawi, Barbara Wiszniewska
Department of Histology and Embryology, Pomeranian Medical University
of Szczecin, Poland, E−mail: [email protected]
The epididymal epithelial cells create epididymal microenvironment essential for the
maturation and storage of spermatozoa. They produce and secrete proteins, glycoproteins,
glycolipids and phospholipids of epididymal fluid. The processes are mainly under control of
androgen. The epididymal epithelial cells in vitro reveal steroidogenic features and are able to
synthesize and release into the culture medium androgens, that are converted to 17β−estradiol
(E2). In my former studies it was documented that similarly to Leydig cells, the synthesis of
E2 is controlled by LH/hCG, due to the presence of LH/hCG receptors in epididymal
epithelial cells. Moreover, during LH/hCG stimulation, the morphology of the cells was
affected. The increase of lipid droplets, glycogen and PAS−positive substances were
observed.
It is known, that the increase of E2 secretion in Leydig cells is followed by disorganization of
cytoskeleton under LH stimulation.
Therefore, the aim of the study was to examine the surface of cultured cells stimulated with
LH/hCG. The experiment was performed on enzymatically isolated cultures of epithelial
epididymal cells of the caput and cauda epididymis in rat. The cells were cultured in
Dulbecco’s medium supplemented with 5% FSC and with/without DHT and without DHT in
supplementation with hCG.
The observation of the cells was carried out in scanning electron microscopy.
The surface of cultured cells depend on the different supplemented medium. The surface of
cells cultured in medium with DHT contains microvilli and granular protrusions, indicating
a high secretory activity of the cells, while the surface of cells cultured without DHT addition
was smooth. However, the stimulation with LH/hCG of cells cultured without DHT caused to
appear microvilli and protrusions.
The studies strongly suggest, that the surface of epididymal epithelial cells cells is changed
and depends on the presence of LH/hCG in the culture medium.
Poster presentation
ANDROGEN RECEPTORS AND AROMATASE IN TESTES OF PATIENT
WITH KLINEFELTER’S SYNDROME - IMMUNOHISTOCHEMICAL
STUDY
Małgorzata Kotula-Balak1, Leszek Bablok2, Stanisław Frącki2 and Barbara Bilińska1
1
Department of Animal Physiology, Laboratory of Endocrinology and Tissue Culture,
Institute of Zoology, Jagiellonian University, Krakow, Poland, 2Department of
Andrology, Insitute of Obstetrics and Gynecology, Medical Academy, Warszawa,
Gonosomal aneuploidies such as Klinefelter’s syndrome (47,XXY) are the most frequent
chromosomal aberration in infertile men, that result in impairments in both spermatogenesis
and testosterone production [1]. Androgens play a crucial role in the initiation and
maintenance of spermatogenesis. Their action is mediated on the molecular level via the
androgen receptor (AR), a transcription factor involved in gene regulation [2]. Recently, it has
been estabilished that also estrogens are necessary for normal male fertility [3, 4].
The aim of present study was to detect cellular distribution of androgen receptors and
aromatase in testes of patient with Klinefelter’s syndrome. Testicular biopsy was obtained
from 31-year-old man with KS diagnosed for ICSI-PESA/TESA procedures. The tissue
sections were processed for morphological and immunohistochemical staining. Additionally,
levels of FSH, LH, PRL, estradiol, and testosterone were measured in the plasma.
Morphological analysis was carried out after haematoxylin and eosin or Masson – Goldner
trichrome stainings. Aromatase immunoexpression was detected by a mouse anti-human
cytochrome P450 aromatase antibody (1:10; Serotec, UK). For ARs immunodetection a
rabbit polyclonal antibody was used (1:10; Novocastra, UK). Hormone levels were measured
enzymatically with a chemilluminescent marker using the automatic Bayer’s apparatus
(ACS:180TM SE; Bayer Co).
Morphological analysis revealed a complete absence of spermatogenesis in the testis of
patient with Klinefelter’s syndrome in comparison with the control one. Germ cells were
absent. In some tubules, nests of apparently degenerating Sertoli cells were found. In the
interstitial area, large clusters of Leydig cells were observed. Using immunohistochemistry, in
the testis of patient with KS, nuclear AR staining was detected in Sertoli and peritubular cells,
whereas in Leydig cells the immunostaining was exclusively cytoplasmic. In control testis,
only nuclear localization of the AR was detected in Sertoli, Leydig, and peritubular cells. In
the testis of patient with KS, cytoplasmic aromatase immmunostaining was observed in
Sertoli cells and Leydig cells, while in control testis a weak aromatase immunostaining was
also present in germ cells. Hormonal analysis revealed increased levels of LH and FSH and
decreased level of testosterone in plasma of patient with KS.
Our study shows that androgen receptors and aromatase are present in testicular cells of
patient with Klinefelter’s syndrome, however, the cytoplasmic staining of AR in Leydig cells
indicates the existence of non-functionally active AR, possibly due to a very low level of
bioavailable, free testosterone.
1. Amory JK, Anawalt BD, Paulsen CA, Bremner WJ (2000) Klinefelter’s syndrome. Lancet 356:
333-335.
2. Grossman ME, Tindall DJ (1995) The androgen receptor is trancriptionally suppressed by proteins
that bind single-stranded DNA. J Biol Chem 18: 10968-10975.
3. Carreau S, Lambard S, Delalande C, Galeraud-Denis I, Bilińska B, Bourguiba S (2003) Aromatase
expression and role of estrogens in male gonad: a review. Reprod Biol Endocrinol 1: 35-42.
4. Hess RA (2003) Estrogen in the adult male reproductive tract: A review. Reprod Biol Endocrinol
1: 52-57
Supported by BW/IZ/ZFZ/2005
MORPHOLOGICAL ANALYSIS AND IMMUNOEXPRESSION OF
AROMATASE IN THE MALE GONADS WITH IMPAIRED
SPERMATOGENESIS
Małgorzata Kotula-Balak1, Józefa Styrna2, Jan K. Wolski3 and Barbara Bilińska1
1
Department of Genetics and Evolution, Institute of Zoology, Jagiellonian University,
Krakow, Poland 3Infertility Center “Novum”, Warszawa, Poland,
2
In the light of recent data estrogens play an important role in the regulation of
spermatogenesis and maintenance of male fertility [1, 2]. The formation of estrogens from
androgens depends on functionally active aromatase (P450arom), a microsomal enzyme [3].
Aromatase has been demonstrated in Leydig cells, Sertoli cells, and germ cells of various
animal species [4].
The aim of the present work was to study the morphology and aromatase immunlocalization
in the testes with impaired spermatogenesis, obtained from mice with mosaic mutation
(Atp7amo-ms), B10. BR-Ydel mice (both strains bred in the Department of Genetics and
Evolution, Jagiellonian University) and from azoospermic men. Mosaic mutation in mice is
associated with disturbances in copper metabolism that affects reproductive function [5]. Ydel mutation in mice is caused by a partial deletion in the long arm of the Y chromosome and
resulted in production of increased number of abnormal spermatozoa [6]. Testicular biopsies
were collected from azoospermic men diagnosed for ICSI-PESA/TESA procedures (Infertility
Center “Novum”, Warszawa).
Morphology and immunohistochemistry were carried out on paraffin-embedded tissues. For
morphology, testicular sections were routinely stained with haematoxylin and eosin.
Aromatase immunoexpression was detected by a rabbit polyclonal antibody against human
placental P450 aromatase (1:400; R-10-2; a gift from Dr. Yoshio Osawa).
Morphological analysis of the mosaic and Y-del mouse testes revealed the presence of many
degenerating seminiferous tubules besides normal-looking ones in comparison to their
respective controls. In the testes of azoospermic man, either undifferientiated germ cells or
total absence of germ cells with Sertoli cells alone were observed inside seminiferous tubules.
Both, in mouse and human testes with impaired spermatogenesis, aromatase
immunoexpression was stronger than in control ones. The strongest immunostaining was
observed in Leydig and germ cells of mosaic and Y-del mouse testes as it was in Leydig and
Sertoli cells of human testes with Sertoli cell only syndrome (SOS). It is possible therefore,
that two different mutations in mice (a single gen mutation in X chromosome, and partial
deletion in the long arm of the Y chromosome), as well as SOS in men that lead to a similar
phenotype in spermatogenesis, induce overexpression of aromatase.
1. Hess RA (2003) Estrogen in the adult male reproductive tract: A review. Reprod Biol Endocrinol
1: 52-57.
2. Carreau S, Lambard S, Delalande C, Galeraud-Denis I, Bilińska B, Bourguiba S (2003) Aromatase
expression and role of estrogens in male gonad: a review. Reprod Biol Endocrinol 1: 35-42.
3. Conley A, Hinshelwood MM (2001) Mammalian aromatase. Reproduction 121: 685-695
4. Lenartowicz M, Sasuła K (2000) Altered copper metabolism in the Mosaic mutant mice. Nutr Res
20: 1467- 1471.
5. Styrna J, Imai HT, Moriwiaki K (1991) An increased level of sperm abnormalities in mice with a
partial deletion of the Y chromosome. Genet Res 1991 57: 195-199.
6. O’Donnell L, Robertson KM, Jones ME, Simpson ER (2001) Estrogen and spermatogenesis. Endocr
Rev 22: 289-318.
Supported by BW/IZ/ZFZ/2005
A SEARCH FOR mRNAs INTERACTING WITH FERTILITY PROTEIN
PUMILIO2 IN THE HUMAN GERM LINE
Anna Spik1, Agata Olszak1 Sławomir Oczkowski2, Maciej Kotecki1, Piotr
Formanowicz2,3, Jacek BłaŜewicz2,3, Jadwiga Jaruzelska1
Institute of Human Genetics, Polish Academy of Science, Poznan; 2Institute of
Computing Science, Poznan University of Technology; 3Institute of Bioorganic
Chemistry Polish Academy of Science, Poznan, Poland,
1
The control of mRNA expression by proteins which bind specific nucleotide motifs in 3’
untranslated regions (3´UTRs) is common in developmental processes of lower organisms. In
Drosophila, translational repressor Pumilio binds short nucleotide elements GUUGU (A) and
AUUGUA (B) in 3’UTR of hunchback (ABAB), cyclinB (ABB) and bicoid (ABB) mRNAs
to regulate body patterning or germ cell development. We identified homologous PUMILIO2
protein in the human male germ cells. The aim of this study was to find out whether potential
mRNA targets of PUMILIO2 bind the same type of motifs.
By electronic searches of the GeneBank we retrieved five candidate mRNAs expressed in
testis tissue including germ cells. In 3’UTRs these mRNAs contain motif ABAB, ABB or
ABAA. Two candidates are morphogenes: 1/ neuronatin, 2/ Cdc42 Effector protein3 – CEP,
while the other three are involved in the regulation of the cell cycle or tumorigenesis: 3/
spindlin, 4/ DMP1, 5/ TSC-22. Using band-shift approach we have demonstrated that all of
them bind PUMILIO2 protein. However, the binding affinity was variable depending on
mRNA type. This variability probably reflects differences in the specificity of PUMILIO2
recognition by RNA nucleotide sequences. The highest binding affinity estimated by band
shift assay was observed in the case of TSC-22. This observation was further confirmed by
competition assay. Preliminary results of this study suggest that PUMILIO2 may regulate
germ cell development by interacting with several target mRNAs.
This study was supported by State Committee for Scientific Research PBZ-KBN084/PO6/2002.
Session V
Nutrition and reproduction
Oral presentation
DIETARY FAT AND ANDROGENS SECRETION AND METABOLISM
Joanna Gromadzka-Ostrowska
Warsaw Agricultural University, Faculty of Human Nutrition and Consumer Sciences,
Department of Dietetics, 02-787 Warsaw, E-mail: [email protected]
Here are described the effects of different dietary fat type, high-fat and low-fat diets as well
as hypercholesterolemic diet on pituitary-gonadal axis activity, androgens secretion, their
tissue metabolism and androgens receptors in different organs. Some molecular aspects of
androgens metabolism such as enzyme activity and its gene expression and their clinical
implications for prostate cancer and atherogenic events are also discussed.
EFFECTS OF PHYTOESTROGENS ON THE HYPOTHALAMUS AND
PITUITARY GLAND IN MAMMALS
Marek Opałka, Barbara Kamińska, Luiza Dusza
Department of Animal Physiology, University of Warmia and Mazury in Olsztyn, 10719 Olsztyn, Poland, E-mail: [email protected]
Phytoestrogens are plant-derived, estrogen-like compounds (with a diphenolic, nonsteroidal
structure) which can be separated into three major classes: isoflavones, coumestans and
lignans. The sources of phytoestrogens are all present in human and animal diets including
fruits, vegetables, legumes, whole-grain and especially soy products,. These estrogen mimics
can selectively bind estrogen receptors (estrogen receptor β greater than estrogen receptor α).
The ability of phytoestrogens to disrupt reproductive function is well established in a number
of species. This disturbance occurs due to direct action on reproductive tracts as well as by
changes in the hypothalamic-pituitary axis function. This review mainly focuses on an
overview of the phytoestrogen effects on: the volume of sexually dimorphic hypothalamus
structures and activity of GnRH puls generator as well as synthesis and release of anterior
pituitary hormones.
A diet of phytoestrogens or administration of genistein changed the volume of the sexually
dimorphic nucleus of the preoptic area and anteroventral periventricular nucleus in
development rats.
Intravenous infusing of coumestrol reduced the frequency of the hypothalamic GnRH pulse
generator and complete suppressed of the LH response to exogenous GnRH. Likewise,
coumestrol inhibited GnRH-induced pituitary LH release in vitro. In contrast, genistein did
affect pulsatile LH secretion. Intracerebroventricular infusion of genistein in the ewes during
the breeding season induced a biphasic response in LH release. In the anoestrus ewes,
genistein decreased both plasma LH concentration and frequency of LH pulses as well as
increased plasma prolactin level. Dietary genistein enhanced plasma prolactin in
ovariectomized rats, too. However, a soy diet (containing genistein and daidzein) for 10-14
days did not have a clear estrogen agonist or antagonist effect on pituitary sensitivity to GnRH
in pre- and postmenopausal women.
The overview of the studies indicates that the effect of phytoestrogens on the hypothalamicpituitary axis depends on the species, reproductive status of the organism, the kind of
phytoestrogens, length of the exposure, the way of treatment administration and treatment
level. Moreover, the mechanisms of phytoestrogen action on the hypothalamus and pituitary
gland remain unclear. Thus, further studies are required, especially in domestic animals.
Research was supported by the State Committee for scientific Research as a Solicited Project
PBZ-KBN-084/P06/2002 from 2003 to 2005 year.
NEUROPEPTIDE Y – A NEUROMODULATORY LINK BETWEEN
NUTRITION AND REPRODUCTION ON THE CENTRAL NERVOUS
SYSTEM LEVELS
Anna Wójcik-Gładysz, Jolanta Polkowska
Nutrition, 05-110 Jabłonna, Poland, E-mail: j.polkowska @ifzz.pan.pl
The relationships between a level of nutrition and reproductive processes in animals are
mainly regulated through the hypothalamo-pituitary gonadotrophic endocrine system. The
both, energy as well as protein deficits in nutrition of many animal species cause a
disturbances in the secretion of the hormones of this axis in growing and adult mammals. As
the most important etiologic factor for the nutritionally induced suppression of pituitaryovarian function, a decreased hypothalamic pulsatile secretion of gonadotrophic hormonereleasing hormone (GnRH) from the hypothalamus has been proposed. This results in the
decrease of, luteinizing hormone (LH) synthesis in the gonadotrophic cells of the pituitary
gland and its release to the circulating blood. These changes are resulted in impaired gonadal
functions, especially in restraining of processes leading to the puberty in young and
disturbances in the course of the oestrous cycles and pregnancy in adult animals. Although the
relationships between nutrition and reproduction are extensively investigated, there is lack
information about the exact mechanisms that couple these two systems on the level of the
central nervous system (CNS). There are searching of the chemical factors situated in the
brain that can play a neuromodulatory link between the nutritional status of the organisms and
reproductive hormones. One of the candidates for such function is neuropeptide Y (NPY). In
the hypothalamus the peptide is synthesised in the arcuate nucleus, and also expressed in
hypothalamic centres taking part in the appetite control processes. We review the distribution
NPY-neurones in the hypothalamus, the contacts with other hypothalamic centres, its
receptors and first of all its orexigenic properties. It has been demonstrated the stimulatory
effect of malnutrition on the secretory activity of the NPY neurones, a participation of NPY in
the regulation of a food intake and relationships with metabolic factors, particularly with
peripheral signals like steroids and leptin. Next, we review the participation NPY in the
regulation of GnRH /LH secretion with stressing its dual role depending on the activity of the
reproductive system. On the example of the sheep, it has been shown its stimulatory effect on
the synthesis of LHβ subunit during the peripubertal period and increase of secretory activity
of arcuate NPY neurones in the preovulatory phase of the estrous cycle.
Taking into consideration orexigenic properties of NPY and its participation in the regulation
of the GnRH/LH secretion it can be supposed that NPY is a candidate for the link between
nutrition and reproduction on the level of the CNS.
REGULATORY ROLES OF LEPTIN IN RUMINANT REPRODUCTION
Dorota A. Zięba 1, Gary L. Williams 2, 3
1
Poland, E-mail: [email protected];
2
Animal Reproduction Laboratory, Texas A&M University Agricultural Research
Station, Beeville, TX, USA; 3 Department of Animal Science, Texas A&M University,
College Station, TX, USA
Reproduction in animals is influenced dramatically by nutrition. The adipose-derived
hormone, leptin, appears to play an integral role in this process by communicating nutritional
status to the central reproductive axis, thereby influencing multiple aspects of reproduction,
including that in ruminants. Leptin administration enhances secretion of LH in fasted sheep
and cattle, directly stimulates GnRH secretion in fasted cows, and has direct stimulatory
effects on bovine adenohypophyseal gonadotrophs. The effects of leptin are dose-dependent,
with highest doses resulting in leptin resistance and failure of the hypothalamic-pituitary axis
to respond with increased gonadotropin release. Full-fed cattle and sheep, and animals in
neutral energy balance, do not respond to leptin. Although circulating leptin increases linearly
from 16 weeks before until the week of pubertal ovulation in yearling heifers, collective
evidence across several species supports the idea that leptin plays a passive or permissive,
rather than causal, role in timing the process of sexual maturation. Moreover, circulating
leptin concentrations increase in sheep from early to mid pregnancy and remain elevated until
late pregnancy. The latter elevations appear to be due to an increase in total adiposity as well
as an increase in leptin mRNA expression per unit of tissue. Leptin receptor mRNA is present
in the placenta of sheep and its concentration increases during pregnancy. It is likely that
leptin receptors in the placenta act to transport maternal leptin to the fetus which may provide
a signal for fetal development. Several lines of evidence indicate that leptin also has direct
actions at the level of the ovary. Bovine ovarian granulosa and thecal cells have leptin
receptors and leptin modulates steroidogenesis of ovine granulosa cells. Finally, the leptin
gene is expressed in the mammary tissue of cows, sheep and goats. Both long and short forms
of the leptin receptor have been shown to be expressed in the ovine mammary gland during
pregnancy and lactation. Thus, leptin and its receptor could be important in regulating
mammary gland growth and development. In summary, in addition to leptin’s ability to
stimulate the hypothalamic-pituitary axis, it is also able to act locally within the placenta,
mammary gland and gonads to modulate physiological events at these sites. Additional
investigations will be necessary to characterize these and other actions more definitively in
ruminants.
Poster presentation
DIETARY FAT TYPE AND DIFFERENT DIETARY VITAMIN E LEVEL
EFFECTS ON GONADAL STEROIDS AND LEPTIN CONCENTRATION IN
RATS
Olga Jabłońska, ElŜbieta Olczak and Joanna Gromadzka-Ostrowska
Warsaw Agricultural University, Faculty of Human Nutrition and Consumer Sciences,
Department of Dietetics, 02-787 Warsaw, E-mail: [email protected]
The purpose of the study was to determined influence of high-fat diets containing different fat
types and different vitamin E level on estradiol, testosterone and leptin plasma concentration
in rats.
The experiment was done with 48 male Wistar rats (body weight 250+10 g) divided into eight
groups differing in a type of dietary fat (fish oil reach in docosahexaenoic acid [22:6], grapesseed oil reach in linoleic acid [18:2 n-6], rapeseed oil reach in oleic acid [18:1] or lard reach
in stearic acid [18:0]) and level of vitamin E in the diets (normal level 50 mg/kg of diet vs
high vitamin E dietary level 500 mg/kg of diet). Animals were kept in individual cages and
environment standard parameters (temperature 22°C, humidity 50-60% and photoperiod L:D
12:12 h). After 3 weeks of feeding rats were bled under penthobarbital anesthesia and
obtained blood plasma were stored in -23°C. Plasma hormones concentration was measured
by radioimmunoassay technique using DSL (for testosterone and estradiol) and Linco Res.
(for leptin) RIA kit. The results were analyzed by one-way analyses of variance (ANOVA)
and simple regression analysis, P<0,05 were considered as significant.
Obtained results showed statistically significant influence of dietary fat type on blood plasma
estradiol and leptin concentration (ANOVA P<0,00001 in both cases) and close relationship
between both hormones (correlation coefficient r = -0,49, p<0,001), whereas any such effects
on testosterone concentration were observed. In rats fed diets with a lard as a source of fat the
highest leptin level and the lowest estradiol concentration were stated (p<0,001). In the same
animals a little higher, but none statistically significant, testosterone concentration were
found. We observed also the lowest leptin and the highest estradiol plasma concentrations in
rats fed a fish oil as a dietary fat source as well as any dietary vitamin E level influence on all
investigated parameters (ANOVA NS).
Data from our studies suggest that high intake of saturated fatty acids are involved in the
stimulation of adipose tissue hormone secretion and inhibition of estrogens secretion. It
seems that last phenomenon is due to changes in aromatase activity in testis and adipose
tissue.
EQUOL AND PARA-ETHYL-PHENOL MODULATE CYTOKINES ACTION
ON BOVINE CORPUS LUTEUM (CL)
Anna Korzekwa, Izabela Woclawek-Potocka, Anna M. Rogozińska, Dariusz J.
SkarŜyński
Research of PAS, Tuwima-St 10, Olsztyn 10-747, Poland,
The mechanisms controlling the regression of bovine CL involve many factors (among them
cytokines) produced both, inside and outside the CL. At the late luteal phase, immune cells
invade bovine CL, and their products (tumor necrosis factor (TNF)α, interferon (IFN)γ, and
interleukin (IL)1 play an important role in mediating the action of prostaglandin (PG)F2α in
luteolysis. Endogenous estrogens control the estrous cycle influencing PGF2α synthesis and
action. Phytoestrogens may act like antagonists or agonists of endogenous estrogens. Daidzein
and genistein are the main isoflavones of soy bean being widely fed on the dairy cows. We
recently found significant levels of equol and p-ethyl phenol, daidzein and genistein
metabolites, respectively, in serum of cows fed with soy-bean. Despite, the evidence that
izoflavones disrupt reproductive processes in many species, their influence on the cytokinedependent mechanisms regulating the regression of bovine CL was studied in vitro. In Exp. 1,
the role of active phytoestrogene metabolites (p-ethyl phenol, equol, each: 10-8M) and 17βestradiol (E2; 10-9M) in the regulation of paracrine mechanism between CL cells was
determined. The different types of CL cells (steroidogenic, endothelial cells and lymphocytes
T) were incubated in cocultures and secretion of luteolysis mediators (PGF2α, nitric oxide NO, leukotrien LTC4) was determined by EIA. P-ethyl-phenol, equol and E2 stimulated NO,
LTC4 and PGF2α synthesis in all kind of cocultures and in the steroidogenic cells (P<0.05). In
Exp. 2, the influence of isoflavones on the luteolytic action of cytokines (TNFα, IL-1β, Fas-L
and IFNγ) was studied comparing to endogenous E2 action. Steroidogenic CL cells were
preincubated with steroids for 24 h. After preincubation, the cells were stimulated with
cytokines (each 10 ng/ml). Isoflavones and E2 augmented the stimulatory effect of cytokines
on PGF2α , LTC4 and NO secretion (P<0.01). In Exp. 3, the influence of p-ethyl phenol and
equol on the cytokine-induced cell death was determined in the steroidogenic CL cells.
Although, cytokines did not influence cell viability in the control (P>0.05), in the cells
preincubated with isoflavones all cytokines revealed the cytotoxic effect (P<0.05; over 30%
of the died cells). In summary, active metabolites of isoflavones: p-ethyl phenol and equol act
like agonists of natural estrogens. They can modulate regression of bovine CL, which depends
on immune cells products and may augment the luteolytic action of cytokines.
Supported by the grant PBZ-KBN 084/P06/2002/Zad5-2
INTRACELLULAR MECHANISM OF PHYTOESTROGENES ACTION ON
BOVINE CORPUS LUTEUM
Dariusz Jan SkarŜyński, Anna Korzekwa, Izabela Wocławek-Potocka, Aleksandra
Bober
Research, PAS, Tuwima-St 10, Olsztyn 10-747, Poland,
Phytoestrogens are plant delivered compounds of natural origin with nonsteroidal structures
that can behave as endogenous estrogens. Feeding cows with the soy bean, that is reach in
phytoestrogens, decreased the rate of successful pregnancies and increased mean insemination
rate. As agonists and/or antagonists of endogenous estrogens, phytoestrogens can disturb
reproductive processes on many different regulatory levels including disorders in the function of
bovine corpus luteum (CL). We have recently shown that phytoestrogens inhibit LHstimulated progesterone (P4) secretion by the bovine CL in vitro and in vivo. The aim of the
present study was to examine the influence of phytoestrogens and their active metabolites on
secretory function of steroidogenic CL cells and to estimate the intracellular, molecular
mechanisms of their action. In Exp. 1 enzymatically isolated steroidogenic cells of bovine CL
(Day 11-17 of the estrous cycle) were stimulated with isoflavones (daidzein and genistein: 108
-10-6M) and their active metabolites (equol and para-etyl-phenol: 10-8-10-6M). In the medium,
the level of P4, testosterone (T) and prostaglandin F2α (PGF2α) was measured by EIA. All used
estrogenic substances had no effect on basal P4 secretion (P>0.05), but stimulated T and
PGF2α secretion (P<0.05). In Exp. 2, the intracellular, molecular mechanism of phytoestrogen
and their active metabolite action was studied. Bovine steroidogenic CL cells were
preincubated (30 min.) with a phospholipase C inhibitor (U-73122; 10-6M), a protein kinase A
inhibitor (staurosporin; 10-6M), an inhibitor of nuclear estrogen receptors (ICI; 10-6M) or with
a translation inhibitor (actinomycin-D; 100 ng/ml) and then stimulated for 24 h with
phytoestrogens (10-7M) or 17β-estradiol (E2; 10-9M). Level of PGF2α was measured in the
medium by EIA. The blockade of nuclear estrogen receptors and inhibition of translation
inhibited isoflavone-stimulated PGF2α synthesis in the cells (P<0.05). However, the effect of
E2 was inhibited by all blockers used (P<0.001). Moreover, in Exp. 3, the effect of isoflavones
(10-7M) and E2 (10-9M) on the intracellular calcium ions mobilization [Ca2+]i was measured
using fluorescent marker Fura-2 and the reverse microscope (OLYMPUS, MicroImage
System). In contrast to E2 action, isoflavones did not cause [Ca2+]i mobilization (P>0.05)
Concluding, isoflavones influence the secretory function of bovine CL stimulating T and
PGF2α production by the steroidogenic cells. In contrast to E2 action, phytoestrogens and their
active metabolite stimulate PGF2α secretion via a classical, genomic and estrogen receptordependent pathway in the CL cells.
Supported by the grant PBZ-KBN 084/P06/2002/Zad5-2
THE EFFECT OF LEPTIN ADMINISTRATION ON THE ACTIVITY OF THE
SOMATOTROPHIC AND THYREOTROPHIC AXIS IN PERIPUBERTAL
FEMALE RATS
Ewa Wolińska-Witort, Lidia Martyńska, Magdalena Chmielowska, ElŜbieta
Wasilewska-Dziubińska, Wojciech Bik, Bogusława Baranowska
Neuroendocrinology Department, Medical Centre of Postgraduate Education,
Marymoncka 99, 01-813 Warsaw, Poland, E-mail:[email protected]
Puberty in mammals is the period in which significant activity changes are observed in
hypothalamo-pituitary axis that controls the synthesis and the secretion of gonadotrophins. It
is necessary for the process of sexual maturation to undergo properly in order to reach an
adequate level of metabolic substrates as well as energy stores contained in the adipose tissue.
The cumulative data suggest that leptin, a satiety factor produced mainly in the adipose tissue,
is an important candidate to the role of a metabolic signal which transmits information to the
hypothalamus that the store of the energy is sufficient to start reproductive processes.
Our previous study, after a long-term leptin administration, has demonstrated an increase of
the activity of the gonadotrophic axis in peripubertal female rats. We found a high abundance
of LH-RH in the medial basal hypothalamus and elevated serum LH concentrations. In these
rats a reduction of the body weight gain was found. It is well known, that the somatotrophic
axis and the thyreortophic axis play a substantial part in the control of metabolic processes
and energy expenditure.
In order to investigate the effect of long-time leptin administration on the activity of
somatotrophic and thyreortophic axis, the action of exogenous leptin in intact and estrogentreated female rats was examined.
Immature female Wistar rats, at the age of 30 days, were divided into four experimental
groups, which received respectively: estradiol/saline, estradiol/leptin, oil/leptin and oil/saline.
Estradiol or oil was administered sc. for 3 days and following this leptin or saline was injected
ip. for 7 days. The day after the final injection, all animals were killed by decapitation. Trunk
blood was collected and rat serum GH, IGF-1, TSH, T3 and T4 levels were assayed by RIA
methods.
Results: Leptin administered alone decreased serum GH level, while a combined therapy i.e.
leptin + estradiol inhibited the release of IGF-1. Leptin increased TSH level compared with
TSH level in the vehicle group. In the group of rats treated with leptin + estradiol a significant
decrease of serum TSH concentrations was observed compared to the animals receiving only
estradiol. No changes in serum T3 and T4 levels in all investigated groups of rats were found.
Conclusion: Leptin significantly influences the activity of somatotrophic and thyreotrophic
axis during sexual maturation, and its effect can be modified by estrogens.
THE EFFECT OF INTRACEREBROVENTRICULAR INFUSIONS OF
LEPTIN ON THE IMMUNOREACTIVITY OF NPY AND GnRH NEURONS
IN THE HYPOTHALAMUS OF PREPUBERTAL SHEEP IN CONDITIONS
OF ACUTE UNDERNUTRITION
Anna Wójcik-Gładysz, Marta Wańkowska, Tomasz Misztal, Jolanta Polkowska.
Nutrition, Polish Academy of Sciences, 05-110, Jabłonna, Poland,
The aim of the present study was to investigate the effect of exogenous leptin on the
expression of immunoreactive (IR) NPY neurons in the hypothalamic nuclei and IR GnRH
neurons in the median eminence of prepubertal sheep in the conditions of acute
undernutrition. Animals were randomly divided into four following groups (each group n=4):
Two groups fed with standard feeds and two groups fasted for 72 h. In one standard and one
fasted groups, the control infusion of Ringer-Locke saline to the 3rd ventricle of the brain were
made. Remaining standard and fasted groups were infused with leptin (10 µl/100µl/h), for 4h
during 3 consecutive days. Immediately after the experiment the sheep were slaughtered,
brains were fixed in situ and prepared for the immunohistological localization of NPY and
GnRH, using the polyclonal antibody anti-NPY and anti-GnRH. Detection of both hormones
in the hypothalamic sections was followed by the image analysis and expressed as the percent
area stained and optical density of immunostaining. In the sheep from all groups three distinct
subpopulations of the IR NPY neurons were localized in the: arcuate, paraventricular and
periventricular nuclei of the hypothalamus. In standard sheep, the expression of IR NPY
neurons was week in all areas investigated. After leptin infusions no changes in this
expression were observed. In fasted sheep a prominent expression of IR material in NPY
neurons located in all subpopulation investigated was observed, most in those detected in the
arcuate nucleus, where considerable number of NPY perikarya appeared. Both, percent area
exhibited positive staining and the density of IR NPY measured in three hypothalamic nuclei
were significantly higher (P<0.001) in fasted than in standard sheep. In fasted sheep infused
with leptin the expression of IR NPY neurons diminished but solely in the arcuate nucleus
without effect in the periventricular and paraventricular NPY neurons. The both parameters
investigated for NPY neurons in this nucleus were lower (P< 0.001) in fasted infused with
leptin compared to fasted sheep. The examination of IR GnRH revealed the augmentation of
the IR material stored in the nerve terminals of the median eminence in fasted in comparison
to standard sheep. After infusion of leptin these stores markedly diminished only in fasted
sheep. Obtained results show that fasting of prepubertal sheep augment the secretory activity
of NPY neurons in the hypothalamus and restrain the release of GnRH. Leptin abolish of
GnRH release suppression through its effect on the subpopulation of NPY neurons located in
the arcuate nucleus. It is suggested that leptin could mediate the restraining of GnRH release
via arcuate NPY neurons in conditions of deficit of nutrients.
Research was supported by the State Committee for Scientific Research as a Solicited Project,
PBZ-KBN-084/P06/2002 from 2003 to 2005 year
Session VI
Toxicology of reproduction
Oral presentation
EFFECT OF 4-NONYLPHENOL ON FISH REPRODUCTION
Krystyna Demska-Zakęś1, Jan Glogowski1,2, Małgorzata Jankun1, Zdzisław Zakęś3
1
Department of Ichthyology, University of Warmia and Mazury in Olsztyn, 10-718
Olsztyn, E-mail: [email protected], 2Department of Molecular Andrology,
Institute of Animal Reproduction and Food Reserch of Polish Academy of Sciences,
10-747 Olsztyn, 3Department of Aquaculture, Inland Fisheries Institute, 10-719
Olsztyn
Estrogens play numerous roles in the fish physiology, including sexual differentiation and the
development of the gonad. Recent studies show that not only endogenous sex steroids but also
environmental contaminants with estrogenic activity (eg. natural and synthetic estrogens, 4nonylphenol) can affect the reproduction and development of fish. Nonylphenol (NP) is a
major industrial byproduct and is found in relatively high concentration in surface water,
sediments, aquatic plants and animal tissues. The concentration of nonylphenol needed to
produce an estrogenic effect has recently been found to be significantly lower than the lethal
concentration in fish.
A few studies have also shown effects of NP on juvenile fish (rainbow trout, common carp,
Japanese medaka and pikeperch). Chronic exposures to aqueous solution of NF, starting
before and including sexual differentiation induced the formation of testes-ova or ovaries in
genetical males. Our results, for the first time, demonstrated that oral administration of NP at
a dose od 0.1 – 100 ppm significantly increased the number of bisexual pikeperch produced,
but permanent feminization effect was not achieved. It is probable that gonad sensitivity to
NP administration depended not only on fish species, dose and time of duration but also on
method of NP application.
Several studies have been performed to evaluate the potential risk of NP exposure on the
endocrine system in adult fish. This agent is able to induce the female specific egg yolk
vitellogenin production in male individuals of various fish species such as rainbow trout,
eelpout and carp. Corresponding morphological effects including an inhibition of testicular
growth and spermatogenesis in adult male rainbow trout, cytological changes of germ and
Sertoli cells of male eelpout, an decrease of the gonadosomatic index in male rainbow trout
were found. We elucidated the effects of oral administration of NP on the reproductive
capacity of adult pikeperch males and studied the transgenerational effects (F1 generation
growth and sex) of this agent on the F1 offspring. The our study indicates, that NP exposure
resulted in decreases in fertility and hatch success. However, we observed no reduction in
survival and growth or any imbalace of the sex ratio of the offspring. In addition, nonylphenol
have not been found to be genotoxic or mutagenic in the SCGE (single cell gel
electrophoresis), chromosome abberation and micronucleus tests.
DEFECTS OF VASCULAR DEVELOPMENT RELATED TO DECREASED
SIGNALING OF Ahr GENE ARE RESCUED BY GESTATIONAL EXPOSURE
TO DIOXIN
Andrzej L. Pawlak
1
Department of Function of Nucleic Acids, Laboratory of Molecular Pathology,
Institute of Human Genetics Polish Academy of Sciences, 60-79 Poznań, Poland,
The biological pathways in which the aryl hydrocarbon receptor (AHR) is involved include
both the metabolic adaptive response to polycyclic aromatic hydrocarbons (PAHs) and the
toxic response to dioxins. As compared to PAHs, dioxins are more potent and pleiotropic
agonists of AHR and induce multiple embryotoxic and teratogenic effects. The developmental
effects of dioxins resemble the metabolic and the toxic effects in the sequence of protein
interactions, as all of them are mediated by the activation of AHR by ligand, its translocation
into nucleus, dimerization with ARNT protein and upregulation of a battery of genes
encoding both the xenobiotic metabolizing (Schmidt V, 1996) and the oxidant responsive
proteins (Nebert DW).
Closing of the embryonic hepatic vascular shunt known as the ductus venosus normally takes
place in the first days after birth. This process is disturbed in the Ahr null mice. In this mice
the ductus remains openand throughout adult life aberrant hepatic blood flow leads to the
phenotype of small liver. Similar phenotypes were seen in the Arnt hypomorphs with the
controlled low expression of this gene (Walisser JA 2004). The mice with hypomorphic
variant of the Ahr gene was produced with the use of the targeting construct, equipped with
the positive and negative selection markers, in which the exon 2 of Ahr d allele is flanked
with the loxp sites. In Ahr hypomorphic mice with the controlled low expression of this gene
the rescue from the patent (open) ductus venosus phenotype was obtained by the
intraperitoneal dioxin injection between the 12,5 and the 18,5 embryonic day. It is presumed
that in these cases the dioxin treatment induces the shifts in the expression of the Ahr and Arnt
genes necessary for the closing of ductus venosus (Walisser JA, 2005).
CHRONIC EXPOSURE TO THE ARYL HYDROCARBON RECEPTOR
AGONIST 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN ACCELERATES
REPRODUCTIVE SENESCENCE IN THE FEMALE RAT
Brian K. Petroff1,2, Anita Franczak1,2,3, Kelli E. Valdez1,2, Kemmy M. Mizinga4
1
Department of Internal Medicine, 2Center for Reproductive Sciences, University of
Kansas Medical Center, Kansas City, KS, U.S.A.; 3Department of Animal Physiology,
University of Warmia and Mazury, Olsztyn, Poland; 4Department of Pharmacology,
Kansas City University of Medicine and Biosciences, Kansas City, MO, U.S.A.
Chronic activation of the aryl hydrocarbon receptor (AhR) can occur in polluted environments
or from smoking-related toxicants. In the current study, female Sprague Dawley rats were
exposed to the potent AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) through
weekly dosing (0, 1, 50, 200 ng/kg/wk p.o.). Dams were dosed during pregnancy and
lactation (gestation days 14 and 21, days 7 and 14 postnatal) and pups were treated directly
thereafter. Reproductive cyclicity was assessed for one week each month through cytologic
examination of daily vaginal smears. Rats were sacrificed once the onset of the transition to
reproductive senescence was evident in TCDD-treated rats at 10 months. The onset of a
prolonged (≥ 6 days) interestrus interval was considered evidence of the transition to
reproductive senescence. Rats were sacrificed on the morning of diestrus once TCDD-treated
animals had exhibited a transition to reproductive senescence and trunk blood and ovaries
were collected. TCDD induced a dose and time-dependent loss of normal cyclicity and
significantly hastened the onset of the transition to reproductive senescence (p<0.05). The
premature onset of the transition to reproductive senescence in TCDD-treated rats was
associated with prolonged estrous cycles and both persistent estrus and diestrus at the highest
dose. The number and size distribution of ovarian follicles was not altered by TCDD.
Diestrous concentrations of progesterone tended (p < 0.09) to be elevated in animals receiving
the two highest doses of the dioxin. There was no consistent effect of TCDD on FSH or LH
concentrations at diestrus although FSH was elevated (p<0.008) vs. controls for the 50 ng/kg
group. These results suggest that chronic exposure to toxicants that activate the AhR can
hasten the loss of reproductive function with age in the female. Data thus far support
endocrine disruption rather than depletion of follicular reserves as a primary mechanism of
the premature transition to female reproductive senescence following chronic activation of the
aryl hydrocarbon receptor pathway in rats.
This work was supported in part by the Kansas City Area Life Sciences Institute, NICHD
HD28934 and NIEHS ES012916.
EFFECT OF PARENT COMPOUNDS (PCB3) AND ITS METABOLITES
(4-OH-PCB3 AND 3,4-DIOH-PCB3) ON FOLLICULAR CELL FUNCTION
Anna Ptak1, Gabriele Ludewig2, Hans-Joachim Lehmler2, Ewa L. Gregoraszczuk1
1
Laboratory of Reproductive Physiology and Toxicology, Department of Physiology,
Institute of Zoology, Jagiellonian University, Ingardena 6, 30-060 Krakow, Poland,
2
Department of Occupational and Environmental Health, College of Public Health,
100 Oakdale Campus #124 IREH, Iowa City, IA 52242-5000, USA
Polychlorinated biphenyls (PCBs) belong to environmental pollutants which exert a variety of
toxic effects on reproductive function. As lipophilic compounds, they accumulate in the testis,
ovary, oviduct and uterus. They were detected in follicular fluid and blood serum. Lipophilic
compounds have a tendency to pass through biological membranes and be stored or enter
phase I and phase II metabolism which makes these compounds even more polar, thus
facilitating biliary and renal excretion. Therefore it must be assumed that not only the PCBs
themselves, but also their metabolites can participate in the toxic effects of PCBs.
Lower chlorinated PCBs are easily metabolized and active forms of their hydroxylated
metabolites (OH-PCBs) are formed. In vivo and in vitro studies have shown that the selected
PCB and PCB metabolites can mimic, enhance or inhibit sex steroid activities, thereby
increasing risk of reproductive disorders. Possible effects of PCBs and their metabolites on
the ovary are particularly important because the ovary is central to female reproductive
function and viability. Moreover, CYP1A1 RNA was detected in porcine granulosa cells,
suggesting that granulosa cells may metabolize PCBs.
Little is known about the effect of lower chlorinated biphenyls and their metabolites as
endocrine disruptors. For this reason, PCB3 was chosen as a prototypical lower halogenated
PCB.
In this lecture, we presented data concerning dose- and time-dependent action of PCB3 and its
mono- and dihydroxylated metabolites on follicular cell function.
Acknowledgements: Supported by grant No. O431/PO4/2004/27 from the State Committee
for Scientific Research. The author is a scholar of Foundation For Polish Science.
THE CIGARETTE SMOKE EXPOSURE IMPROVES THE REARING
CAPACITY OF THE AhR bd C57BL CONGENIC FEMALE MICE
Ewa Strauss1, Ewa Florek2, Andrzej L. Pawlak1
1
Department of Function of Nucleic Acids, Laboratory of Molecular Pathology,
Institute of Human Genetics Polish Academy of Sciences, 60-79 Poznań, 2Department
of Toxicology, Medical University, Poznan, E-mail: [email protected]
The effects of the cigarette smoke (CS) exposure on the mothers rearing capacity were studied
in the C57BL congenic mice, differing in the ahr genotype. The rearing capacity of the
mothers has been assessed by the survival of progeny estimated at the 21-st day of life in
series of pairings either between the unexposed animals or the cigarette smoke (CS) exposed.
This parameter was shown unchanged by cigarette smoke (CS) exposure in the ahr bb
mothers, whereas in ahr bd mothers it was found enhanced by CS exposure. Following CS
exposure the survival index D0/D21 did not change in the ahr bb mothers (0,87 vs. 0,895),
whereas the survival index of the progeny of CS exposed ahr bd mothers was significantly
higher (0,76; 25/33) as compared to that seen in unexposed animals (0,45; p<0,02). The
reported finding, should be related mainly to the differences in the ahr genotypes of the
female mice. It is assumed that the studies should be extended to the wider set of experimental
conditions to find out when the expected negative effects of the activation of aryl hydrocarbon
pathway on the female neuroendocrine ovarian functions would occur.
Poster presentation
A MIXTURE OF DIOXINS AND FURANS ACTS ON CYP 1A1 ACTIVITY
BUT DOES NOT INDUCE APOPTOSIS OF PLACENTAL HUMAN
CHORIOCARCINOMA JEG-3 CELLS
Katarzyna Augustowska, Ewa L Gregoraszczuk
Laboratory of Physiology and Toxicology of Reproduction, Department of Animal
Physiology, Institute of Zoology, Jagiellonian University, Krakow, Poland,
Polychlorinated dibenzodioxins (PCDD) and dibenzofurans (PCDF) are contaminants that
accumulate in the environment because of their lipophilic properties and stability. So far, only
individual congeners of dioxins and furans were investigated in several experiments
concerning their endocrine disrupting effects or other toxic actions.
JEG-3 cell line from malignant placental tissue has been used as an in vitro model for
investigation of the toxic effects of xenobiotics on placenta. These cells are morphologically
similar to the trophoblast of the normal first-trimester placenta, and produce many peptides
and steroid hormones found in normal trophoblast cells, such as hCG, GhRH, progesterone.
Taking into consideration that in the environment dioxins and furans are present in the form
of the mixture, the present study was conducted to define the action of a mixture, obtained by
extraction and purification of real fly ash, which is spread-out into atmosphere from thermal
industrial process, on some specific endocrine toxicity endpoints such as (1) expression of
cytochrome P450 1A1 (CYP 1A1), (2) DNA damage, (3) cell apoptosis.
The results show CYP 1A1 activation after 24 and 48-hour exposure to the mixture. The
activation of CYP1A1 was not correlated with DNA single or double strand breaks. The 24hour incubation of JEG-3 cells with the PCD/PCDF mixture did not induce DNA damage as
evaluated by the comet assay. PCDD/PCDF mixture had no effect on apoptosis assessed by
three parameters: 1) identification of viable, apoptotic and necrotic cells by acridine orange/
ethidium bromide uptake (100% of viable cells in control and toxicant-treated cultures), 2)
caspase –3 activity (104.6% in PCDD/PCDF-treated cells compared to the control), and 3)
apoptotic bodies index (0% in control and toxicant-treated cultures).
In conclusion, CYP1A1 induction is considered to be one of the most sensitive biochemical
markers of action of dioxin-like compounds.
Supported by grant 0563/P05/2003/25 from the State Committee for Scientific Research
(KBN).
THE INFLUENCE OF ZEARALENONE ON THE MEIOTIC MATURATION
OF PIG OOCYTES
Zuzana Kuthanová1, Jaroslav Petr2, Radko Rajmon1, Michal Ješeta1, Eva
Chmelíková1, Markéta Sedmíková1
1
Department of Veterinary Science, Czech University of Agriculture, Kamýcká 129,
Prague 6 - Suchdol, 165 21, Czech Republic, E-mail: [email protected]
2
Research Institute of Animal Production, Přátelství 815, Prague 10 - Uhříněves, 104
01, Czech Republic
Mycoestrogens belong to the group of environmental estrogens. They represent a group of
mycotoxins which can act as endogenous estrogens due to their binding ability with estrogen
receptors, and thereby affect animals. Estrogenic mycotoxins are produced by moulds of the
genus Fusarium and Giberella ssp. (Betina, 1990).
Mycoestrogens include zearalenone and its metabolites. Zearalenone can cause reproductive
disturbances in vivo – for example, disappearance of estrus, early embryonic mortality,
malformation of the fetus, abortion, infertility, etc. (Kummer, 2001). Zearalenone can also to
influence meiotic maturation of bovine oocytes in vitro – oocytes cultivated in the presence of
30 µg of zearalenone per 1 ml of the medium to metaphase II (MII) were inhibited between
metaphase I (MI) and telophase I (TI), and many chromosomal abnormalities were observed
(Minervini et al., 2001). However, there are no reports concerning its influence on pig
oocytes.
The aim of our study was to verify zearalenone effects on meiotic maturation of pig oocytes.
Oocytes were cultivated in the M 199 medium under constant conditions of 39 ºC and 5 %
CO2 in the air for 24 hours to the MI stage. Various amounts of zearalenone (0, 6, 18, 36, 72
µg/ml) were added to the medium.
Zearalenone inhibited maturation of pig oocytes in the stage of germinal vesicle (GV) and late
diakinesis (LD) in a dose-dependent manner. The highest effectiveness was observed in the
concentration 36 µg/ml of the medium (GV 52 %, LD 37 %). The zearalenone dose of 72
µg/ml already had a toxic effect, as many degenerate oocytes were present.
The results of our study show that zearalenone can negatively affect maturation of
mammalian oocytes and thus disturb fertility of farm animals in this way.
References:
Betina, Vl. (1990): Mykotoxíny. Alfa, Bratislava, pp.284.
Kummer, Vl., Faldíková, L., Herzig, I., Laníková, A., (2001): Účinky mykotoxinů na zdraví a
reprodukci zvířat, diagnostika a prevence mykotoxikóz. Veterinary Research Inst., Brno, pp.
43.
Minervini, F et al.. (2001): Toxic effects of the mycotoxin zearalenone and its derivatives on
in vitro maturation of bovine oocytes and 17β-estradiol levels in mural granulosa cell
cultures.Toxicology in vitro (15), 489-495.
This work was supported by grants NAZV QF3025, GACR 523/03/H076.
EFFECTS OF PERINATAL EXPOSURE TO DBP, DINP AND DEHA ON
HYPOTHALAMIC GENE EXPRESSION AND SEXUAL BEHAVIOR IN
RATS
Hwi-Cheul Lee , Keitaro Yamanouchi and Masugi Nishihara
Department of Veterinary Physiology, Veterinary Medical Science, The University of
Tokyo, Tokyo 113-8657, Japan, E-mail: [email protected]
Di-n-butyl phthalate (DBP), diisononyl phthalate (DINP) and di-(2-ethylhexyl) adipate
(DEHA) are ubiquitously distributed chemicals that are widely used as plasticizers. These
chemicals are suspected to interfere with the endocrine system as environmental endocrine
disruptors having estrogenic properties or antiandrogenic effects. Our previous research has
identified granulin (grn) and p130 genes as sex steroid-inducible genes in the rat
hypothalamus, which might be involved in sexual differentiation of the brain. The present
study assessed the effects of perinatal exposure to DBP, DINP and DEHA on grn and p130
mRNA expressions in the hypothalamus at postnatal day (PND) 7 and sexual behaviors after
maturation in both sexes. Maternal rats were given a phytoestrogen-free diet containing
different doses of DBP (0, 20, 200, 2,000 and 10,000 ppm), DINP (0, 40, 400, 4,000 and
20,000 ppm) and DEHA (0, 480, 2,400 and 12,000 ppm) from gestational day 15 to PND 21.
In both male and female pups, DBP and DINP exposure during the perinatal period resulted in
an increase in hypothalamic grn and p130 mRNA levels, but DEHA exposure decreased
expression levels of these genes, though the effects were not dose-dependent. On the other
hand, after maturation, male and female rats that were exposured to DBP, DINP and DEHA
during the perinatal period displayed a tendency to decreased copulatory behaviors. These
results suggest that inappropriate expression of grn and/or p130 genes in the brain of male and
female neonatal rats by perinatal exposure to these chemicals may exert permanent effects on
the hypothalamus thereby decrease sexual behaviors after maturation.
EFFECT OF A FUNGICIDE (FUNABEN T) ON REPRODUCTION OF BANK
VOLES (CLETHRIONOMYS GLAREOLUS)
Anna Marchlewska-Koj1 , Marta Labocha1,Zbigniew Burgieł2
1
Department of Mammalian Reproduction, Institute of Environmental Sciences,
Jagiellonian University, 30-387 Krakow, 2Department of Plan Protection, University
of Agriculture 31 425 Krakow, Poland, E-mail: [email protected]
Among pesticides, fungicides constitute a group of preparations broadly employed in recent
years to fight fungal diseases of plants important for agriculture. In Poland commonly used
for dressing of seed corn is Funaben T, a mixture of 20% carbendazim (belonging to
benzimidazoles) and 45% of thiram (belonging to dithiocarbaminianes). Each of these
compounds have been separately studied in respect of toxicity toward selected laboratory
animals. It has been concluded that both compounds are not particularly toxic for mammals.
However, recent studies provide information about various metabolic disturbances concerning
a detoxication complex of the liver and reproductive system in rodents. The purpose of
investigations presented here is to elucidate how Funaben T added to the diet of bank vole
(Clethrionomys glareolus) affects the reproductive processes in this species. Bank vole is one
of the most common rodents occurring in Europe. It inhabits forests but can be also found in
the fields, gardens and parks. Bank voles feed on seeds of grass, corns and trees, but their diet
includes also some invertebrates. As the experimental model we used bank voles deriving
from the Department of Mammalian Reproduction, Institute of Environmental Sciences,
Jagiellonian University, where they have been reared for over 20 years as an outbred colony.
As indicated by our preliminary results presence of Funaben T in diet of bank voles did not
affect sexual preferences of females, duration of pregnancy, number of fertilized and
implanted embryos. However, critical period for toxic effects was observed during
postimplantation period. Number of newborn bank voles delivered by females exposed to
Funaben T was lower than by control mothers. Toxic effect was also observed during the first
24 hours after birth and was manifested by the presence of a high number of dead bank vole
pups found in nests of Funaben T-treated females. Detailed data concerning influence of
Funaben T on postnatal development of bank vole offspring will be presented and discussed.
1
The project was supported by Center of Excellence EU ; EVK2-CT-2002-80009
EFFECT OF POLYCHLORINATED BIPHENYLS (PCBs) ON OXYTOCIN
SECRETION FROM THE OVARIAN CELLS IN COW. POSSIBLE
PARTICIPATION OF GLUCOCORTICOID RECEPTORS.
Jarosław Janusz Młynarczuk, Jan Kotwica
Institute of Animal Reproduction and Food Research; Polish Academy of Sciences,
PCBs added to the culture increased (P<0.05) secretion OT from both granulosa and luteal
cell. Some distinct effect was observed after treatments with PCB77 or 153 (P<0.05). RU486
decreased (P<0.05) PCBs-stimulated secretion of OT from granulosa cells of both size
follicles and from luteal cells but the effect was different for individual congeners of PCBs.
Most evident effect was observed in granulosa and luteal cells incubated with PCB 77, where
RU486 decreased OT secretion (P<0.05) to the control values. In contrast, there was no
influence of RU486 on OT secretion stimulated by PCB 126 from granulosa cells obtained
from the follicles >1cm. RU486 markedly (P<0.01) decreased OT secretion stimulated by
cortisol. In cultures of granulosa cells obtained from follicles >1 cm PCB77-stimulated
release of OT was decreased by TX but it did not affect stimulatory effect of PCB 126 and
153. In granulosa cells from follicles <1 cm, TX decreased (P<0.05) response of this cells on
PCB77 stimulation but increased stimulatory effect PCB153 and 126 (P<0.05). Addition of
TX to the luteal cells decreased OT secretion stimulated by PCB77 and 153 but it did not
affect response of cells treated with PCB 126 (P<0.05). TX alone increased OT secretion from
granulosa cells (P<0.05) while it did not affect luteal cells response.
PCBs are recognized as environmental pollutants since they are resistant for the degradation
and can be accumulated in tissues. They can impair many reproductive functions via estradiol
(E2) or arylhydrocarbon receptors (AhR). Oxytocin (OT) produced in bovine ovary affects the
secretion of progesterone (P4) from corpus luteum (CL) and the release of E2 from follicles.
In turn, both P4 and E2 can influence on OT secretion from bovine CL and from follicle.
Therefore the aim of the present investigations was to study the effect of the PCBs (126, 77 or
153) at a dose (1, 10 or 100 ng/ml) on OT secretion from the luteal (2,5x105/ml) and
granulosa (3x105/ml) cells, with or without glucocorticoid (GC) receptor blocker (RU486; 105
M) and 4-hydroxytamoxifen (TX, 10-5M) - E2 receptor blocker. Luteal cells from day 5-10 of
the estrous cycle, were obtained by enzymatic dispersion of CL while granulosa cells were
aspirated by means of syringe from the follicles of two sizes: >1 and < 1cm. Cortisol (10-5M)
was used as a positive control. After 48 hrs of incubation (n=8), concentrations of OT in
medium was measured by EIA method.
This data suggest that: (a) PCB77 and 153 can stimulate OT secretion from ovarian cells via
GC receptors, (b) Moreover, E2 receptor is stimulated by PCB77 in both luteal and granulosa
cells and by PCB 153 in luteal cells, (c) Effect of PCBs on granulosa cells from different sizes
of follicles was less evident
Supported by grant (PBZ-KBN-084/P06/2002).
PHYTOESTROGENS ACTION IN GRANULOSA CELLS DERIVED FROM
SMALL OVARIAN FOLLICLES IN PIGS: STEROIDOGENESIS,
PROLIFERATION AND EXPRESSION OF ESTROGEN RECEPTORS
Anna Nynca, Olga Kraszewska, Maria Słomczyńska, Renata Ciereszko
Institute of Zoology, Jagiellonian University, Krakow, Poland,
Phytoestrogens are plant-derived phenolic compounds which exhibit estrogen-like activity.
The most examined phytoestrogens are genistein and daidzein. Moreover, genistein is known
as a tyrosine-protein-kinase (PTK) inhibitor. The objective of the study was to investigate the
effect of genistein and daidzein on: 1) in vitro production of progesterone (P4) and estradiol
(E2) by porcine granulosa cells harvested from small ovarian follicles (3-6 mm); 2) granulosa
cell proliferation; and 3) expression of estrogen receptor α (ERα) and β (ERβ) proteins in the
cells. Moreover, the effect of lavendustin C (LAV), the non-phytoestrogen PTK inhibitor, on
steroidogenesis was examined. Granulosa cells (300 000/ml/well) were first cultured for 72 h
(37ºC, 95% air, 5% CO2) in Eagle medium. Then, the cells were cultured for 48 h with E2
(100 ng/ml), FSH (100 ng/ml), IGF-I (30 ng/ml), genistein, daidzein (0.05-50 µM) and/or
LAV (0.05-50 µM). Medium concentrations of P4 and E2 were determined by
radioimmunoassay. Cell proliferation was measured by Alamar Blue assay. Expression of
ERα and β in cultured granulosa cells was evaluated by immunocytochemistry. FSH
stimulated P4 but not E2 production by granulosa cells originated from small ovarian follicles
in pigs. IGF-I did not affect follicular steroidogenesis. In addition, P4 production was not
affected by E2. Genistein and daidzein inhibited, in a dose-dependent manner, basal P4
production. Similarly, both phytoestrogens decreased P4 production in the presence of FSH
and IGF-I. Estradiol production both in the presence and in the absence of FSH and IGF-I was
stimulated by genistein. None of the examined doses of LAV affected steroid hormone
production. Daidzein did not affect E2 production by granulosa cells. ERα was not found in
granulosa cells harvested from small porcine follicles. In contrast, expression of ERβ was
demonstrated in nuclei of granulosa cells in all examined preparations. Estradiol, genistein
and daidzein appeared to amplify nuclear ERβ immunostaining. In conclusion, genistein and
daidzein inhibited P4 production in granulosa cells originated from small ovarian follicles in
pigs. The inhibitory effect of genistein did not appear to be associated with PTK activity since
LAV (PTK inhibitor) did not affect steroidogenesis. Phytoestrogen action on porcine
granulosa cell function, however, seemed to be mediated by ERβ.
Supported by PBZ-KBN-084/P06/2002 and UWM 522.0206.0206, Poland.
GENISTEIN ACTION IN GRANULOSA CELLS DERIVED FROM LARGE
OVARIAN FOLLICLES IN PIGS: STEROIDOGENESIS, PROLIFERATION
AND EXPRESSION OF ESTROGEN RECEPTORS
Anna Nynca, Magdalena Macewicz, Maria Słomczyńska, Barbara Kamińska, Renata
Ciereszko
Genistein (GEN), a phytoestrogen, may affect the female reproductive system by imitating or
antagonizing estradiol action. However, GEN mechanism of action is not recognized in most
species including pigs. The objective of the study was to investigate the effect of GEN on: 1)
in vitro production of progesterone (P4) and estradiol (E2) by porcine granulosa cells
harvested from large preovulatory ovarian follicles (>7 mm); 2) proliferation of granulosa
cells; and 3) expression of estrogen receptor α (ERα) and β (ERβ) proteins in the cells.
Granulosa cells (150 000/ml/well) were first cultured for 72h (37ºC, 95% air, 5% CO2) in
Eagle medium. Then, the cells were cultured for 24 or 48h with estradiol (10, 100 ng/ml),
FSH (100 ng/ml), LH (100 ng/ml) and/or GEN (0.05-50 µM). Medium concentrations of P4
and E2 were determined by radioimmunoassay. Cell proliferation was measured by Alamar
Blue assay. Expression of ERα and β in cultured granulosa cells was evaluated by
immunocytochemistry (ICC). The ICC detection of ERα and β was performed using
monoclonal mouse antihuman ER antibodies. Color reaction was detected by using
biotinylated anti-mouse IgG, avidin-biotin peroxidase complex and developed in
DAB/peroxidase reaction. Both gonadotropins stimulated P4 production by granulosa cells
irrespective of culture time. FSH, in contrast to LH, did not increase E2 production.
Progesterone production was decreased or increased by E2 following 24h or 48h culture,
respectively. Regardless of culture time, GEN inhibited, in a dose-dependent manner, both
basal and gonadotropin-stimulated P4 production by granulosa cells. Estradiol was not
affected by GEN. Cell proliferation was slightly reduced solely by the highest dose of GEN.
In the current study, ERα was not found in porcine granulosa cells. In contrast, expression of
ERβ was demonstrated in nuclei of granulosa cells in all examined preparations. Estradiol and
GEN appeared to intensify nuclear ER immunostaining. In conclusion, GEN did not immitate
E2 action on porcine granulosa cells. Progesterone production was inhibited by E2 after 24h
culture and enhanced after 48h of culture. In contrast, GEN, regardless of the length of
granulosa cell culture, decreased both basal and gonadotropin-stimulated P4 production. It
appears that the inhibition was not caused by changes in cell number and viability. In
addition, E2 and GEN increased ERβ expression in granulosa cells originated from large
ovarian follicles in pigs.
Supported by PBZ-KBN-084/P06/2002 and UWM 522.0206.0206, Poland.
2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD) AFFECTS
PROGESTERONE SECRETION BY CHICKEN PREOVULATORY
FOLLICLES
Andrzej Sechman, Anna Hrabia, Janusz Rząsa
Agricultural University of Cracow, Department of Animal Physiology, Al.
Mickiewicza 24/28, 30-059 Krakow, Poland; E-mail: [email protected]
Several lines of evidence indicate that in mammals TCDD affects secretion of sex hormones
from the ovarian cells. In birds, it has been shown that TCDD is a potent endocrine disruptor,
however, in the available literature there are no data on the effect of TCDD on steroid
hormones secretion by cells of avian ovarian follicles. Therefore, the aim of this study was to
examine the effect of TCDD on basal and LH-induced progesterone (P4) secretion by the
chicken preovulatory follicles.
Isa Brown laying hens (n=6; 25-weeks old) which were maintained in standard conditions and
fed ad libitum were used in the experiment. The chickens were decapitated 2 h after ovulation
and three largest preovulatory follicles (F3-F1; F3<F2<F1) were isolated form the ovary. The
granulosa layers separated from the preovulatory follicles were divided into 6 equal fragments
and were incubated for 24 h at 38°C in 1 ml Eagle’s medium supplemented with TCDD (at
dose: 1, 10 or 100 nM), ovine LH (10 ng/ml) and TCDD (10 nM)+LH (10 ng/ml). Control
granulosa layers were incubated in medium without the dioxin or hormone. Concentration of
P4 in collected medium was measured by means of RIA method; protein level in the
granulosa was determined by Lowry’s method.
TCDD at a dose 10 nM significantly increased P4 secretion from granulosa layer of F3 and F2
preovulatory follicles by 110% and 54%, respectively (P<0.01). However, the highest applied
TCDD dose (i.e. 100 nM) diminished P4 secretion from F3, F2 and F1 follicles by 11% (NS),
24% and 34% (P<0.05), respectively. In comparison to control group, LH significantly
(P<0.001) increased P4 secretion from all examined ovarian follicles (F3, F2 and F1).
Addition of TCDD to medium supplemented with LH significantly inhibited the stimulatory
effect of LH by 20%, 36% and 29%, respectively (P<0.05-0.01).
The results obtained indicate that: (i) TCDD affects progesterone secretion from chicken
preovulatory follicles, (ii) the effect of TCDD depends on the applied dose, (iii) TCDD
diminishes the LH-induced progesterone secretion from the granulosa layer of all examined
hierarchical follicles. The further studies are needed to elucidate the mechanism of the dioxin
action in the chicken preovulatory follicles.
Supported by grant: DS-3243/KFZ.
EFFECT OF 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD) ON
ESTRADIOL LEVEL IN CHICKEN EMBRYOS
Andrzej Sechman1, Marcin W. Lis2, Anna Hrabia1, Jerzy Niedziółka2, Janusz Rząsa1
1
Department of Animal Physiology, 2Department of Animal Hygiene and Breeding
Environment, University of Agriculture, al. Mickiewicza 24/28, 30-059 Krakow,
In order to study the effect of TCDD on estradiol level in blood of chicken embryos, three
hundred fertilized eggs of ISA 215 parental broiler flock were incubated in Masalles type 65
DIGIT incubator in standard conditions. Eggs were divided randomly into 3 equal groups and
on day 7 of embryogenesis they were injected with 0, 2.5 and 5.0 ng TCDD per egg dissolved
in 50 µl of peanut oil. Blood samples were collected from chick embryos on day 14 (E14), 18
(E18), 20 (E20; external pipping), and on day 21 of incubation, i.e. just after hatching (D1).
After blood sampling, embryos and chicks were sexed and blood samples allocated according
to sex of the embryo. Estradiol level in blood plasma was measured radioimmunologically.
It was found that TCDD significantly affected estradiol levels in blood plasma of both female
and male chicken embryos. In female embryos of each group, the level of estradiol gradually
decreased in blood plasma from E14 toward D1 (in control group from 147 ± 15 to 55 ± 9
pg/ml; P<0.01). In comparison with control group, both applied doses of TCDD decreased
estradiol level on E14 by 27 and 31% (P<0.05), respectively. On E18 the effective was only
the higher dose of TCDD which lowered estradiol level by 43% (P<0.05). On E20 the level of
estradiol did not differ among groups, but on D1 it was by 48% higher in blood plasma of
embryos treated with 2.5 ng TCDD (P<0.05).
In male embryos, the level of estradiol in control group did not changed significantly and was
fluctuated from 40 ± 7 pg/ml on E14 to 29 ± 6 pg/ml on D1. In both TCDD treated groups the
level of estradiol was on the average by 66 and 191% higher on E14 and E18, respectively
(P<0.01). There were no significant differences in estradiol level on E20 and D1 among
control and TCDD treated groups.
The results obtained indicate that during embryogenesis TCDD decreases the level of
estradiol in blood plasma of female chicken embryos, on the other hand in male embryos the
effect of TCDD is reverse. The further experiments are necessary to explain the effect of
dioxin on gonadal function during chicken embryogenesis.
Studies supported by grants: BW-2223/KFZ and DS-3210/ZHZiŚH.
ACTION OF TWO DIFFERENT PCBs MIXURE (DELOR 103 AND DELOR
106) ON LH SECRETION BY DISPERSED PITUITARY CELLS INCUBATED
IN VITRO
Magdalena Socha1, Mirosława Sokołowska-Mikołajczyk1, Paweł Szczerbik1, Piotr
Epler1, R Grabic2
1
Department of Ichthyobiology and Fisheries, University of Agriculture, 30-199
Krakow, Poland, E-mail: [email protected]
2
Institute of Public Health in Ostrava, National Reference Laboratory for Analysis
of POPs, Ministry of Health CR, Partyzanske nam. 7, 702 00 Ostrava, Czech Republic
Polychlorinated biphenyls (PCBs) are persistent and bioaccumulative environmental toxicant,
which are able to impair various aspects of reproduction in both mammals and fish. There are
a few data showing that in vivo exposition to these xenobiotics change secretion of many
hormones e.g. gonadotropin, steroids, what suggest that the hypothalamic-pituitary-gonadal
axis could be one of the targets of PCBs.
The aim of this study was to investigate the possible direct effect of polychlorinated biphenyls
on the LH secretion from dispersed pituitary cells taken from female goldfish (Carassius
auratus gibelio Bloch). Two PCBs mixture: low-chlorinated (Delor 103) and high-chlorinated
(Delor 106) at the concentration: 1, 5, 10, 50 and 100 ng ml-1 were added to cultured medium
of experimental groups. The LH levels were measured after 5 and 24 hours of incubation in
the cultured medium by ELISA method. The lowest tested concentration (1 ng ml–1) of both
mixture had no effect on LH secretion from goldfish dispersed pituitary cells at 5 and 24
hours of culture. Significant increase in LH release (p<0,05) was observed in the groups
treated with PCBs in doses from 5 to 100 ng ml –1 medium after 5 and 24 hours of incubation.
The increase was dose dependent, the highest LH (70-80 ng ml –1 medium) level was
observed in groups in which the concentration of Delor 103 and 106 was 50 and 100 ng ml –1
medium.
These results indicate that endocrine disrupters such as PBCs could disrupt reproduction in
goldfish by acting at the level of pituitary, directly on the gonadotrophs, affecting LH
secretion.
INFLUENCE OF SOME ENVIRONMENTAL STRESSORS ON COMMON
CARP (CYPRINUS CARPIO) PITUITARY CELL FUNCTIONS
Paweł Szczerbik, Tomasz Mikołajczyk, Mirosława Sokołowska-Mikołajczyk,
Magdalena Socha, Jarosław Chyb, Piotr Epler
Department of Ichthyobiology and Fisheries, University of Agriculture,
30-199 Krakow-Mydlniki, Poland, E-mail: [email protected]
The influence of cadmium, di(2-ethylhexyl)phthalate (DEHP) and dibutylphthalate (DBP) on
the spontaneous in vitro LH secretion from dispersed pituitary cells of common carp
(Cyprinus carpio L.) was investigated. Cadmium is one of the most toxic heavy metals,
known for its ability to persistent accumulation in tissues of living organisms and also in
sediments. Phthalates (phthalic acid esters) have a variety of industrial uses. Di(2ethylhexyl)phthalate and dibutylphthalate belong to common plasticizers, used among others
in PVC (polyvinyl chloride) production. They do not persistently accumulate in animal tissues
but fish are exposed to these substances continuously for their common appearing and instant
influx into the waters (they leak out from PVC and other items). Little is known about the
influence of phtalates on endocrine functions of fish organisms.
Cadmium was applied at the concentrations of 0, 2x10-5 M and 2x10-4 M, DEHP and DBP at
0, 10-6 M and 10-5 M. LH level in the incubation media was measured with ELISA method.
We found no influence of DEHP and DBP on spontaneous secretion of LH from dispersed
pituitary cells. On the other hand cadmium at a higher concentration (2x10-4 M) significantly
stimulated spontaneous LH secretion. We suppose, that the effects of Cd ions on the pituitary
cells come from the chemical similarity of cadmium to calcium. Thus the influence of
cadmium on gonadotropic cells can be interpreted (among others) as the competition between
ions of these metals. Obtained results suggest that cadmium modify the functions of common
carp pituitary cells and probably can influence the reproductive success of fish.
It is possible that DEHP and DBP do not have a direct effect on the pituitary cells but they act
at the level of hypothalamus, modulating the release of the factors which control LH
secretion.
EFFECT OF 2,2’,4,4’,5,5’-HEXACHLOROBIPHENYL (PCB 153) ON THE
FSH-STIMULATED ESTRADIOL SECRETION AND LH-STIMULATED
PROGESTERONE SECRETION BY PIG OVARIAN FOLLICULAR CELLS
Anna Wójtowicz, Anna Stankiewicz and Ewa Ł. Gregoraszczuk
Laboratory of Physiology and Toxicology of Reproduction, Department of Animal
Physiology, Institute of Zoology, Jagiellonian University, Krakow, Poland,
E-nail: [email protected]
Introduction: Polychlorinated biphenyls (PCBs) are the most widespread environmental
contaminants. Because of their resistance to environmental degradation and bioaccumulation
in the tissue, they affect a number of physiological processes including reproduction. One of
the most prominent congener is the ortho-substituted non-coplanar 2,2’,4,4’,5,5’hexachlorobiphenyl (PCB153).
In our previous paper, we demonstrated that the exposure of porcine follicular cells to
PCB153 affected ovarian steroidogenesis, and that the action of this congener depended on
the stage of follicular development. The presence of LH receptors and granulosa FSH
receptors in the late preantral to early antral stages of development makes the follicles
dependent on gonadotropin support. The increased estrogen secretion by developing follicles
triggers a positive feedback mechanism and, as a consequence, the LH surge necessary for
initiating the ovulation.
Aim: The objective of this study was to determine whether PCB153 could be responsible for
premature development of follicles and, consequently, premature sexual maturation by
stimulating estradiol secretion by ovarian follicles in prepubertal animals and synergistic
action with gonadotropins
Methods: To investigate whether PCB 153 had a synergistic action with gonadotropins on
ovarian steroidogenesis, granulosa and theca cells were isolated from ovarian follicles taken
from prepubertal pigs. Cells were initially cultured for 24 h to allow their attachment to the
plates. After 24h, the media were changed and cells were cultured with the addition of 100
ng/ml of PCB 153 alone or in combination with 100ng/ml of FSH or 100 ng/ml of LH. After
48-h culture, the media were collected for progesterone and estradiol analysis using EIA.
Additionally, the activity of LDH in the medium and caspase-3 in the cells were measured as
markers of cytotoxicity and apoptosis. Proliferation of follicular cells was determinate using
Alamar Blue test.
Results: PCB 153 had no effect on basal estradiol secretion but additional increase in
estradiol secretion was observed in the FSH-treated cells. Moreover, it caused a decrease in
caspase-3 activity and produced no effect on cell viability and proliferation .
Conclusion: Stimulatory action of PCB 153 on the FSH-stimulated estradiol secretion with
parallel action as anti-apoptotic agent, observed in the presented study, could be one of the
principal mechanisms engaged in the action of these compounds as factors advancing sexual
maturation. It is possible to propose a hypothesis that earlier age at menarche among girls and
higher rates of precocious puberty is due to prenatal exposure to xenoestrogens.
The work was supported by Jagiellonian University grant DS/IZ/FZ/2004
INFLUENCE OF POLYCHLORINATED BIPHENYLS (PCBs) ON THE
CONTRACTION OF BOVINE UTERUS IN VITRO
Michał Wróbel, Krzysztof Kamiński, Jan Kotwica
Spontaneous uterine contractions are calcium ([Ca2+]i) dependent and this is amplified by
oxytocin (OT). PCBs are persistent environmental pollutants due to their low rate of
degradation and ability to accumulate in tissues. Hence the aim of these studies was to
investigate the effect of PCBs on (a) viability of myometrial cells, (b) uterine contractions and
(c) [Ca 2+]i concentrations.
Uteri were collected from slaughtered cows at peri-ovulatory period. In Exp.1, myometrial
cells were obtained by enzymatic digestion of tissue and pre-incubated (3x105/ml) for 96h in
medium (DMEM/F-12 HAM) with 10% FCS. Thereafter medium was replaced by fresh with
0,1% BSA, and cells were incubated for 48h with mixture of PCBs (Ar1248) or with one of
PCB congeners -77,-126,-153 (10, 100 ng/ml). Using TOX1-kit viability of myometrial cells
was assigned. In Exp.2 myometrial strips were incubated (4oC; atmosphere: of 95% air and
5% CO2) with Ar1248 or with PCB-77,-123 or -153 (10, 100 ng/ml) for 24, 48 or 72h.
Thereafter strips were mounted in the water-jacketed bath at 38oC and aerated with mixture of
95% O2 and 5% CO2 for the measurement of spontaneous and OT-stimulated (10-7M)
contractions. In Exp.3 myometrial cells were pre-incubated as in Exp. 1. The cells (5x104/ml)
were incubated for 48h with either PCB-77,-126, or -153 (10, 100 ng/ml). Changes of [Ca2+]i
concentrations before and after OT stimulation (10-7M), were monitored in cells loaded with
the fluorescent Ca2+-sensitive probe FURA-2AM (5µg/ml), and analyzed by computer
software (Micro Image 4.0; Olympus Optical Co.).
PCBs pre-treatment did not affect the viability of cells compared to control. The force and
frequency of strip contraction was changed by PCBs (P<0.05). Mixture of PCBs increased the
force of contraction after 24h and decreased after 48h. PCB congeners increased the basal
force of contraction of myometrium after 48h. In response to OT all uterine strips increased
its contraction force. However, PCBs (100 ng/ml) suppressed the effect of OT (P<0.05)
before ovulation. Moreover, individual PCBs tended to stimulate frequency of spontaneous
and OT-stimulated contractions of myometrial strips. Ar1248 diminished the effect by OT
(P<0.05). PCBs (10ng/ml) substantially increased Ca2+ concentrations in myometrial cells.
Different congeners at different doses suppressed or delayed (P<0.05) increase [Ca2+]i. within
short time after supplementation of medium with OT.
Data suggest that effect of PCBs on basal concentrations of [Ca2+]i in myometrial cells can
impair the spontaneous and OT-stimulated contraction of the uterus. PCBs in the used doses
do not affect viability of myometrial cells.
Supported by grant (PBZ-KBN-084/P06/2002)
Session VII
Andrology II
Oral presentation
RELATIONSHIP BETWEEN AROMATASE IMMUNOEXPRESSION AND
ANTIOXIDANT ABILITY IN LH, IGF-I AND PRL - TREATED BANK
VOLES
Monika Gancarczyk1, Magdalena Kuklińska2, Jerzy StrzeŜek2, and Barbara Bilińska1
1
2
Department of Animal Biochemistry and Biotechnology, University of Warmia and
Mazury, Olsztyn, Kortowo, Poland
Aromatization and antioxidant strategies in the male gonads are important processes, which
control normal fertility. The ability of the testis to convert irreversibly androgens into
estrogens is related to the presence of the cytochrome P450 aromatase (P450arom).
Aromatase expression has been shown not only in testicular somatic cells, but also in meiotic
and post-meiotic germ cells. Testicular tissue is vulnerable to reactive oxygen species (ROS)
attack, because they are generated by Leydig and Sertoli cells, germ cells and inflammatory
cells. Protection against ROS is provided by antioxidant enzyme - superoxide dismutase
(SOD). The SOD activity has been found in the interstitial tissue, as well as in seminiferous
tubules. It is well known that various hormones and environmental factors modulate steroid
production and germ cells maturation. In this context the question arises whether luteinizing
hormone (LH), prolactin (PRL), insulin-like growth factor (IGF-I), and the length of
photoperiod are able to exert the effect on aromatase immunoexpression and antioxidant
concentration in testes of bank voles, seasonally breeding rodents.
Mature bank voles, reared under short light cycles (6L:18D) or long light cycles (18L:6D)
were used in this study. The animals were injected intraperitoneally with PRL or LH or IGF-I
(separately or together) at a dose of 0.1µg/g body weight dissolved in 30µl PBS. The
injections were performed twice a day during three days. Aromatase was detected
immunohistochemically by a rabbit polyclonal antibody against human placental P450
aromatase (1:400; R-10-2; a gift from Dr. Yoshio Osawa, Buffalo NY, USA). Antioxidant
ability in the homogenized testicular tissue was determined by biochemical analyses. The
SOD activity was measured according to the method of Beauchchamp and Fridovich. The
concentration of antioxidants was measured using the Total Antioxidant Status Kit (Randox
Laboratories Ltd., UK). Androgen and estrogen levels in the homogenized testicular tissue
were measured radioimmunologically.
In testicular sections of the control group most of seminiferous tubules exhibited small lumens
with undifferentiated germ cells, and a moderate immunoreactivity for aromatase. In turn,
after LH, PRL, and IGF-I treatment advancement of spermatogenesis and intensified
immunostaining of aromatase was observed. Decreased SOD activity and increased total
antioxidant concentration in voles treated with LH, PRL, and IGF-I suggest a protective
influence of administered hormones against toxic oxygen radical. In animals treated with LH,
PRL, and IGF-I significant increase in testosterone and estradiol levels was observed in
comparison with a control group. The strongest influence of administered hormones on
aromatase immunoexpression, antioxidant ability, and steroid hormones levels was observed
when LH was injected together with IGF-I, indicating a synergistic action of these hormones.
Moreover, both testicular processes studied were found to be clearly photoperiod-dependent.
Beneficial effect of photoperiod was noted in voles kept under long light cycles.
The present study demonstrates significant relationship between aromatization, antioxidation
and the length of photoperiod in bank vole testes.
Supported by PBZ/KBN 084/PO6/2002
ANTIOXIDANT SYSTEM OF BOAR AND STALLION SEMINAL PLASMA –
COMPARATIVE STUDIES
Magdalena Kuklińska1, Monika Gorazd2, Agata Nowacka1, Kazimierz KosiniakKamysz2, Jerzy StrzeŜek1
1
Mazury, 10-718 Olsztyn-Kortowo, 2Department of Horse Breeding, University of
Agriculture, 30-059 Krakow, Poland, E-mail: [email protected]
The main components of the defence system of animal semen against reactive oxygen species
(ROS) are enzymes of spermatozoa and seminal plasma as well as low-molecular weight
antioxidants. The occurrence and concentrations of individual antioxidants are speciesspecific. For example, stallion spermatozoa possess an array of antioxidant enzymes
(superoxide dismutase, SOD; glutathione peroxidase,GPx; catalase,CAT) compared with boar
spermatozoa. Boar spermatozoa possess high SOD activity, which is contributed by one
molecular form of copper/zinc–SOD (Cu/Zn-SOD) and residual enzymes of the glutathione
cycle. Moreover, catalase activity is not present in boar spermatozoa.
The aim of this study was to compare the antioxidant systems of boar and stallion seminal
plasma, which are implicated in the sperm defence system against ROS. Boar seminal plasma
and secretory fluid of the second fraction of stallion ejaculate were used in this study. The
activity of the antioxidant enzymes, the content of low-molecular weight antioxidants. (Lglutathione, L-ergothioneine and L-ascorbic acid) and total antioxidant status (TAS) in boar
or stallion seminal plasma were determined. Total protein content was measured using
different analytical methods.
The enzymatic antioxidant defence system in boar seminal plasma is provided by one Cu/Zn
molecular form of extracellular SOD (EC-SOD). Furthermore, the content of the enzymes of
the glutathione cycle was negligible, whereas catalase activity was not detected in boar
seminal plasma. In contrast, in the stallion seminal plasma, it was confirmed the presence of
basic enzyme activity of the antioxidant system, in which CAT and GPx showed the highest
and lowest activity, respectively. The dominant low-molecular weight antioxidant is
glutathione, which in combination with ergothioneine forms the thiol groups, resulting in the
stabilization of the SH-groups in protein structures. In addition, glutathione content in stallion
seminal plasma was approximately 10-fold higher than that in boar seminal plasma. The low
content of non-enzymatic antioxidants in the seminal plasma of boar is recompensed by the
presence of SOD activity and its specific antiperoxidant properties of low-molecular seminal
plasma proteins. Moreover, the total protein content in the seminal plasma may determine the
antioxidant status of boar semen. The confirmed correlations between the total protein content
and the antioxidant systems of boar or stallion seminal plasma may have a practical
application in assessing the secretory efficiency of the accessory sex glands, particularly the
defence capability against oxidative stress caused by the reactive oxygen species.
Supported by funds from University of Warmia and Mazury in Olsztyn (No. 0103.0803).
ANTIOXIDATIVE ENZYMES PROTECTING THE EPIDIDYMAL
SPERMATOZOA
Mariola Marchlewicz, Barbara Wiszniewska
70-111 Szczecin, Al. Powstańców Wielkopolskich 72, Poland,
The causes of male infertility may be difficult to recognize. According to some
authors oxidative stress is one of the main causes of the phenomenon of worsening of male
fertility. During transport through the epididymal duct the sperm cells may be exposed to
endo- or exogenic factors that increase the concentrations of reactive oxygen species (ROS).
This is very dangerous for sperm cells since they are not equipped satisfactorily in enzymes
for the antioxidative protection. In addition, during the transport through the epididymis the
amount of polyunsaturated fatty acids increase within their cellular membrane. The latter
increases the liquidity of the cellular membrane thus facilitating the capacitation and
acrosomal reaction; however it also results in higher susceptibility to peroxidative damage.
Thus, the enzymatic proteins synthesized by the epithelial epididymal cells play an important
role in the degradation of ROS. These proteins include superoxide dismutase (SOD), catalase,
glutathione peroxidase (GPX) and glutathione-associated enzymes: glutathione reductase and
transferase, γ-glutamyl transpeptidase. Superoxide dismutase, an enzyme that catalyzes the
reaction of superoxide anion radical to H2O2 dismutation, occurs in large quantities along the
whole epididymis of the rat. The fluid from the cauda of the rat epididymis revealed the
presence of epididymal extracellular superoxide dismutase (E-SOD) that is influenced by the
gonadal factors. The presence of various glutathione peroxidases (GPX 1, 3, 4, 5) is proven in
the epididymis; their expression depends on region and the developmental stage of the
epididymis. It has been proven that expression of GPX 3 and GPX 5 was an androgen
controlled process. The presence of GPX 3 was revealed in the cytoplasm of the epithelial
cells of the cauda of epididymis i.e. in the part of the duct where sperm cells were stored for
several days. The latter correlates with the need for the epithelial cell protection from the
sperm cell-induced peroxidative damage. GPX 5 is an epididymal-specific, seleniumindependent enzyme and does not contain selenocysteine in the catalytic centre. It is likely
that that enzyme shows affinity to phospholipid peroxides or plays another, non-enzymatic
role. The expression of the GPX 5 gene was shown in the epithelial cells of the epididymal
duct before the sperm cells were present. That phenomenon suggests that GPX 5 prepares the
epididymal environment for the “sperm cell oncoming”. GPX 5 secreted in the head of the
epididymis is bound up with the head of the sperm cells traveling along the epididymis and
protects them from free radicals and preterm acrosomal reaction. This coupling also stabilizes
the enzyme itself in the epididymal environment. That enzyme is released from the membrane
of the sperm cells head in the female reproductive system and plays a special role during
fertilization. The enzymes functionally associated with glutathione (GS) play an important
role in the protection of sperm cells in the epididymis. According to some authors glutathione
belongs to the most important antioxidants synthesized in the epididymis. Glutathione Stransferases (GST) are enzymes that can bind glutathione to various, sometimes toxic for the
organism, compounds. High activity of GST was found in the basal cells of the cauda of
epididymis, moderate activity was found in the main cells of the head of epididymis.
Glutathione reductase (GR) is an enzyme that restores the reduced form of the glutathione.
Among tissues of the male rat reproductive system the highest levels of mRNA GR and
enzymatic protein, and also the highest activity of that enzyme, were found in the epididymis.
γ- glutamyl transpeptidase (GGT) restores indirectly the proper level of GSH.
The research project Nr PBZ-KBN-084/P06/2002 supported by Polish Scientific Committee
was performed between 2003 and 2005.
PROTEOMICS OF BOAR SEMINAL PLASMA – CURRENT STUDIES AND
POSSIBILITY OF THEIR APPLICATION IN BIOTECHNOLOGY OF
ANIMAL REPRODUCTION
Jerzy StrzeŜek, Paweł Wysocki, Władysław Kordan, Magdalena Kuklińska,
Mogielnicka Marzena, Daniel Soliwoda, Leyland Fraser
Mazury in Olsztyn, 10-718 Olsztyn-Kortowo, Poland, E-mail: [email protected]
The major goal of proteomics as science is making inventory of all proteins encoded in the
genome: PROTEin products expressed by the genOME.
Generally, proteomics can be defined as the qualitative and quantitative comparisons of
proteomes under different conditions to understand cellular mechanism underlying biological
processes. Moreover, several subcategories of proteome research have been developed in the
past couple of years.
Proteomics technologies are just beginning to have an impact in reproductive biology. The
identification of the properties and functions of proteins involved in the mechanism regulating
the activity of the reproductive tract can be used in male fertility assessment and clinical
diagnosis of the physiological state of individual reproductive organs. Furthermore, the
interactions between proteins of the seminal plasma and spermatozoa could be provide a
useful model for studying ligand-cell interactions occurring at the sperm cell surface. These
phenomena may have applicative significance in the preparation of different technologies of
semen preservation and the use of modern techniques in embryonic engineering.
The main aspects of proteomics of the seminal plasma, including our studies based on boar
seminal plasma, were been addressed in this study. Also, some aspects of expressive
proteomics regarding glycosylation of seminal plasma proteins were been presented. We have
suggested that the process of sex differentiation in the boar is synchronized with glycosylation
reactions accompanying protein secretions and, with the age of the animal, in relation to its
growth and secretory activity of the accessory sex glands. Only some selected elements of
functional proteomics components of boar seminal plasma proteins, particularly those related
to the basic physiological function of spermatozoa in mammalian reproductive process, have
been discussed. Special attention has been given to spermadhesins, which act as a
multifunctional protein of the seminal plasma. We have proposed that aggregation of the
seminal plasma proteins is probably an important phenomenon in the fertilization process,
particularly in the reproductive tract function of the boar.
Our research team has undertaken complex studies on the system of boar seminal plasma
proteins, particularly those secreted by the seminal vesicle glands. The localization and
detection of the proteins in the polypeptide profile was conducted using western blot analysis
of boar seminal plasma proteins, with specific polyclonal antisera, and two-dimensional (2D)
electrophoresis. Besides boar seminal plasma proteins, these methods have enabled the
identification of the molecular forms of a protein tyrosine acid phosphatase (AcP), platelet
activating acetylhydrolase (PAF-AH) and superoxide dismutase (SOD), which have been
highly purified in our laboratory. The biochemical properties and biological functions of these
enzymes were been discussed.
Some applicative aspects of proteomes of seminal plasma include the possibility of
application of seminal plasma proteins as a marker of fertilizing ability of semen, addition of
some isolated seminal plasma proteins to extended semen, with the aim of increasing the
viability of spermatozoa sorted for sex selection and post-thaw sperm survival, effects of
semen dialysis on viability of spermatozoa following preservation in liquid and frozen states,
were been addressed.
In summary, it can be suggested that further improvement in the biotechnological methods in
animal reproduction needs to elucidate the action of seminal plasma proteins in the
reproductive process. This indicates the necessity of further studies on proteomics of the
seminal plasma.
Supported by funds from University of Warmia and Mazury in Olsztyn (No. 0103.0803).
Poster presentation
THE PRESENCE OF FUNCTIONAL ENZYME, P450 AROMATASE,
IN RODENT AND HUMAN TESTICULAR CELLS
Barbara Bilińska1, Amalia Carpino2 Monika Gancarczyk1, Anna Hejmej1, Małgorzata
Kotula-Balak1
1
University, Krakow, Poland, E-mail: [email protected]
2
Department of Cell Biology, University of Calabria, Cosenza, Italy
In order to visualize immunoreactive aromatase or demonstrate its presence in rodent and
human testicular cells, specific anti-aromatase antibodies (a polyclonal rabbit anti-human
aromatase (a gift from Dr Yoshio Osawa, Hauptman-Woodward Medical Research Institute,
Buffalo NY, USA) or a monoclonal, mouse anti-human cytochrome P450 aromatase (Serotec
Ltd, Oxford, UK) were used.
A cytochrome P450 aromatase is responsible for the conversion of androgens into estrogens.
Recently, aromatase has been immunolocalized in germ cells of mouse, brown bear and rooster.
Our studies extended the number of species to rat, inbred strains of mouse, bank vole, and
human germ cells. In these species aromatase is functionally active not only in somatic cells but
also in the cells of spermatogenic line [1, 2].
In order to improve our understanding of the testicular site of aromatization in rodents several
complementary techniques have been used. Immunoreactive aromatase was found within
immature rat testis in spermatocytes [3] whereas in adult rat it was localized in spermatocytes,
round and elongated spermatids [4]. In these cells, the presence of 55kDa protein was
demonstrated, whereas in the bank vole the aromatase mol wt was 52kDa on Western blots [5].
Moreover, using RT-PCR, the amplified product from adult rat germ cells was detected in
pachytene spermatocytes, round spermatids and spermatozoa, as well as in somatic cells such as
Sertoli and Leydig cells [4].
In isolated sperms of the mouse, B10. BR-Ydel, and the bank vole, immunoreactive aromatase
was found in cytoplasmic droplets of sperm flagella [6]. At the ultrastructural level the gold
particles were distributed over the cytoplasm of elongated spermatids and residual bodies [7].
More recently, immunolocalization and expression of aromatase has also been detected in
human testicular cells and ejaculated sperms [8, 9, 10]. All together these data indicate that germ
cells represent an additional source of estrogens within the male gonad.
1. Carreau S, Genissel C, Bilińska B, Levallet J. Int J Androl 22: 211-223, 1999
2. Carreau S, Lambard S, Delalande C, Denis-Galeraud I, Bilińska B, Bourguiba S. RBE 1: 35-45, 2003
3. Carpino A, Pezzi V, Rago V, Bilińska B, Ando S.Tissue Cell 33: 349-353, 2001
4. Levallet J, Bilińska B, Mittre H, Genissel C, Fresnel J, Carreau S. Biol Reprod 58: 919-26, 1998
5. Bilińska B, Schmalz-Frączek B, Kotula M, Carreau S. Mol Cell Endocrinol 178: 189-198, 2001
6. Kotula-Balak M, Grzmil P, Styrna J, Bilińska B. Acta Histochem 106: 55-64, 2004
7. Kotula-Balak M, Słomczyńska M, Bourguiba S, Tabarowski Z, Bilińska B. Eur J Histochem 47:
55-62, 2003
8. Kotula-Balak M, Bablok L, Frącki S, Jankowska A, Bilińska B. Folia Histochem Cytobiol 42:
215-220, 2004
9. Rago V, Bilińska B, Palma A, Ando S, Carpino A. Folia Histochem Cytobiol 41: 23-27, 2003
10. Lambard S, Galeraud-Denis I, Bouraima H, Bourguiba S, Chocat A, Carreau S. Mol Hum Reprod
9: 117-24, 2003
Supported by the State Committee for Scientific Research as a Solicited Project PBZ-KBN084/PO6/2002 realized from 2003 to 2005.
The fruitful scientific cooperation with Dr. Serge Carreau (University of Caen, France) is
highly appreciated.
OPTIMIZATION OF COMET ASSAY FOR BOAR, BULL, AND TURKEY
SPERMATOZOA
Andrzej Ciereszko, Halina Karol, Mariola Kotłowska, Grzegorz J. Dietrich, Mariola
Wojtczak
Semen Biology Group, Institute of Animal Reproduction and Food Research, 10-747
Olsztyn, Poland, E-mail: [email protected]
Preservation of DNA integrity is essential for the protection of sperm quality. Sperm DNA
contains basic proteins providing a high degree of packing within the spermatozoon head
which minimizes damage from exogenous agents and keeps the genome transcriptionally
inactive. Sperm DNA, however, is not fully protected against damage. The main causes of
DNA damage in the spermatozoa have been identified as oxidative stress and deficiencies in
natural processes such as chromatin package and abortive apoptosis. Lack of repair
mechanisms in spermatozoa makes sperm DNA vulnerable to damage and this damage is
linked to male infertility. Single-cell gel electrophoresis (SCGE, comet assay) is a simple,
rapid, visual, and sensitive method for detecting primary DNA damage at the individual cell
level. The preliminary results indicated difficulties in sperm lysis and lack of the positive
control of the assay as the two main obstacles for comet assay in mammalian and birds sperm
due to ineffectiveness of H2O2 in inducing of DNA fragmentation. The objective of this study
was to optimize comet assay analysis for boar, bull, and turkey spermatozoa.
Semen samples from turkey toms (extended semen for 2-4h, N = 4), boars (extended semen
for 2-3 days, N = 5), and bulls (cryopreserved semen, N = 5) were used. The neutral comet
assay protocol as described by the Trevigen (Gaithersburg, MD, USA) was used. This
protocol had been modified and two versions were worked out. The first version (CA1)
included prolonged (to 3h) lysis at 37 °C with 40 mM dithiothreitol and proteinase K (0.1
mg/ml) into lysis solution. Electrophoresis was carried out for 20 min. The second version
(CA2) included an additional unwinding step in alkali solution (15 min at room temperature).
Proteinase K was omitted from CA2 for boar and bull, but not turkey sperm. Electrophoresis
was carried out for 10 min. Potassium permanganate was used for positive control, instead of
H2O2. Although both protocols produced comet pictures, CA2 seems to be more appropriate
for studies of DNA fragmentation in the three species. For this version, a dose-dependent
relationship was found between KMnO4 concentrations (0 - 25 mM for boar and bull and 0 –
50 mM for turkey) and comet parameters (head DNA, tail moments, but not tail length). On
the other hand, CA1 may be useful for analysing changes in the comet tail length of boar
spermatozoa. Both CA1 and CA2 are not time-consuming and can be performed within one
day. In conclusion, our results suggest that comet assay in the CA2 version is more useful for
the studies of DNA fragmentation of boar, bull, and turkey semen.
This study was supported by the State Committee for Scientific Research (KBN) as a Solicited
Project PBZ-KBN-084/P06/2002 from 2003 to 2005.
EFFECT OF SYNTHETIC ANTIOXIDANT ADDITION ON BIOLOGICAL
PROPERTIES OF BOAR SPERMATOZOA FOLLOWING LIQUID
STORAGE AT 5º AND 16ºC
Anna Dziekońska1, Magdalena Kuklińska1, Anatoly S. Erokhin2, Jerzy StrzeŜek1
1
Department of Animal Biochemistry and Biotechnology, University of Warmia &
Mazury, 10-718 Olsztyn, Poland, 2Research Institute of Animal Breeding, Box Lesnye
Polyany, Pushkin District, Moscow, Russia, E-mail: [email protected]
Disturbances in the metabolic processes of spermatozoa during a long-term liquid storage of
semen can be caused by lipid peroxidation of polyunsaturated fatty acids, which are
components of boar sperm plasmalemma. This phenomenon is associated with increased
extracellular enzyme leakage, which could lead to impaired metabolic activity and reduced
motility spermatozoa.
The aim of the study was to investigate the effects of the addition of a synthetic antioxidant,
bromid-NN-dimetyl-N-hexadecyl-4oxy-3,5-dibutyl-benzyl-amine (1), to semen extenders on
the biological properties of boar spermatozoa during storage at 5º and 16ºC.
Semen, collected from 3 boars, was diluted in a standard extender, Kortowo 3 (K3), or K3
extender containing lipoprotein fractions extracted from hen egg yolk (LPFh) or ostrich egg
yolk (LPFo) and stored for 7 days at 5º and 16ºC. The antioxidant was added to each
extender prior to storage. Extended semen samples without the addition of the antioxidant
were served as the control. Sperm motility was assessed microscopically. The percentage of
viable spermatozoa with functional mitochondria was assessed using a combination of
fluorochromes, JC-1 and propidium iodide (PI), an exclusion stain. The metabolic activity of
spermatozoa was evaluated
by measuring adenosine triphosphate (ATP) content
(bioluminescent method) and oxygen uptake (Z02) using the Clark-type electrode. Superoxide
dismutase (SOD) activity was determined in the supernatants of K-3 extended sperm samples
using the nitroblue tetrazolium (NBT) reduction method with the xanthine and xanthine
oxidase system.
The sperm parameters analyzed (sperm motility and metabolic activity as well as SOD
content in spermatozoa) for extenders supplemented with the antioxidant were higher
compared with the control, irrespective of the extender type and storage temperature. Higher
percentage of spermatozoa exhibiting positive JC-1 staining was more marked in LPF-based
extenders after 72 h of storage. The deterioration of sperm plasmalemma was accompanied by
increased SOD leakage in samples. There were individual variations among the boars.
The results of this study indicate that the addition of antioxidant to semen extenders, prior to
storage, had a beneficial effect on the biological properties of boar spermatozoa.
Supported by funds from University of Warmia and Mazury in Olsztyn (No. 0103.0206) and
within the framework of a joint collaborative agreement between the University of Warmia
and Mazury and Research Institute of Animal Breeding, Moscow, Russia.
1. Erokhin A. S. et. al. (1999) 3rd ESDAR Conference, Nov. 26 –27th, pp. 51-52.
COMPARISONS OF DNA INTEGRITY OF SPERMATOZOA FROM
WHOLE SEMEN AND RICH FRACTIONS OF BOAR EJACULATE
FOLLOWING FREEZING-THAWING IN DIFFERENT EXTENDERS
Leyland Fraser, Jerzy StrzeŜek
One of the research areas that has been studied intensively during the past decade as a cause
of male infertility is the DNA integrity of mature ejaculated spermatozoa. Sperm DNA
integrity is a requisite for best reproductive outcome and its maintenance is a task of vital
importance to the sperm cell. The aim of this study was to analyze the effects of freezing
extenders on DNA integrity of spermatozoa from whole semen and rich fractions of boar
ejaculate following freezing-thawing in different extenders.
Whole semen or sperm-rich fractions, collected from four Polish Large White boars, were
frozen were frozen under controlled conditions in 10-ml aluminum tubes or 5-ml straws after
extension in cryopreservation extenders consisting of 11% lactose and 20% whole hen egg
yolk (HEY), whole ostrich egg yolk (OEY) or 5% lyophilized lipoprotein fractions, extracted
from ostrich egg yolk (LPFo), with 3% glycerol and 0.5% Equex STM. Sperm samples frozen
in a standard boar semen extender, Kortowo-3, (K-3) without protective substances and
glycerol, served as the control. The neutral comet assay was employed to evaluate boar sperm
DNA integrity. Fluorescent microscopy was used to analyse comets and images were
evaluated for the percent of tail DNA and tail length DNA, using special imaging analysis
software (Komet System). Besides sperm motility, sperm plasma membrane integrity was
assessed using fluorescent methods.
Post-thaw sperm DNA damage was significantly increased (p≤0.05) compared with fresh
semen, regardless of the ejaculate collection type. However, in all boars the percentage of
post-thaw DNA damage was lower (p≤0.05) in spermatozoa from the whole semen compared
with those from the rich fractions frozen, irrespective of the extender type and packaging
materials. Moreover, higher DNA damage was observed in sperm samples frozen in K-3
extender (control). It was observed that post-thaw sperm DNA damage in extender containing
OEY was more marked in extenders containing HEY and LPFo, particularly for spermatozoa
from the rich fractions. Inter-boar variations in post-thaw DNA damage were more
pronounced in sperm samples frozen in freezing extender containing HEY or LPFo. In each
boar, the comet-analyzed, percent tail DNA and tail length DNA, were significantly higher
(p≤0.05) in the control. Throughout this study the deterioration in post-thaw sperm DNA
damage was accompanied by increased plasma membrane destabilization. The results of the
current show that components of egg yolk or lyophilized lipoprotein fractions are
indispensable for maintaining DNA integrity of frozen-thawed boar spermatozoa.
Supported by the State Committee for Scientific Research, Project No. PBZ-KBN084/PO6/2002.
DIALYSIS OF BOAR SEMEN AND ITS EFFECT ON POST-THAW SPERM
QUALITY
Leyland Fraser, Jerzy StrzeŜek
Cryopreservation of boar semen is known to be associated with a decrease in post-thaw sperm
quality. The initial quality of the semen is one of the factors that may affect its freezability.
This study was undertaken to determine whether the dialysis process of whole boar semen
would improve the post-thaw sperm quality, irrespective of the extender type and packaging
materials.
Whole semen, collected from three Polish Large White boars, was subjected to macroscopic
and microscopic analyses. Besides motility evaluation, the percentage of spermatozoa with
intact plasma membrane (SYBR-14 and propidium iodide - PI) and functional mitochondria
(rhodamine 123/PI) was assessed. Lipid peroxidation was measured by determining the
production of thiobarbituric acid reactive species (TBARS) and the neutral comet assay was
employed to assess sperm DNA integrity. One portion of the semen samples was subjected to
a 5-h period dialysis against standard boar semen extender, Kortowo-3 (K-3), using cut-off
membrane of 12-14 kDa (Visking Dialysis Tubing), whereas the other portion was diluted in
K-3 extender and cooled at 16°C. Non-dialyzed and dialyzed semen samples were frozen in
10-ml aluminium tubes or 5-ml straws after extension in cryopreservation extenders
consisting of 11% lactose and 20% whole hen egg yolk (HEY) or 5% lyophilized lipoprotein
fractions extracted from ostrich egg yolk (LPFo), with 3% glycerol and 0.5% Equex STM.
Prior to freezing, enhanced sperm quality including motility, plasma membrane integrity and
mitochondrial function, was evident after dialysis of the semen. However, the proportions of
spermatozoa with intact plasma membrane (SYBR-14/PI) and functional mitochondria
(R123/PI) declined with a corresponding increase in TBARS levels and DNA damage in postthaw semen samples, regardless of the type of pre-freezing preparation technology. Moreover,
dialysis significantly improved (p≤0.05) the post-thaw sperm quality, irrespective of the
extender type and packaging materials. A higher proportion of motile spermatozoa was
concomitant with increased percentage of positive SYBR-14 and R123 stained cells in postthaw dialyzed samples frozen in aluminum tubes or straws. Post-thaw sperm TBARS levels
and DNA integrity were lower in dialyzed samples and there were wide variations among the
boars. No significant differences in post-thaw sperm quality were found between freezing
extenders containing HEY and LPFo. The results of this study show that dialysis of boar
semen is beneficial for prolonged sperm survival following freezing-thawing. This is due to
the removal of low-molecular weight substances (ions, peptides and peroxides) that probably
can affect the structural and functional characteristics of boar spermatozoa during
cryopreservation.
Supported by the State Committee for Scientific Research, Project No. PBZ-KBN084/PO6/2002.
MORPHOLOGICAL CHANGES WITHIN SEMINIFEROUS TUBULES IN
IMMATURE BANK VOLES TREATED EITHER WITH 17β ‫־‬ESTRADIOL
OR A PURE ANTI-ESTROGEN ICI 182,780
Monika Gancarczyk1, Anna Hejmej1, and Barbara Bilińska1
1
It has been known that estrogen administration or deprivation can affect development and/or
maintenance of male gonadal functions. Testicular estrogens play a significant role in germ
cells development, especially spermatid production and epididymis sperm maturation.
Treatment of animals with a pure anti-estrogen ICI 182,780, which blocks estrogen receptors
α and β, has been shown to be a useful model for the examination of estrogen action in the
male reproductive tract. The aim of the present study was to check a dose-dependent effect of
17β-estradiol (E2) and ICI on testicular structure and spermatogenesis in the sexually
immature males.
Immature bank voles (seasonally breeding rodents) were derived from our own colony, and
since 10 generations they have been reared under short light cycles (6L:18D) or long light
cycles (18L:6D). The animals were injected intraperitoneally with two doses of either
17β ‫־‬estradiol (0.1 or 10 µg/g body weight dissolved in 20 µl sesame oil or with ICI 182,780
(10 or 100 µg/g body weight) in the same solvent. Control groups received 20µl sesame oil
only. The injections were performed twice a week during 2 weeks. These doses were selected
on the basis of results of a preliminary dose-dependency study. For morphological analysis,
testicular sections from both control and experimental groups were routinely stained with
haematoxylin and eosin. To check for the presence of apoptotic cells in tissue sections In Situ
Cell Death Detection Kit was used.
In testicular sections from control groups most of seminiferous tubules exhibited small
lumens with undifferentiated germ cells and a scant interstitial tissue. Exposure to the low
dose of estradiol induced acceleration of the onset of spermatogenesis. This was particularly
apparent in voles kept under short light cycle conditions. On the other hand, when males were
treated with the high dose of estradiol or ICI, disruptions of testicular structure and tubular
atrophy were observed. Apoptosis of germ cells was observed either after exposure to the high
dose of estradiol or ICI. An increased number of apoptotic cells were apparently visible in
comparison with the low dose of E2 as well as with control groups. We like to suggest that
spermatogenesis in the immature bank vole is upregulated by low doses of estrogens as in
mice, rats, and hamsters.
Therefore, it is concluded that bank voles, as seasonally breeding rodents, are a good model
for studying the role of estrogens in the functioning of the testis.
Supported by PBZ/KBN 084/PO6/2002
IMMUNOEXPRESSION OF Cu/Zn − SOD IN EPIDIDYMAL EPITHELIAL
CELLS OF RATS WITH DHT DEFICIENCY
Agnieszka Kolasa, Mariola Marchlewicz, Lidia Wenda-RóŜewicka, Barbara
Wiszniewska
70-111 Szczecin, Poland, E-mail: [email protected]
The mammalian spermatozoa are immature when they leave the testis. It is necessary for them
to pass through the epididymis to develop characteristics which are essential to achieve the
capacity for motility and to fertilize the oocyte. However, the epididymis apart from providing
the microenvironment for the maturation of spermatozoa, it takes part in their transport,
storage and maintaining sperm viability. During the transit through the epididymis, they are
exposed to reactive oxygen species (ROS) which are generated in low quantities as byproducts of normal cellular metabolism. The epididymal epithelial cells produce many
antioxidant enzymes, like superoxide dismutases (SODs) or glutathione peroxidases (GPXs).
The expression of majority epididymal genes is under control of androgen, mainly
dihydrotestosterone (DHT). DHT is formed by the irreversibly conversion of testosterone due
to the 5α-reductase type 2 (5α-red2) activity. In previous study we showed that the expression
of E-SOD and GPX5 mRNAs are modulated in epididymis of rats with DHT deficiency. The
aim of the study was to estimate the expression of copper/zinc superoxide dismutase (Cu/ZnSOD) in epithelial cells of the epididymis in rats with DHT deficiency.
The experiment was performed in adult, male Wistar rats. The rats were randomly divided
into control (C) and experimental groups (5 animals each). The inhibitor of 5α-red2
(finasteride; Proscar®, MSD Sweden) was administered orally to the animals from the
experimental group for 56 days (the time period of one spermatogenesis) in daily doses
amounting to 5mg/kg of body weight. After experiment period, the rats epididymis, both
control and experimental group, have been used for estimating copper/zinc superoxide
dismutase (Cu/Zn-SOD) expression by immunohistochemistry (Cu/Zn-SOD mouse
monoclonal antibody, clone 30F11, IgG1, Novocastra Lab. Ltd).
In physiological condition, the Cu/Zn-SOD immunoexpression was observed in epithelial
cells throughout whole epididymal duct. In the cytoplasm of epithelial cells of caput and
cauda epididymis of rats with DTH-deficiency we noticed the decrease of Cu/Zn-SOD
immunoexpression in comparison to caput and cauda epididymis of intact, control rats.
In summary our results confirmed that the expression of the antioxidant enzymes, like
a Cu/Zn-SOD, could depend on actual hormonal status within the epididymis.
The study was granted approval by the Local Ethics Committee and Animals Research.
Research was supported by the State Committee for Scientific Research as a Solicited Project
ESTIMATION OF DIFFERENCES IN SPERM MORPHOLOGY OF CATTLE
AND DOMESTIC PIG MALES
Stanisław Kondracki, Dorota Banaszewska, Anna Wysokińska, Joanna Chomicz
Department of Animal Hygiene and Veterinary Prophylaxis, University of Podlasie,
08-110 Siedlce, ul. B. Prusa 14, Poland. E-mail: [email protected]
The experiments were conducted on 44 ejaculates collected from 11 bulls of Black-andWhite breed and 44 ejaculates collected from 11 boars of Polish Landrace breed. The sires
were kept in the Mazovian Centre of Animal Breeding and Reproduction. Bulls semen was
taken by means of the artificial vagina method, whereas boars semen using the gloved-hand
method. Semen samples were collected from each ejaculate and microscopic slides were made
in order to estimate sperm morphology. Then, the slides were tested by Nikon E-400
microscope using immersion lens with 100 times enlargement. The frequency of occurrence
of morphological changes basing on microscopic tests of 500 spermatozoa in each slide was
analysed. Spermatozoa with proper morphology and with main and secondary changes were
identified according to Blom classification. Morphometrical measurements of randomly
chosen spermatozoa were made in each slide. The spermatozoal measurements were
performed manually using a set for the picture analysis made by the computer (Screen
Measurement v. 4.1). Measurements of spermatozoa were established as follows: the area of
the spermatozoon head, the length of the spermatozoon head, the width of the spermatozoon
head, the length of the spermatozoon flagellum and the total length of the spermatozoon. The
results of morphometrical measurements were used to calculate the following indexes of
spermatozoal morphology:
- the width of the spermatozoon head/head length ratio,
- the length of the spermatozoon head/the total length of spermatozoon ratio,
- the length of the spermatozoon head/the length of spermatozoal flagellum ratio,
- the size of the spermatozoon head/the total length of spermatozoon ratio.
Phenotypic correlations between the morphometrical traits and the frequency of occurrence of
developmental anomalies of spermatozoa were also calculated.
It was found that there were considerable and statistically significant differences in
spermatozoal morphology in bulls and boars. Bull spermatozoa were larger than boars, which
was above all proved by larger area of microscopic picture of the spermatozoon head by 3.19
µm2 and longer flagellum by 15.99 µm. Bull spermatozoa were longer than boars by
approximately 17 µm. No significant differences between species in the shape of the
spermatozoal head were found. However, the correlation between the frequency of
morphological changes and morphometrical traits of boar spermatozoa was noticed. More
morphological anomalies of spermatozoa in ejaculates containing longer spermatozoa were
also proved.
VARIABILITY OF MALES SPERMIOGRAM IN DOMESTIC PIGS IN
RELATION TO DIFFERENT BREEDS AND CROSSBREEDING VARIANTS
Stanisław Kondracki, Anna Wysokińska, Dorota Banaszewska, ElŜbieta Woźniak
Department of Animal Hygiene and Veterinary Prophylaxis, University of Podlasie,
08-110 Siedlce, ul. B. Prusa 14, Poland, E-mail: [email protected]
The experiments were carried out on 405 ejaculates taking from 74 boars. All boars were kept
in one sow insemination station, which belongs to the Mazovian Centre of Animal Breeding
and Reproduction, and they were used in similar environmental conditions. 136 ejaculates
collected from 31 boars of Polish Landrace, 46 ejaculates from 11 boars of Polish Large
White, 69 ejaculates from 7 boars of Pietrain, 21 ejaculates from 4 boars of Belgian Landrace,
19 ejaculates from 3 boars of Duroc and 17 ejaculates from 2 boars of Hampshire were tested.
97 ejaculates collected from two-breed crossbreds, including: 65 ejaculates from 7 boars of
Duroc x Pietrain crossbred and 32 ejaculates from 5 Hampshire x Pietrain crossbred were also
analysed. The ejaculates were taken by means of the gloved-hand technique twice a week.
Immediately after the collection, there were prepared slides from each ejaculate. The
microscopic assessment was used to estimate morphological structure of 500 spermatozoa in
each slide and to show the number of spermatozoa with proper morphology or with
morphological changes, distinguishing forms with major and minor abnormalities according
to Blom classification.
The classification of spermiogram quality in the 6-degree scale was also worked out in order
to compare sperm morphology in an individual male. The scale enabled the authors to mark
the ejaculates from 0 – for ejaculates with the worst sperm morphology – up to 5 – for
ejaculates with the best sperm morphology: Spermiograms of the tested ejaculates were
estimated and assigned to one of the six classes of the above classification. The frequency of
spermiograms in each class for the whole tested material and within each breed were also
calculated.
It was found that male spermiograms in domestic pigs were characterized by large variability
between breeds and within breeds. The estimation of semen quality, only in regard to the
mean frequency of occurrence of particular morphological forms of spermatozoa, was not
effective to evaluate an individual boar or a group of boars rated basing on the analysis of
many ejaculates. However, the method was effective to estimate particular ejaculates. The
quality classification of the spermiogram was essential to evaluate ejaculates because it
enabled the authors to prove the frequency of occurrence of ejaculates with good and average
usefulness for insemination, ejaculates with questionable usefulness or totally useless for
insemination within a group.
EFFECTS OF SUPPLEMENTATION OF PLATELET ACTIVATING
FACTOR (PAF) ON SELECTED MOTILITY CHARACTERISTICS AND
PLASMALEMMA INTEGRITY OF BOAR SPERMATOZOA FOLLOWING
LIQUID STORAGE
Władysław Kordan, Jerzy StrzeŜek, Anna Kolendo
Mazury in Olsztyn, 10-718 Olsztyn –Kortowo,
E-mail: wladyslaw.kordan @uwm. edu.pl
Platelet activating factor, PAF, (1-O-alkyl-2-acetyl-sn-glycero3-phosphorylcholine), a
membrane phospholipid, is present in the male and female reproductive systems. Our earlier
studies showed that PAF had a stimulating effect on the motility apparatus of boar
spermatozoa during liquid storage.
The aim of this study was to analyze the effect of supplementation of synthetic PAF to a
standard boar semen extender, Kortowo-3 (K-3) or K-3 extender containing lipoprotein
fractions extracted from hen egg yolk (LPFh) or lyophilized lipoprotein fractions extracted
from ostrich egg yolk (LPFo) on selected motility characteristics and plasmalemma integrity
at different regions of the spermatozoa during storage at 5o and 16oC. PAF was added to each
extended semen sample prior to storage at 5o and 16oC. Extended semen samples without the
supplementation of PAF were used as the control. Sperm motility was assessed
microscopically and using a computer-assisted semen analysis (CASA) system (SM-CMA,
Strımberg-Mika, Germany). The percentage of motile, locally motile and immotile
spermatozoa was determined. Motile spermatozoa were classified as circular, non-linear and
linear. The integrity of the plasmalemma middle-piece was assessed by determining the
activity of aspartate aminotransferase (AspAT) from spermatozoa subjected to cold shock
treatment. Fluorochrome, Hoechst 33258 (Hoechst 258), a DNA-binding stain, was used to
assess the sperm plasmalemma integrity overlying the acrosomal region.
It was confirmed that the supplementation of exogenous PAF to K-3 extender containing
LPFh or LPFo had a beneficial effect on sperm quality during storage at 5o or 16oC compared
with the control. This phenomenon was manifested in enhanced sperm motility and
survivability as well as plasmalemma integrity in PAF-treated samples. The results of this
study indicate that the supplementation of lipoprotein fractions extracted from hen egg yolk or
lyophilized lipoprotein fractions extracted from ostrich egg yolk to boar semen extender had a
protective effect on the plasmalemma integrity overlying the middle-piece and acrosomal
region of spermatozoa, regardless of the storage temperature.
This study was supported by funds from Warmia and Mazury University in Olsztyn
(No.0103.0206)
COMPARATIVE ANALYSIS BETWEEN ALKALINE PHOSPHATASE
ACTIVITY AND OTHER COMPONENTS IN STALLION SEMEN
Kazimierz Kosiniak-Kamysz, Monika Gorazd, Zenon Podstawski, Anna Bittmar
Department of Horse Breeding, University of Agriculture, Al. Mickiewicza 24/28,
30-059 Krakow, E-email: [email protected]
The alkaline phosphatase (AP) activity has been found in stallion semen however the role and
dynamic of their activity changes is still under discussion. After Turner and McDonell (2003)
we can state that the activity of this enzyme originates from epidydimis and testicles and it
can be use as an marker of testicular epithelium activity, however this enzyme is produced too
in accessory sex glands especially in epididymis in sexually normal stallions and dogs
(Kutzler et al. 2003).
It was found too that spermatozoa are not a source of this enzyme activity however Gordon
and Dandekar (1977) estimated this enzyme activity in sperm membrane collected from cauda
epidydimis and from sperm’s membrane after capacitation but there were not observed AP
activity in sperm membrane after ejaculation.
This enzyme activity was observed too in cytoplasmatic droplets as well as in postacrosomal
part of sperm head, neck, mid-piece and in the end of sperm tail (Angelowa 1967).
It is still interested the relation between alkaline phosphatase activity and indices on the base
of which semen is examine as volume, sperm motility concentration and morphology. This
correlation was presented by Roussel and Stallcup (1966) in bull semen and Glogowski
(1988) in boar semen plasma who suggested that the low activity of this enzyme in boar
semen with oligospermia is a result of sperm induction on secretion of AP in epididymal duct.
During our research work based on semen of 224 stallions which were qualified to the group
with full fertility and to the group which represent very low sperm concentration and low
number of motile spermatozoa we observed many correlations between AP activity and basic
indicators of semen value as well as biochemical components of semen plasma.
It was found that the activity of alkaline phosphatase in stallion’s semen plasma was
significantly negatively correlated with volume of ejaculate, Aspartate aminotransferase
(AspAT) activity (r=-0,4) and TAS (Total Antioxidant Status) (r=-0,26) but positively
correlated with sperm concentration and Lactate dehydrogenase (LDH) activity (r=0,6).
Correlation was observed between AP activity and sperm morphology especially in relation
with cytoplasmatic droplets.
On the basis of these observations we can state that the estimation of AP activity in stallion
seminal plasma can be used as important marker of the stallion semen quality examination.
A LOCAL PATHWAY FOR INCREASE OF TESTOSTERONE SUPPLY
FROM THE TESTIS TO THE EPIDIDYMIS, VAS DEFERENS AND SEX
ACCESSORY GLANDS OF RAT
Tadeusz Krzymowski1, Stanisława Stefańczyk-Krzymowska1, Przemysław Gilun2,
Michał Radomski1, Marek Koziorowski2
1
Department of Local Physiological Regulations, Institute of Animal Reproduction
and Food Research of the Polish Academy of Sciences, Tuwima 10, 10747 Olsztyn,
Poland, E mail: [email protected]
2
Department of Animal Physiology and Reproduction, University in Rzeszów, Rejtana
16c, 35-959 Rzeszów, Poland
It has been well documented that a morphological adaptation in the periovarian vascular
complex in females mesovarium produces the local increase of the concentration of ovarian
steroids in arterial blood, supplying not only the ovary (retrograde transfer) but also the
oviduct and uterus (destination transfer).
A resemblance of the morphology between male and female perigonadal vascular complexes
suggested a possible participation of the spermatic cord and vasculature of the mesofuniculus
of spermatic cord in the local transfer of steroid hormones from the testis to the sex accessory
glands, epididymis and vas deferens. Radiolabeled testosterone (3H-T) was infused into the
testis or mesofuniculus of spermatic cord (106 dpm) or injected into the testis (2x106 dpm) of
rats. The concentration of 3H-T was estimated in the tissue samples collected 15 or 10 min
after 3H-T infusion or injection, respectively, from the prostate, seminal vesicle, cauda and
caput epididymis, vas deferens and from the mesofuniculus of spermatic cord. The abdominal
aorta and posterior vena cava were cannulated and artificial circulation of blood in the
posterior part of the body (with different blood volume and pressure) was used for study of
3
H-T transfer from the testis to venous blood and other male genital organs. Reduction of
blood pressure as well as partial blocking of blood supply to the genital organs (after
ligatured of both spermatic interna arteries or the terminal part of the abdominal aorta)
elevated, but removing of the mesofuniculus of spermatic cord with the vas deferens
decreased the concentration of 3H-T in the accessory glands and cauda epididymis.
It was demonstrated that both arterial trunks: spermatic interna arteries, and common iliac
arteries are connected by anastomoses in target organs so effectively, that supplying the male
genital organs with blood only by one of them make possible to assure the intensive transfer
of testosterone from the testis to the cauda and caput epididymis, vas deferens, mesofuniculus
of spermatic cord as well as prostate and seminal vesicle.
We conclude that lymphatic vessels of the spermatic cord and mesofuniculus of spermatic
cord as well as venous and arterial vasculature of the mesofuniculus of spermatic cord create
till unknown local pathway for increase of testosterone supply from testis to the epididymis,
prostate, seminal vesicle and vas deferens of rat.
EFFECT OF WARM-REARING AND WARM-ACCLIMATION ON
REPRODUCTION IN MALE RATS
Beata Kurowicka1, Genowefa Kotwica1, Alina Gajewska2, Anita Franczak1,
Agnieszka Oponowicz1
1
Department of Animal Physiology, Faculty of Biology, University of Warmia and
Mazury, 10-719 Olsztyn, Poland, E-mail: [email protected], 2Institute of
Physiology and Nutrition, Polish Academy of Science, Jabłonna/Warsaw, Poland.
Reproductive functions of adult males are affected by high ambient temperature. It is
manifested by lower plasma concentrations of LH, FSH, T4 and PRL during acute heat
treatment and T4 and PRL after chronic exposure to elevated ambient temperature. However
little is known about the effect of heat treatment of the males during postnatal development on
its reproductive processes at adulthood. Thus, the aim of this study was to compare the effect
of warm-rearing and warm- acclimation of male rats on plasma LH, FSH, PRL, T4, A4 and E2
concentrations and on the steroidogenic ability of isolated Leydig cells. Four groups of male
rats were used in this study: Group CR (control, n=9) and WR (n=9) - had been reared from
birth to adulthood at ambient temperature of 20°C and 34°C respectively; Group WA (n=6) had been acclimated to temperature of 34°C during 45 days of their adult life, and Group DA
(n=6) – had been reared in ambient temperature of 34°C for the first 45 days and than
acclimated to temperature of 20°C for the next 45 days. The rats were bleeding at age of 90
days for analysis of plasma hormones concentration and isolation of testes for in vitro
experiment on Leydig cells. Warm-rearing and warm-acclimation caused: 1) higher (p<0.05)
testis mass (g/100g body weight) in WR and DA groups compare to CR and WA rats, 2)
lower (p<0.05) body weight of all heat treated groups 2) plasma concentration of LH did not
differ between CR, WR and DA males but in WA group LH level was higher (p<0.01), 3)
FSH level was the highest (p<0.05) in DA males, 4) plasma PRL concentration was not
affected in all groups, 5) plasma T4 level was the lowest (p<0.05) in WA group but in vitro
Leydig cells secreted comparable T4 concentration to that observed in other groups, 6) plasma
A4 concentration was the lowest (p<0.05) in WR rats and in vitro secretion of this hormone
was lower (p<0.05) in WR and WA groups compare to CR rats, 7) plasma E2 concentration
was not affected by temperature regime but Leydig cells from WR rats secreted higher E2
level (p<0.05) than the cells from all other studied groups. We concluded that heat
acclimation during postnatal development of male rats coused better adjustments in hormonal
milieu than warm acclimation of adult males. Heat acclimation of adult rats lowered the
sensitivity of testicular T4 production in response to LH stimulation.
This work was supported by grant 0206-0207 (University of Warmia and Mazury in Olsztyn)
PROLIFERATIVE ACTIVITES IN THE EPITHELIAL CELLS OF THE RAT
PROSTATE LOBES
Maria Laszczyńska, Marian Wylot, Małgorzata. Piasecka
al. Powstańców Wielkopolskich 72, 70-111 Szczecin, Poland,
Cell proliferation is constant event in epithelial tissues of all organs. Proliferative activities
are strongly dependent on influence of hormones and paracrine factors. The influence of
testosterone (T) and dihydrotestosterone (DHT) in the prostate is direct and well established.
Other hormones such as growth hormone (GH), prolactin (PRL), thyroxine (T4), and
estrogens (E) are known to exert influence too. Furthermore subsequent cross reaction and
reciprocal influence between testosterone, prolactin and estrogens have been described.
Studies were performed on sexually mature 3, and 18 month old male rats of Wistar strain.
Animals were divided into experimental and control groups. The experimental rats were
administered haloperidol (HAL) intraperitonealy in a dose of 2.0mg/kg of the body mass, for
14 days, whereas control groups received 0.9%NaCl. Prolactin (PRL) and testosterone (T)
serum concentrations were measured with immunoenzyme method (SpiBio France) and
radioimmunoassay (Orion Diagnostica Finland) respectively. Tissue sections were routinely
obtained for light and electron microscopy. For immunohistochemical detection of the
proliferation in the prostate epithelium the anty-Proliferating Cell Nuclear Antigen antibody
(PCNA), and in situ detection system EnVision System Horseradish Peroxidaze – AEC
(DAKO, Denmark) were used.
In 3 and 18 month old rats of experimental group receiving HAL for 14 days the mean PRL
concentration was about 2 fold higher, and the mean T concentration was over 2 fold lower as
compared to the control groups.
At light and electron microscopy proliferation evidence in the epithelial cells of the prostate in
experimental group (mainly in 18 month old rats) was observed.
The androgen dependent epithelial cells of prostate express modified proliferation under
decreased serum testosterone concentration especially in the lateral lobe of prostate. There
also has been noticed focal exerted proliferation strongly exhibited in 18 month old rats
correlating to age related prostate epithelium hyperplasia. Evident increased proliferation has
been confirmed with immunohistochemical PCNA detection: staining occurred especially in
patches of cells clusters and fingerlike outgrowths of epithelial cells in the lateral lobe of
prostate as compared to the control group.
PROPERTIES OF SEMINAL PLASMA PROTEIN COMPLEXES WITH
LIPOPROTEINS EXTRACTED FROM BIRD EGG YOLK
Marek Lecewicz, Jerzy StrzeŜek, Paweł Kaźmierczak
The aim of this study was to determine whether the components of the extender: lipoprotein
fractions extracted from hen egg yolk (LPFh), lyophilized lipoprotein fractions extracted from
ostrich egg yolk (LPFo) or seminal plasma proteins suppress induced ascorbate-Fe2+ lipid
peroxidation (LPO).
The determination of the production of thiobarbituric acid reactive species (TBARS) was used
as a measure of lipid peroxidation. The production of TBARS determined in different variants
of incubation media was as follows: 1) LPFh or LPFo; 2) LPFh or LPFo with whole seminal
plasma; 3) spermatozoa with whole seminal plasma; 4) spermatozoa with LPFh or LPFo; 5)
spermatozoa and whole seminal plasma with addition of LPFh or LPFo; 6) spermatozoa and
LPFh or LPFo with addition of whole seminal plasma. For the incubation media, a
concentration of 1 x 108 spermatozoa, 1ml of LPFh or LPFo and about 8 mg of seminal
plasma proteins were used. The degree of Fe2+ and ascorbate binding to the seminal plasma
proteins and the lipoprotein fractions were analyzed. It was observed that LPFo samples were
highly susceptible to induced lipid peroxidation compared to LPFh samples during incubation
at 37°C. The rate of TBARS production was three times higher than that of LPFh. However,
the presence of seminal plasma in the incubation media markedly reduced LPFo susceptibility
to lipid peroxidation. Individual variations regarding the antiperoxidant activity of seminal
plasma were observed.
In the incubation media consisting of seminal plasma and spermatozoa, there was about 40%
reduction in TBARS production compared with the incubation media consisting of only
spermatozoa. Furthermore, the addition of LPFh or LPFo significantly suppressed lipid
peroxidation, as evident in reduced TBARS production. The incubation of spermatozoa with
seminal plasma or LPFh or LPFo has confirmed the inhibitory action of the lipoproteins and
seminal plasma on lipid peroxidation process. The production of TBARS was approximately
twenty times lower in the incubation media when LPFh or LPFo was added compared with
that containing seminal plasma. Moreover, both lipoprotein fractions exhibited higher
neutralization action against Fe2+ than the seminal plasma. On the other hand, the seminal
plasma showed more pronounced effect on ascorbate reduction in the incubation media than
the lipoprotein fractions. The results of this study show that egg yolk lipoproteins and seminal
plasma can have an important role in protecting the plasmalemma lipid of boar spermatozoa
against reactive oxygen species (ROS) and may prevent plasmalemma destabilization. It can
be suggested that the addition of egg yolk lipoproteins to extenders may have a practical
application for boar semen preservation.
Study supported by funds from Warmia and Mazury University in Olsztyn (No. 0103.0206).
USE OF TWO-DIMENSIONAL (2-D) ELECTROPHORESIS TO IDENTIFY
HEPARIN-, Zn2+- AND PHOSPHORYLCHOLINE (PCH)- BINDING
PROTEINS OF BOAR SEMINAL PLASMA
Marzena Mogielnicka, Jerzy StrzeŜek
Mazury, 10-718 Olsztyn, Poland, E-mail: [email protected]
The seminal plasma of many mammalian species contains proteins that are secreted by the
accessory sex glands and adsorbed on the sperm plasmalemma at ejaculation. These proteins
are implicated in important reproductive function, such as including capacitation, gamete
recognition and acrosome reaction. The multifunctional properties of seminal plasma proteins
are facilitated by their interactions with various substances, such as heparin, Zn2+-ions and
phosphorylcholine (PCH). The concentrations of a few proteins in the seminal plasma of boar,
bovine or equine, identified using the two-dimensional (2-D) electrophoresis method, were
highly correlated with fertility parameters
The aim of this study was to separate and analyze proteins of boar seminal plasma, which
possess affinity for heparin, Zn2+-ions and phosphorylcholine, using the two-dimensional
polyacrylamide gel electrophoresis. Seminal plasma was obtained following centrifugation of
boar semen. The FPLC system was used to isolate heparin-binding proteins (HEPBP) on
Heparin Sepharose CL6B and phosphorylcholine-binding proteins (PCHBP) on pAminophenyl Phosphoryl Choline Gel. Zn2+-binding proteins (ZNBP) were separated on
Chelating Sepharose Fast Flow-Zn2+ employing the Bio-Pilot system. All protein fractions
were finally separated in 15% polyacrylamide gel, using 2-D electrophoresis.
The seminal plasma protein fractions, which showed affinity for heparin, Zn2+ and PCH were
isolated using liquid chromatography methods. The use of 2-D electrophoresis showed the
presence of approximately 165, 148 and 155 polypeptides that belong to the HEPBP, ZNBP
and PCHBP groups, respectively. Most of the polypeptides that belong to these 3 groups had
isoelectric points (pI) at neutral and basic pH range (7.0 – 9.0). Only a few polypeptides had
pI at acid pH range (3.0 – 7.0). Among the HEPBP, ZNBP and PCHBP, peptides with
molecular weights ranging from 12 – 20 kDa belong to the spermadhesin family. It is
interesting that a few proteins of HepBP fraction were highly correlated with boar fertility.
Boar seminal plasma in denaturating conditions showed a broad range of proteins and
peptides of highly differentiated molecular weights (10 to 100 kDa) and pI at neutral to basic
pH range. These proteins and peptides possess ability to bind heparin, Zn2+ and PCH, which
facilitate their multifunctional properties.
The analysis of seminal plasma proteins could lead to better insight into the biological
propertis of boar semen.
Supported by funds from University of Warmia and Mazury in Olsztyn (No. 0103.0803)
EXPRESSION OF THE StAR PROTEIN IN OVARIAN AND TESTICULAR
CELLS OF THE PIG AND BANK VOLE, RESPECTIVELY
Maria Słomczyńska, Małgorzata Duda, Jerzy Galas, Monika Gancarczyk, Barbara
Bilińska
University, Krakow, Poland, E-mail: [email protected]
Recent advances in the knowledge of the acute regulatory mechanisms during
steroidogenesis, concern the biochemical and molecular characteristics of steroidogenic acute
regulatory protein (StAR), which plays a key role in the mitochondrial step of steroid
hormone biosynthesis [1, 2, 3]. This protein is responsible for cholesterol transport from the
outer to the inner mitochondrial membrane of the steroidogenic cell. Synthesis of steroid
hormones is regulated by signals from the anterior pituitary gland that act on specific
steroidogenic cells in adrenals, ovaries, and testes. In response to these signals the StAR is
synthesized, playing an essential role in adrenal and gonadal steroidogenesis [4, 5, 6].
The aim of this study was to show the expression of the StAR protein in porcine granulosa
cells and in bank vole Leydig cells in vitro [7]. To examine the influence of insulin and LH on
porcine granulosa cells, progesterone production and the StAR accumulation within cells
were studied. LH and insulin alone exerted a small effect on progesterone secretion however
it depended on the size of follicles used as the source of granulosa cells. In contrast, insulin in
combination with LH increased both progesterone secretion and StAR expression as shown
by RIA, immunocytochemistry and Western blot using a specific antibody against the StAR
protein (a gift from Dr DM Stocco) [8]. The in vitro study indicates that insulin and LH may
contribute to steroidogenic differentiation during follicular maturation. To show whether the
StAR protein is co-localized with mitochondria a selective staining of mitochondria in living
Leydig cells was performed using a Mito Tracker Red CMXR probe [9]. Superimposed
images from double-fluorescence staining showed a remarkable degree of similarity in the
distribution of the StAR protein and mitochondria, indicating mitochondrial localization of
the StAR in Leydig cells in vitro. Immunofluorescent double-staining seems to be a good
technique for visualization of the StAR protein within cell mitochondria [10].
1. Stocco DM, Sodeman TC. J Biol Chem 1991; 266: 731-738.
2. Clark BJ, Wells J, King SR, Stocco DM. J Biol Chem 1994; 269: 314-322.
3. Bilińska B. Post Biol Kom 1997; 24:183-202
4. Clark BJ, Parker KL, Stocco DM. Mol Endocrinol 1995; 9:1346-1355.
5. Lin D, Sugawara T, Clark BJ, Stocco DM. Science 1995; 267:1828-1831.
6. Stocco DM. W: The Leydig cell. L. Russell [ed.], Cache River Press, Vienna Il, 1996; 242-251.
7. Słomczyńska M, Duda M, Galas J, Bilińska B. J Histochem Cytochem 2004; 52: P12-4
8. Stocco DM, Clark BJ. Endocr Rev 1996; 17:221-244.
9. Kotula M, Kozieł E, Gancarczyk M, Bilińska B. Folia Histochem Cytobiol 2001; 39: 169-170
10. Bilińska A B. W: Molekularne podstawy rozrodu. M. Kurpisz [red], 2002;179-183, Termedia, Poznań
Supported by DS/IZ/ZFZ/2005
OSMOTIC EFFECTS ON MOTILITY AND PLASMA MEMBRANE
INTEGRITY OF SPERMATOZOA FROM RICH FRACTIONS OF DOG
EJACULATE
Rafał StrzeŜek, Leyland Fraser, Jerzy StrzeŜek
Canine spermatozoa may exhibit widely different osmotic responses when exposed to media
varying in osmolality. The differences in osmotic tolerance of the sperm cell may affect its
freezing sensitivity and survivability. The aim of this study was to investigate the effects of
different osmolalities (150 – 1120 mOsm/kg) on the structural and functional characteristics
of canine spermatozoa
The rich fractions of ejaculate from 4 dogs of mixed breed were used in this study. Following
macroscopic and microscopic analyses, the ejaculates were diluted in Tris-citric acid-fructose
solution varying in osmolality (150, 300, 350, 550, 800 and 1100 mOsm/kg) and held for 10
min at room temperature. Motility evaluation was conducted simultaneously with fluorescent
microscopic assessment of plasma membrane integrity using 2 flurochromes,
carboxyfluorescein diacetate (CFDA) with propidium iodide (PI). The percentage of viable
spermatozoa with functional mitochondria was assessed using rhodamine 123 (R123), a
cationic fluorescent molecule, with PI. The functional integrity of the sperm plasma
membrane overlying the tail region was assessed using 2 different types of hypo-osmotic
swelling tests, HOS and 'water'. The solution used for HOS test was prepared with fructosesodium citrate, whereas distilled water was used for the 'water test'. In hypo-osmotic
conditions (150 mOsm) the percentage of membrane-intact spermatozoa (CFDA/PI) was
significantly higher (p≤0.05) than that of motility of spermatozoa of each dog. The
proportions of positive R123-stained cells were also higher (p≤0.05) than motility estimates
in hypo-osmotic conditions. In isosomotic conditions (300 mOsm) and in 350 mOsm solution
there were no significant differences (p≥0.05) in sperm motility, plasma membrane integrity
and mitochondrial activity. Furthermore, in 550 and 800 mOsm solutions, the reduction
(p≤0.05) in positive CFDA- and R123-stained sperm cells was accompanied by lower sperm
motility. Spermatozoa exposed to hyper-osmotic conditions (>1000 mOsm) were completely
immotile, but a few spermatozoa retained their membrane integrity and functional
mitochondria. It was observed that sperm mitochondrial activity was more sensitive to
anisosmotic conditions than the plasma membrane integrity. The mean values of swollencoiled spermatozoa (intact tail membrane) in hypo-osmotic swelling test and water test were
83% and 78%, respectively. There were wide variations in terms of motility and plasma
membrane integrity of spermatozoa when exposed to different osmolalities. The results of
this study indicate that the structural and functional integrity of plasma membrane of canine
spermatozoa was more resistant to changes in osmotic conditions than the motility apparatus.
It seems that the different responses of spermatozoa to anisosmotic conditions are indicative
of the presence of subpopulations of spermatozoa within the rich fractions of dog ejaculate.
Study supported by funds from Warmia and Mazury University in Olsztyn (No. 0103.0206).
THE EXPRESSION OF ANDROGEN RECEPTOR IN THE EPIDIDYMIS OF
RATS WITH DIHYDROTESTOSTERONE (DHT) DEFICIENCY
Grzegorz Trybek, Agnieszka Kolasa, Mariola Marchlewicz, Barbara Wiszniewska
70-111 Szczecin, Poland, E-mail: [email protected]
The morphology and function of epididymis is maintained and regulated by androgens –
testosterone (T) and dihydrotestosterone (DHT). Both, T and DHT exert their effect
throughout specific intracellular androgen receptor (AR). However, DHT is regarded as the
most potent androgen of epididymis, which is formed in the conversion of T by the steroid
5α-reductase activity. Two steroid 5α-reductase genes have been cloned, and they encode
different isozymes (types 1 and 2). In the epididymis 5α-reductase type 2 (5α-red2) is more
abundant than type 1. In the experiment, the activity of 5α-red2 was inhibited using steroidbased inhibitor – finasteride (Proscar®, MSD Sweden). The finasteride (5mg/kg b.w.) was
administerd per os into adult male Wistar rats for time of one course of spermatogenesis (56
days). The aim of the study was to estimate the influence of DHT deficit on the expression of
androgen receptor (AR) in the epididymis of rats. To detect AR immunoreactivity, the
monoclonal mouse anti-androgen receptor antibody (Dako, USA) was used.
There were no morphological changes in the morphology of epididymis of rats with DHT
deficiency. In caput epididymis of control rats, the majority of epithelial cells showed the
immunoexpression for AR in nuclei but cytoplasmic reaction was observed in interstitial
tissue cells. In rats with DHT-deficit, there was weak immunoexpression in few nucleus and
mainly in the cytoplasm of most epithelial cells of caput epididymis. Moreover, weak
cytoplasmic immunoreaction shown cells of interstitial tissue. In control cauda epididymis,
only single nucleus of epithelial cells showed the immunoexpression for AR, however the
diffusive reaction in the cytoplasm of the cells was observed. There was no immunoreaction
in nuclei but cytoplasm of epithelial cells of epididymis rats with DHT-deficit was
immunopositive. Interstitial tissue of cauda epididymis, both control and DHT-deficiency rats
was immunonegative.
The results indicated that the expression of androgen receptor is modulated when the balance
between testosterone and dihydrotestosterone is changed.
The study was granted approval by the Local Ethics Committee and Animal Research.
BIOCHEMICAL CHARACTERISTICS OF EPIDIDYMAL MOLECULAR
FORMS OF ACID PHOSPHATASE ISOLATED FROM BOAR SEMINAL
PLASMA
Paweł Wysocki, Jerzy StrzeŜek
Electrophoretic studies on boar seminal plasma showed the presence of four molecular forms
of acid phosphatase which, in different variants, occur in the epididymal, prostatic and
vesicular fluid. The activity of the dominant epididymal forms represented about 90% of total
acid phosphatase activity of the seminal plasma. The kinetic analysis of purified epididymal
isoenzyme migrating faster to the anode showed the ability for phosphotyrosine
dephosphorylation. This epididymal form of acid phosphatase exhibited lower affinity for
phosphotyrosine substrate (Km = 2,1x 10-3M) compared with the vesicular form of acid
phosphatase (Km = 0,37 x 10-3M). Furthermore, the epididymal form did not show any
sensitivity to inhibitory action, which is characteristic for the activity of acid phosphatase
isolated from the fluid of the vesicular glands. Moreover, both epididymal and vesicular forms
of acid phosphatase have numerous similarities (molecular weight, thermal stability,
glycoprotein structure). Protein complex of high-molecular weight with acid phosphatase
activity was purified from the caudal epididymal fluid and whole seminal plasma using PAGE
and electroelution. The protein complex isolated from the caudal epididymal fluid consists of
3 proteins, with molecular weights more than 60 kDa. On the other hand, the protein complex
isolated from the seminal plasma is characterized by higher heterogeneity and consists of
protein fractions with molecular weights lower than 20 kDa, which probably originate from
the vesicular glands. Polypeptides with molecular weights of 67 and 100 kDa are stable
component of the protein complex originating from the epididymis and seminal plasma. The
polypeptides present in the epididymal form of acid phosphatase are replaced by other
polypeptides occurring in the seminal plasma. The results of this study show that changes in
the protein composition of high molecular weight protein complex with acid phosphatase
activity during ejaculation may affect its biological activity. There was immunological
similarity between epididymal and vesicular forms of acid phosphatase. The use of antibody
immobilization on chromatography column enabled efficient isolation of both epididymal and
vesicular forms of acid phosphatase from boar seminal plasma. Acid phosphatases, with
molecular weights approximately 45 kDa and 50 kDa, and pI 7.0, were detected in the boar
seminal plasma using two-dimensional (2D) electrophoresis and immunoblotting.
Supported by the State Committee for Scientific Research, Project No. PBZ-KBN084/P06/2002.
Session VIII
Progress in treatment of infertility
Oral presentation
IMPORTANCE OF BASIC SCIENCE FOR IMPROVEMENT OF CULTURE
OF HUMAN OOCYTES AND EMBRYOS IN IVF PROGRAMS
Wojciech Głąbowski
Department of Histology and Embryology, Pomeranian Medical University, Szczecin,
The great improvement has been done over last decade at the area of assisted reproductive
techniques (ART), however the success rates are still unsatisfactory. The main goal of in vitro
fertilization (IVF) programs is to increase the pregnancy rates and especially “take home
baby” rates. On the other hand, the very important objective is to reduce the risk of
complications such as ovarian hyperstimulation syndrome or multiple pregnancy. There are a
number of techniques facing these problems that have been developed in last decade.
Culture and transfer of human blastocyst is one of the most important developments in ART
programs. Extended culture protocols use sequential media culture systems that match
different nutrients and growth factors requirement of the embryo at different stages of
development. The improvement of pregnancy rates after blastocyst culture and transfer was
reported, however the utilization of this technique is still limited by costs of culture media and
relatively low rates of embryos reaching the blastocyst stage.
Another procedure of increasing importance is in vitro maturation of oocyte (IVM). This
technique includes culture of immature human oocytes to the metaphase II stage and
subsequent in vitro fertilization of these oocytes. There are advantages of this method such as
lower gonadotropin doses required for ovarian stimulation, reduction of frequency of ovarian
stimulation and risk of ovarian hyperstimulation syndrome. The disadvantage is that the
retrieval of intact pre-antral or small antral follicles (easier to be grown to metaphase II stage)
is mach more difficult and laborious than retrieval of mature oocytes. Thus, the in vitro
maturation of granulosa-oocyte complexes harvested from bigger antral follicles is more
available. On the other hand, the latter method requires much more complex and yet not well
defined culture conditions including composition of media, optimal density of granulosa cells
and presence of regulating factors. Moreover, due to large size and the presence of spindle
apparatus the oocytes at more advanced stages are susceptible to damage caused by low
temperatures used in cryopreservation.
The advanced assessment of embryo quality is the next area that really influences the success
rates in IVF programs. The zygote, cleaving embryos and blastocyst scoring systems
established and introduced in last years are very helpful in selection the best embryos for
transfer. Additional advanced method is the biopsy of blastomere of day 3 embryo for
preimplantation genetic diagnosis (PGD). Using fluorescent in situ hybridization (FISH) to
detect most often found types of aneuploidy or polymerase chain reaction (PCR) in case of
risk of particular mutations we are able to exclude genetic abnormalities and transfer
genetically normal embryos at blastocyst stage (only if extended culture is applied).
IMMUNOLOGICAL INFERTILITY – FACTS AND MYTHS
Rafał Kurzawa
Department of Reproductive Medicine and Gynecology, Pomeranian Medical
University, Szczecin, Poland, E-mail: [email protected]
Immunologic factor may be involved in repeated failures of fertilization, implantation
(including failures of IVF), early and late pregnancy losses (RPL) as well as in the pathology
of pregnancy. Unexplained infertility may also be included in the group. Common
pathological mechanisms may be associated both with immunologic infertility and repeated
losses of early pregnancies.
The implantation of embryo constitutes a unique immunological process. In the luteal phase
of the menstrual cycle, during the implantation window, various factors are expressed by the
endometrium in the time dependent manner. The most important ones are adhesion molecules
- integrins (particularly αvβ 3), glycoproteins – glycodelin, MUC-1 and cytokines –
interleukins 1 (IL-1α, IL-1β, IL-1ra), leukemia inhibitory factor (LIF) or colony stimulating
factor-1 (CSF-1). The factors are regulated by complex hormonal and cytokine networks.
Theoretically, immunological disorders leading to infertility or RPL, may be triggered by the
presence of (1) antisperm antibodies (ASA) in males or females; (2) antiphospholipid (APA),
antinuclear (ANA), and thyroid (ATA) antibodies in females; (3) ovarian antibodies and
premature ovarian failure; (4) hyperactivity of natural killer (NK) cells in the endometrium
and (or) peritoneal cavity; (5) altered balance between Th1 and Th2 humoral response in
favor of proinflammatory (Th1) cytokines; and finally (6) high compatibility between paternal
and maternal human leukocyte antigens (HLA).
On the basis of randomized controlled trials (RCT) only the involvement of ASA or APA has
been clearly confirmed to be responsible for infertility or RPL respectively.
The presence of sperm bound ASA reduces the number of freely moving spermatozoa by
agglutination or by triggering cytotoxic reaction. A vast majority of men with more than half
of sperm coated with the antibodies have reduced fertility. ASA are classical indication to IVF
(ICSI). The presence of ASA does not affect the outcome of treatment with IVF.
There is no link between APA and infertility. Although APA are apparently more numerous
in couples undergoing IVF, the antibodies do not affect the effectiveness of treatment. It has
been established that APA are mainly responsible for RPL. In such cases, RCT showed that
number of live births might be significantly improved by a combined treatment with low-dose
aspirin and heparin. Similarly, there has been no sufficient evidence to suggest that either
ATA or ANA affect the outcome of infertility treatment with IVF. Autoantibodies directed
towards ovarian tissue are associated with premature ovarian failure. Beside the obvious
destructive effect of the antibodies on the ovarian tissue and functions, it remains unclear
whether the antibodies alone may cause infertility.
Hyperactive NK cells may theoretically affect gamets, preimplantation embryo, implantation
or pregnancy. This attractive hypothesis could explain some cases of idiopathic infertility and
early RPL. However, no correlation between peripheral blood and endometrial NK cells
concentrations has been established so far. Therefore, one must acknowledge that the true role
of NK cells in reproduction remains unclear. Unbalanced Th1 vs. Th2 humoral response may
lead to implantation failures and RPL. The Th1 proinflammatory cytokines trigger apoptosis
in gametes, embryos and decidua, affecting fertilization, implantation and maintenance of
pregnancy. Th1 are thought to be responsible for recurrent failures of IVF.
HLA sharing (HLA A, HLA B, HLA C, HLA G, HLA DQ, and HLA DR) may generate
inadequate immune response towards the implanting embryo and may be responsible for
some cases of unexplained infertility including repeated IVF failures. However, despite
numerous studies the obtained results have been inconclusive and the exact significance of
high paternal and maternal compatibility of HLA still awaits explanation.
DIAGNOSTIC EVALUATION OF SPERM MITOCHONDRIA
Małgorzata Piasecka
70-111 Szczecin, Poland,E-mail: [email protected]
The sperm mitochondria (sperm type mitochondria) are the unique organelles somewhat
different from the somatic mitochondria. Their morphological, biochemical and molecular
transformations take place during spermatogenesis and they are involved in the expression of
specific proteins which are related to markers of their maturation (e.g. cyt cT, LDH-C4,
sulphydryl oxidase, LON protease, hsp 60). The energy of sperm mitochondria is used not
only to maintain sperm motility but also for sperm hyperactivation, acrosome reaction and
finally sperm-oocyte interaction. Therefore, their morphological and functional defects
contribute to male infertility (including idiopathic) expressed as asthenozoospermia and very
often resulting from developmental failure in spermatogenic remodeling process. The
mitochondrial defects can be identified with precise and comprehensive diagnostic methods:
1/ cytochemical screening NADH-dependent NBT assay. The test is an adequate marker of
sperm mitochondria activity and sperm maturation. It can be used to identify and visualise
subtle and drastic deformations of sperm mitochondrial sheath, immature sperm forms with
extensive cytoplasmic retention, sperm conglomerates related to apoptotic bodies and
mitochondrial NADH-dependent oxidoreductase system; 2/ microscopic fluorescence and
cytofluorometric tests using mitochondrial probes (e.g. JC-1, Mito Tracker, rhodamine 123,
DiOC6 (3)). Microscopic studies allow to evaluate the presence and arrangement of active
(with high ∆Ψm) or dysfunctional mitochondria in sperm. Moreover, mitochondrial
fluorochrome are used to visualise the sperm mitochondria after fertilization in order to
discover their fate and their ubiquitin-dependent degradation inside dividing zygote. In turn,
the flow cytometry of mitochondrial probe stained spermatozoa is a reliable and valuable
method which enables on the basis of fluorescence values and computer statistical program to
evaluate a large number of spermatozoa and to establish a real number of “healthy”
spermatozoa; 3/ immunocytochemical tests to display defective - ubiquitinated sperm
mitochondria or spermatozoa with lower expression of mitochondrial keratinous capsule
proteins, phospholipid hydroperoxide glutathione peroxidase and LDH-C4; 4/ electron
microscopy, biochemical and genetic tests showing mitochondrial and nuclear genome
mutations (t haplotype) resulting in disorders of mitochondrial respiratory chain activity and
diverse ultrastructural defects of sperm midpiece and flagellum.
The mitochondrial assay helps explain pathogenesis of asthenozoospermia and can be a
component of male fertility diagnostics. It should be emphasized that mitochondrial defects
in some cases of conception (in vivo and in vitro) may deactivate their own elimination in a
fertilized egg cell. In idiopathic infertility they may trigger the spontaneous, pre-implantation
embryo abortion. The mitochondrial diagnostics is of considerable prognostic and predictive
value for in vivo and in vitro fertilization.
THE EFFECTIVENESS OF RECONSTRUCTIVE OVIDUCT SURGERY
DEPENDING ON THE MEDICAL AND PHYSIOLOGICAL FACTORS
Andrzej Starczewski 1, Agnieszka Brodowska 1, Maria Laszczyńska 2
1
Department of Reproductive Medicine and Gynecology, Pomeranian Medical
University, Szczecin; 2Department of Embriology and Histology, Pomeranian Medical
University, Szczecin, Poland
The progress in the treatment of infertility (connected with oviduct abnormalities) took place
especially thanks to development of microsurgery and operative laparoscopy. Currently the
role of these methods is diminished and limited to best-result methods, what is connected with
the use of ART (assisted reproductive techniques). The best effects carrying: partial resection
of isthmical and intramural part of fallopian tube, istmico-isthmical anastomosis, tubal
fimbrioplasty and reconstruction an abdominal ostium. Surgical treatment gives better results
than IVF in the case of occlusion the proximal part of oviduct. Besides, this method almost
permanently restores fertility. Cornual anastomosis results with about 60% of normal
pregnancies and isthmico-isthmical with even 80%. The condition of such good results is
correct anatomical state of oviduct outside the occlusion place.
The pathological changes of abdominal opening of the oviduct are the most frequent cause of
infertility. This is connected with the lack of fimbriae and anatomical changes in the oviduct
wall. The intensity of these changes depends on the time of occlusion. The results of
reconstructive microsurgery are connected with the correct qualification of patients. Among
some factors decreasing fertility, we should take into consideration anatomical state of
oviduct, especially endosalpinx. In case of significant anatomical changes in distal part of
fallopian tube it is necessary to remove it.
Comprehensive evaluation of anatomical state of oviducts is possible thanks to the use of
laparoscopy and salpingoscopy. The salpingoscopy lets to assess the endosalpinx state and let
to predict the results of reconstructive surgery. It also enables to select the patients for whom
reconstructive surgery will give better results than IVF. Perioviductal adhesions have also
influence on the results of reconstructive surgery. Postoperative administration of β-oestradiol
accelerates the regeneration of endosalpinx and influences the morphology of oviducts,
especially in cases of long-lasting occlusion. Proper qualification enables to receive 30-60%
of pregnancies after surgery of oviduct openings. This causes that microsurgical methods have
still their place in the treatment of infertility connected with anatomical disorders of oviducts.
Poster presentation
(no abstracts were received)
Session IX
Immunology of reproduction
Oral presentation
ANALYSIS OF THE RELATIONSHIP BETWEEN UREAPLASMAL
INFECTION AND THE OUTCOME OF PREGNANCY
Kayoko Harada, Hiroyuki Tanaka, Yoshiyuki Tsuji and Koji Koyama
Department of Obstetrics and Gynecology, Hyogo College of Medicine, Japan,
Object : Ureaplasma has recently been reported to be a pathogenic organism for sexually
transmitted diseases and also for chorioamnionitis, premature delivery and neonatal
pneumonia. In this study, we examined whether the ureaplasma infection is related to
premature delivery.
Method: Vaginal swabs were obtained from 85 pregnant women (including 63 with normal
delivery and 23 with premature delivery), and cultured in Ureaplasma culture medium. The
swabs were also analyzed for bacteria flora using the conventional culture method. DNA was
extracted from the grown ureaplasma colonies to determine the biotype by PCR direct
sequence method.
Results: Ureaplasma infection was found in 51.6 and 52.6% of full-term and preterm delivery
women, respectively, indicating that ureaplasma infection had no relation to premature
delivery. The biotype of ureaplasma had no relevance to preterm delivery, too. However, the
interval between hospitalization due to threatened premature delivery and actual delivery was
8.4 days for ureaplasma-infected patients, as compared to 18 days for non-infected patients,
suggesting that ureaplasma infection accelerates the process of premature delivery or shorter
time to premature delivery..
Conclusion: Our study found no association between ureaplasma infection or biotype and
premature delivery. However, we found that ureaplasma-infected patients were not as readily
treatable for threatened preterm delivery as non-infected patients.
ANALYSIS OF ANDROGENIC EFFECT DURING SPERMATOGENESIS IN
TESTIS
Shinji Komori 1,2, Hiroyuki Kasumi1, Kazuko Sakata1, Koji Koyama1,2
1
Department of Obstetrics and Gynecology, Hyogo College of Medicine; 2Laboratory
of Developmental Biology and Reproduction, Hyogo College of Medicine, Japan,
The identification of proteins induced by androgens in Sertoli cells is a major issue in
spermatogenesis. In this study, we analyzed protein profiles in TM4 Sertoli cells treated with
dihydrotestosterone using SELDI-TOF mass spectrometry. We found increase in expression
of a 5.0 kDa protein at 15 min, a 11.3 kDa protein at 24 hrs and 4.3, 5.7, 5.8, 9.95 and 9.98
kDa proteins at 48 hrs. On the other hand, expression of 6.3 and 8.6 kDa proteins were
decreased at 30 min, and 4.9, 5.0, 12.4 and 19.8 kDa proteins at 48 hrs. The 11.3 kDa
molecule was identified as macrophage migration inhibitory factor. The 9.98 kDa increased
molecule was identified as calgizzarin known to bind to Ca2+. The 19.8 kDa molecule was
identified as translationally controlled tumor protein (TCTP) known to bind to tubulin and
Ca2+ and regulates the cell proliferation. The timing of proteins expression suggests that
these are involved in late androgen regulation of spermatogenesis, in Sertoli cells. The late
change in the expression of these proteins suggests that androgenic effects on
spermatogenesis can be exerted possibly through these proteins.
QUANTITATIVE ANALYSIS THE INTERLEUKIN 1 GENE SYSTEM IN
NORMAL OR IMPAIRED HUMAN SPERMATOGENESIS AND
TESTICULAR TUMORS
Natalia Rozwadowska1, Dorota Fiszer1, Piotr Jedrzejczak2, Włodzimierz Kosicki3,
Maciej Kurpisz1
1
Institute of Human Genetics, Pol. Acad. Sci, Poznan, 2Clinic of Infertility and
Reproductive Endocrinology, School of Medicine, Poznan, 3Clinic of Urology,
District Regional Hospital, Poznan, Poland, E-mail: [email protected]
Homeostasis in the seminiferous epithelium requires permanent balance between proliferation
and apoptosis. Altered equilibrium between them could lead to testicular pathologies such as
fertility disorders or cancerogenesis. IL-1 is a pleiotropic cytokine that may play a role
contributing to the specific immune environment of mammalian testis. IL-1 family forms a
complex system with tight regulatory mechanisms. We can distinguish among this family
members: ligands - IL-1α, IL-1β, IL-1RA and IL-18; receptors - IL-1RI , IL-1RII, IL-18Rα;
receptor associated proteins - IL-1RAcP and IL-18β. Additionally, caspase-1 or interleukin1β converting enzyme (ICE) is responsible for cleavage of precursors of IL-1β and IL-18 and
it is also important modulator of apoptosis.
We determined transcriptional activity of IL-1 gene family members applying real-time PCR
(10 genes and one reference gene - β-actin) in physiological testis (n = 8), testis with
spermatogenetic arrest (n = 27) and testicular tumors (n=10). We observed a general tendency
for high expression of genes coding for IL-1α and IL-1RA in the intratubular compartment of
the testis. There was another pattern of expression found in the interstitium – we determined
the intense transcription of IL-1β gene and high level of mRNA for anti-inflammatory factors
(IL-1RA and IL-1RII). The analysis of mRNA expression in tissue homogenates from testis
with maturation arrest, Sertoli cell only syndrome (SCOS) and obstructive azoospermia
showed the inversely related balance between IL-1α and IL-1RA. The oligobiopsies from
SCOS patients showed the exceptionally high expression for IL-1α in proportion to IL-1RA
mRNA. The maturation block of spermatogenesis (azoospermia) was connected with almost
equal expression of both compared genes. When a spermatogenesis was close to normal
(obstructive azoospermia) it was noted the prevalence of IL-1RA mRNA over the IL-1α.
mRNA quantitative analysis in tumor testicular tissues revealed the increased level of
transcription of anti-inflammatory genes (IL-1Ra and IL-1RII) and surprisingly the genes
connected with apoptosis i.e. IL-18 and ICE. The found expression profile could be a
prognostic factor for testicular patophysiology.
A MALE FACTOR: LINK BETWEEN INNATE IMMUNITY AND
OXIDATIVE STRESS
Dorota Sanocka , Maciej Kurpisz
Institute of Human Genetics Polish Academy of Sciences, 60-479 Poznań,
ul. Strzeszyńska 32, Poland, E-mail: [email protected]
Oxidative stress commonly defined as an imbalance in pro- and antioxidant levels is involved
in the etiopathology of various inflammatory and autoimmune diseases, but also in male
infertility. Oxidative stress in male semen may be caused by several factors; ischemiareperfusion injury, ageing, carcinogenesis and most commonly – inflammation.
Activation of seminal plasma white blood cells during genital tract inflammation or triggering
the cellular reactions against microbial agents may provoke a release of variety of products
such as cytokines and reactive oxygen species (ROS). The aim of the study was to evaluate
whether a panel of selected cytokines (IL1−β, IL-6, IL-8, TNF-α) detectable in seminal
plasma during the male genital tract inflammation could be considered as interactive factors
between the semen parameters and pro- and antioxidant substances. Performed studies using
chemiluminescence, spectrophotometry and ELISA indicated, that proinflammatory
cytokines such as IL-1β, IL-6, IL-8 and TNF−α may modulate pro- and antioxidant activities
in infected semen. The obtained data also suggest that the function of pro- and antioxidant
systems in semen may directly influence basic semen parameters and such affected long-term
genital tract inflammation may lead to persistent male infertility. The presented data provide
the evidence, that evaluation of number of seminal plasma leukocytes during the male genital
tract infection without associated contribution of cytokines and semen antioxidant’s capacity
appeared to be of a little prognostic value in evaluation of male fertilization potential.
Poster presentation
IMMUNOREACTIVITY OF PDGF AND PDGF RECEPTORS IN THE
PORCINE PARAOVARIAN VASCULAR PLEXUS IN DIFFERENT STAGES
OF THE OESTROUS CYCLE
Marcin Chruściel, Aneta Andronowska, Katarzyna Jankowska, Teresa Doboszyńska
Department of Reproductive Histophysiology, Institute of Animal Reproduction and
Food Research PAN in Olsztyn, Poland, E-mail: [email protected]
Paraovarian vascular plexus (PVP) is the vascular thick band composed of the blood and
lymphatic vessels leaving the ovary. All these vessels participate in the countercurrent transfer
of hormones and the other biological active substances. PDGF is one of the angiogenic factors
regulating their function eg. proliferation and migration of vascular endothelial cells,
vasculogenesis. The aim of the present study was to determine the distribution of PDGF-A,
PDGF-B, PDGF-Rα and PDGF-Rβ immunoreactivity in the endothelium of blood vessels of
the PVP during the oestrous cycle. The middle part of PVP was taken from various phases of
oestrous cycle, fixed in 4% PFA in 0.1 M PB (pH=7.4). The adjacent cryostat sections were
stained immunohistochemically using ABC method with the specific polyclonal antibody
directed against PDGF-A, PDGF-B, and PDGF receptors (Santa Cruz Biotechnology, USA).
The intensity of immunohistochemical reaction was estimated by measuring the optical
density under the light microscope using DP SOFT software (OLYMPUS).
The significant increase (p < 0.001) of PDGF-A and PDGF-B staining was observed in the
endothelium of PVP arteries at the middle luteal phase. The lowest immunoreactivity of
PDGF-A was noted in the late luteal phase and for PDGF-B at early luteal phase. In the
endothelium of utero-ovarian vein the highest PDGF-A and PDGF-B staining occurred at
early luteal phase, the lowest at follicular phase. The highest (p < 0.001) PDGF-Rα
immunoreactivity in the PVP arteries was found at the middle luteal phase. The lowest
(p<0.001) PDGF-Rα staining occurred at follicular phase. The optical density of PDGF-Rβ
staining remained stable through the oestrous cycle except of significant (p<0.001) decrease at
the late luteal phase. In the utero-ovarian vein PDGF-Rα immunoreactivity achieved the
highest (p<0.001) level at the middle luteal phase. The lowest reactivity for PDGF-Rα was
observed at follicular phase. The optical density of PDGF-Rβ in utero-ovarian vein did not
show statistical differences in staining throughout studied stages of the oestrous cycle. The
lower (p<0.05) immunostaining for PDGF-A and both type of receptors was found in the
endothelium of the utero-ovarian vein in comparison to PVP arteries.
The differential immunoreactivity of PDGF-A, PDGF-B and PDGF receptors in the blood
vessels of paraovarian vascular plexus during oestrous cycle in the pig suggests its possible
role in the control of vascular bed function.
The study was supported by Grant KBN nr 3P06D 01122
THE ROLE OF CYTOKINES IN REGULATION OF SECRETORY AND
MOTOR FUNCTION OF OVIDUCT
Katarzyna M. Deptuła, Dariusz J. SkarŜyński
Department of reproductive Immunology, Institute of Animal Reproduction and Food
Research, 10-747 Olsztyn, Poland, E-mail: [email protected]
The oviduct plays a critical role in reproduction being the site of the gamete maturation and
transport, fertilization and early embryonic development. It has been shown that the secretory
and motor activities of the oviduct are regulated by several ovarian and uterine factors
including prostaglandins (PG)s. In addition to endocrine regulators, oviductal functions
undergo both temporal and regional modulation by the product of immune cells – cytokines.
In several tissues cytokines were found to be the most potent regulators of PGs secretion and
actions. However, the auto/paracrine mechanisms regulated by the locally produced PGs in
the bovine oviduct are controversial. Previously reports revealed that tumor necrosis factor
(TNF)α gene is expressed in mouse oviductal epithelial cells, and in human embryo.
Moreover, human oviductal fluid contains TNFα protein. In cow, the developing embryo is
capable of stimulating PGs production by the oviductal epithelial cells. The embryo at
oviductal transport stages expresses TNFα. Therefore, TNFα may stimulate the PGs
production by oviduct. We have recently shown that cytokines (TNFα, IL-1,6, IFNγ, IFNτ)
regulate differently PGs and nitric oxide (NO) production in ampulla and isthmus of the
bovine oviduct. In our study, TNF-α and S-NAP (a NO donor) stimulate PGF2α output which
was two-fold stronger at the postovulatory phase than at the estrus. Production of PGF2α in
ampullary segments was two-fold higher than in the isthmus of the oviduct. In contrast, PGE2
output was higher in the isthmus than in ampulla of the oviduct. Moreover, TNFα stimulated
production of NO in the bovine oviduct, and NO is a potent mediator of TNFα action in the
oviduct. AMT (a competitive inhibitor of inducible isoform of NO synthase) inhibited output
of both PGs, i.e., PGF2α in ampulla and PGE2 in the isthmus of the oviduct and strongly
reduced production of NO2/NO3 at both examined phases of the cycle. Also the other
cytokines (IL-1, IFNγ) stimulate production of PGF2α and PGE2 in oviductal epithelial cells.
In the last study we established the physiological significance of these findings in terms of the
regulation of bovine oviductal motility at the time of gamete transport and early embryo
development. Although TNFα and NO did not affect oviductal contraction, PGE2 relaxed but
PGF2α stimulated the contraction of the ampulla and isthmus. These results indicate that
cytokines are potent regulators/modulators of PGF2α and PGE2 secretion in the bovine oviduct
(NO may be involved in this process) and thereby modulate oviductal contraction to regulate
transport of the gametes and embryo. This controlled local system enables the embryo to
develop and migrate into the uterus at optimal timing.
Supported by the basic fund of Polish Academy of Sciences
INFLUENCE OF IMMUNISATION, MELATONIN AND SEROTONIN
TREATMENT, AND PINEALECTOMY ON HYPOTHALAMIC-GONADAL
AXIS IN BIRD
Mykola Dzerzhinsky, Andriy Pustovalov
Department of Cytology, Histology and Developmental Biology, Kiev Taras
Shevchenko University,64 Vladimirskaya St., Kiev, 01033 Ukraine.
This study investigated the interacting effects of a immunisation, melatonin, serotonin
treatment and pinealectomy in birds. It was shown, that both immunisation and exogenous
melatonin caused activation of hypothalamic-gonadal system of five-week male Gallus
domesticus. At the same time both immunisation and exogenous melatonin was applied to
increase suppression of reproductive system caused by exogenous serotonin or pinealectomy.
Combined immunisation and melatonin treatment caused less significant changes, than
immunisation or exogenous melatonin alone. It is possible that immunisation influenced the
reproductive system indirectly by melatonin. It was shown that day pineal synthetic
production caused activation of gonads by hypothalamus-thyroid axis by way of oppression of
activity of paraventricular nucleus neurocytes. Immunisation or combined melatonin and
serotonin treatment could provoke a decrease it daily pineal synthetic production. It was
shown that both exogenous melatonin and pinealectomy caused an increase of antibody titre
in birds. Birds body weight increased after immunization and decreased after immunization of
pinealectomised birds in spite of melatonin treatment. Gonad weight and ratio of gonad
weight/body showed generally insignificant changes. The increase of these indices was shown
only after combined melatonin and serotonin treatment in immunization absence.
ET-1 IMMUNOREACTIVITY IN THE ENDOTHELIUM OF SUBUTERINE
PRECOLLECTOR LYMPHATICS IN THE LIGAMENTUM LATUM UTERI
IN THE COW
Katarzyna Jankowska, Aneta Andronowska, Teresa Doboszyńska, Hubert
Niewęgłowski, Anna Postek, Marcin Chruściel
Institute of Animal Reproduction and Food Research of the Polish Academy of
Sciences in Olsztyn, Poland, Email: [email protected]
Precollector lymphatics leaving the uterus participate together with the blood vessels in the
transportation of biologically active substances in the area of the ligamentum latum uteri.
Endothelin 1 (ET-1) is one of the factors regulating of the blood flow. Its presence caused the
decrease of the cross-section size of lymphatic lumen.
The aim of the study was to assess immunoreactivity of ET-1 in the endothelial cells of
precollector lymphatics connected with lymph outflowing from the uterine horn during the
estrous cycle in a cow.
Tissue samples form uterine mesometrium in different stages of the estrous cycle were used.
Cryostat sections (12µm) were stained immunohistochemically using specific rabbit
polyclonal antibody against ET-1 (ABC method). Obtained colored product of reaction,
visible in the endothelial cells of precollector lymphangions, revealed the antigen localization.
The intensity of staining was confirmed by measuring the optical density (DP SOFT,
Olympus). The highest ET-1 activity (174.6 ± 0.75) was detected in lymphangions in the early
luteal phase and the lowest activity (184.2 ± 0.62) - during the middle luteal phase. Statistical
analysis of optical density measurement revealed the significant differences (P<0,001)
between studied phases of the estrous cycle and early luteal phase. No significant differences
between follicular and late luteal phases were detected.
Differential expression of ET-1 in the endothelium of the lymphatic vessels during the estrous
cycle in the cow may suggest its influence on lymphatic vessels contractility and participation
in the regulation of lymph flow in the ligamentum latum uteri.
EXAMINATION OF CD25 ANTIGEN ON DECIDUAL MONONUCLEAR
CELLS (DMC) IN THE CASES OF SPONTANEOUS ABORTIONS
Tomasz Maj1, Alicja Halberstadt2, Jarosław Pająk2, Marian Stanisław Gabryś2, Anna
Chełmońska-Soyta1
1
Institute of Immunology and Experimental Therapy, 53-114 Wrocław, Poland,
2
II Department of Gynecology, University School of Medicine, Wrocław, Poland
Objective: Participation of mechanisms of innate immunity in spontaneous abortions is
poorly understood. Interleukin 2 (IL-2) is absent in normal decidua, however it can be present
in case of abortion. The aim of the study was to determine the percentage of activated CD2+
and CD56+ cells in DMC isolated from the decidua in cases of spontaneous abortions.
Materials and methods: Decidual mononuclear cells (DMC) were obtained through
mechanical dissociation of decidua (10 women) and centrifuged on Ficol gradient. After
washing, surface DMC and PBMC (4 women) antigens (CD2, CD25, CD56) were doublelabeled and analysed by flow cytometry.
Results: In the all examined cases among CD56+ two populations: CD56dim and CD56bright
were present. CD56bright dominated over CD56dim. However the percentage of activated cells
(CD25+) was higher among CD56dim cells than among CD56bright and on CD2+ cells CD25
antigen was hardly present. There was observed pronounced representation of CD25+/CD56/CD2- cells. On the contrary, in peripheral blood of 4 examined patients dominated
CD56bright/CD25+ and CD2+/CD25+ cells.
Conclusions: Our results show that mechanism of innate immunity can be associated with
spontaneous abortion. Moreover, it seems that cells connected to this phenomenon are
CD56dim cells and CD25+/CD56-/CD2-. The further research is in a due course.
REGULATION OF PROSTAGLANDIN (PG) SYNTHESIS
BY INTERLEUKIN-1 (IL-1) IN BOVINE ENDOMETRIUM
Katarzyna K. Piotrowska1, Anna Korzekwa1, Michiyo Tanikawa2, Mamadou M. Bah1,
Tomas. J. Acosta2, Kiyoshi Okuda2, Dariusz J. SkarŜyński2.
1
Department of Reproductive Immunology, Institute of Animal Reproduction and
Food Research PAS, 10-747 Olsztyn, Poland, E-mail: [email protected];
2
Laboratory of Reproductive Endocrinology, Okayama University, Okayama 7008530, Japan.
Interleukin-1 plays a critical role in the generation of inflammatory response and in the
regulation of many physiological events. IL-1 may also participate in endocrine and local
regulations of many reproductive functions. In the present study, we examined IL-1 role in
autocrine/paracrine regulations of the bovine endometrium. In Exp. 1, IL-1α, IL-1β and IL-1
receptor type 1 (IL-1RT1) gene expression, in the bovine endometrium was determined.
Bovine uteri were classified into five stages of the estrous cycle (early luteal I: Days 2-3;
early luteal II-developing luteal: Days 5-6; mid luteal: Days 8-12; late luteal: Days 15-17; and
follicular: Days 19-21). A semi-quantitative RT-PCR revealed that IL-1α gene expression
was greater at the early luteal stage than at the other stages (P<0.05). IL-1β gene expression
was higher at the early and mid luteal stages than at the other stages (P<0.05), and the lowest
at the follicular stages. IL-1RT1 gene expression was greater at the late luteal stage than at the
other stages (P<0.05). In Exp. 2, IL-1α effects on PGE2 and PGF2α output by the
endometrium was studied. Endometrial slices (20-30 µg) taken from five stages of the estrous
cycle, were exposed to 0.6 nM IL-1α for 4h. IL-1α stimulated PGE2 output with the highest
response during the mid-luteal stage, but it did not stimulate PGE2 output during the follicular
stage. IL-1α significantly enhanced PGF2α output with the highest response at the follicular
stage (P<0.001). IL-1α increased PGE2:PGF2α ratio at the mid luteal stage, and decreased it at
the late luteal and follicular stages. In Exp. 3, IL-1α effect on PGF2α synthesis in the bovine
endometrial cells was determined. Cultured epithelial and stromal cells were exposed to IL1α (0.006-0.6 nM) for 24hrs. Although, IL-1α stimulated PGF2α synthesis in the stromal cells
in a dose-dependent manner (P<0.05), it did not affect PGF2α in the epithelial cells. In Exp. 4,
the intracellular mechanism of IL-1α action was established. The stromal cells were exposed
to a phospholipase (PL) A2 inhibitor (ACA) or PLC inhibitor (U-73122) with or without IL1α. Although, U-73122 inhibited neither basal nor IL-1α-induced PGF2α synthesis, ACA
completely stopped IL-1α action (P<0.05), indicating that IL-1α-induced PGF2α synthesis
was mediated by PLA2.
Concluding, IL-1 is produced in the bovine endometrium throughout the estrous cycle, and
plays some roles not only in maintenance of CL, but also in luteolysis by regulating the
endometrial PGF2α:PGE2 ratio.
Supported by the PAS and the Joint Polish-Japanese JSPS-PAS project
Session X
Biotechnology of reproduction
Oral presentation
SEX REGULATION IN MAMMALIANS BY SEPARATION OF X AND Y
SPERMATOZOA
Michał Bochenek, Zdzisław Smorąg, Tomasz Herjan
Animal Reproduction, 32-083 Balice, Poland, E-mail: [email protected]
From economical point of view the possibility of sex regulation in animal is very attractive
idea. The only reliable and relatively easy method is X and Y sperm separation by flow
cytometry. Now it is possible to successfully separate spermatozoa of many species – humans,
cattle, horses, swines, goats, rabbits, dogs, gorillas, dolphins and others. History of
development of the method is described: from first sperm DNA volume measurement in the
early 80-ies up to large scale commercial application today.
A number of technical aspects of sperm sorting is discussed in details: optical and
hydrodynamic characteristics of sperm heads, instrument modification and calibration,
relationship between sorting speed and purity, semen handling after sexing, sperm viability
after preparation and sorting, freezing methods and insemination doses. At present it is
possible to sort 15-25mln spermatozoa of each fraction per hour, with purity 90-96% for X
fraction and 85-90% for Y fraction. Motility immediately after sorting is 90-95% and 50-70%
after freezeing/thawing. The sexed semen is frozen in straws with 2.0-2.5 mln
spermatozoa/straw.
At the beginning of 2003 National Research Institute of Animal Production started to
introduce this method into cattle production. First calf in Poland after insemination with sexed
semen was born in Balice in September 2003. Now there are more than 90 calves born after
inseminations with sexed semen with ab. 87% of expected sex (females). The best fertility
rate achieved after insemination was 72.73%.
EMBRYONIC STEM CELLS – THE HOPE OF BIOTECHNOLOGY ?
Jolanta Karasiewicz, Jacek A. Modliński
Department of Experimental Embryology, Institute of Genetics and Animal Breeding
PAS Jastrzębiec, 05-552 Wólka Kosowska, Poland, E-mail: [email protected]
Most sophisticated interventions are now possible using mouse embryonic stem cells (mESC),
which comprise cloning mice and making chimaeras to produce strains with genetic
modifications at request or to direct the fate a given line of modified cells within a wild host
throughout development. Human ESC (hESC) follow, but in this case the aim is not to clone
people but to differentiate those cells and replace a diseased tissue with them.
Large domestic animals lag behind due to poor quality of their ESC. Bovine, ovine, goat or
even rabbit ESC do not keep undifferentiated phenotype in vitro but become epithelioid
instead. Even more severe vice is that the prove of their entering the germ line in chimaeras is
scarce. This is why they are often referred to as ESC-like cells. Sporadic cases of their use for
cloning have not delivered data encouraging enough and these are somatic cells, not ESC, that
are now massively used as nuclear donors.
There is a line of evidence, however, that species specific differences between the conditions
to derive ESC are very prominent. The detailed comparison of mESC and hESC have shown
that the main signaling pathways supporting their proliferation and renewal differ, and thus
should culture media supplements and culture design. This is the first indication that new
methods of deriving farm animals ESC could be developed. The second indication comes
from embryology. Large animals have in their development an embryonic disc more like birds
than like house mouse in which implantation as soon as at the fourth day after coitus
interferes with germ layers formation by adding maternal tissue interactions instead. Bird ESC
are morphologically and also by their origin - from a multithousand-cell embryonic disc closer to native epiblast cells than to mouse ESC. Therefore, new trials might be diverging far
from those copied after mouse species.
Even as imperfect as they are now, ESC in large animals can better conform to the demands
of being genetically modified than do somatic cells. ESC survive definitely more cell
divisions in vitro than cells from primary cultures of tissue explants like ear skin or alike,
giving time for cell clone selection after genetic modification.
In our Department ovine ESC-like cells were used with limited success for cloning. Effective
chimaera formation was possible with these cells. A line of pig ESC-like cells has been also
derived to be genetically modified in vitro. New trials at generating ovine ESC follow.
SOMATIC CELL CLONING IN MAMMALS – PRESENT POSSIBILITIES
AND LIMITATIONS
Maria Skrzyszowska, Marcin Samiec
Department of Animal Reproduction Biotechnology, National Research Institute of
Animal Production, 32-083 Balice/Krakow, Poland,
The possible propagation of mammalian individuals by somatic cell nuclear transfer (SCNT)
has important economical implications in biotechnology and biomedicine as so far has been
shown by generation of cloned transgenic animals with the ability to produce valuable
recombinant human proteins. Swine embryo engineering (somatic cell cloning combined with
transgenesis) is a particularly important research field of assisted reproduction technologies
due to increasing role of the porcine organs in xenotransplantology or creation of animal
bioreactors providing with biopharmaceuticals. However, high early-, mid- and late-gestation
mortality rates of nuclear-transferred embryos/foetuses as well as numerous malformations of
resultant cloned offspring appear still often. The studies on mammalian somatic cell cloning
confirmed that pre- and postimplantation development of nuclear-transferred
embryos/foetuses depends to a high degree on the preservation of appropriate requirements in
all steps of SCNT procedure. One of the most important factors which determine the somatic
cell cloning efficiency is structuro-functional quality of nuclear donor cells. Moreover, right
coordination between nuclear donor cell phenotype and cell cycle stage, maternal
chromosome elimination method, oocyte reconstruction technique and artificial activation
system seem to be also significant. Transcriptional activity of donor nuclear genome during
pre- and/or postimplantation embryo- as well as fetogenesis is correlated with the frequency
of remodelling/reprogramming for epigenetic modifications including DNA
methylation/demethylation and histone deacetylation/acetylation. Generally, the cause of low
somatic cell cloning efficiency may be an incomplete recapitulation of donor cell genomic
DNA-associated methylation pattern and thus impaired restoration of totipotency/pluripotency
of embryonic cell lines. It has been ascertained that somatic cell nuclei should undergo the
wide DNA cytosine residue demethylation changes throughout early development of cloned
embryos to reset its own epigenetic and parental genomic imprinting memory which have
been established by specific pathway of somatic and germ cell lineage differentiation.
Understanding the molecular mechanisms of epigenetic transcriptional reprogramming of
donor nuclear genome will be helpful to solve the problems resulting from somatic cell
nuclear transfer and open new possibilities for common application of this technology in
human biomedicine.
This research was supported by the State Committee for Scientific Research as a Solicited
Project number PBZ-KBN-048/P05/2001/08 from 2002 to 2005 year.
EXPRESSION OF GENE CONSTRUCTS IN ANIMAL TRANSGENESIS
Ryszard Słomski1,2, Daniel Lipiński2, Joanna Zeyland1, Wojciech Juzwa1, Andrzej
Pławski2, Robert Kalak1, Marlena Szalata1,2, Karolina Wielgus1, Jacek Jura3, Maria
Skrzyszowska3, Zdzisław Smorąg3, Marek Pieńkowski4
1
Department of Biochemistry and Biotechnology, University of Agriculture, 60-637
Poznań, 2Institute of Human Genetics Polish Academy of Sciences, 60-479 Poznań,3
National Research Institute of Animal Production, Balice, Poland; 4Allergic Diseases,
Asthma and Immunology Clinic, P.C., Knoxville, TN 37919, USA.
Current modifications of animal genomes are oriented towards two main directions involving
the use of mammary gland as bioreactor and tissue engineering for application in humans. The
advent of transgenic technology provided the methods for production of pharmaceuticals by
isolation of these proteins from transgenic animals. Transgenic animals can be obtained
mainly by introduction of genetic constructs by direct embryo microinjection or by various
delivery strategies to cultured in vitro recipient cells. The mammary gland has been focused
on as a bioreactor since milk is easily collected from lactating animals and protein production
can be expressed at very high level including hormones and enzymes. We generated
transgenic rabbit producing human growth hormone in milk after microinjection of genetic
construct containing mammary gland-specific promoter and entire human growth hormone
gene (hGH). The growth hormone was detected in the milk, but not in serum, of the
transgenic rabbit females up to the level of 10 µg/ml. Ectopic expression of transgene in brain,
heart, kidney, liver and salivary gland was not observed. Biological activity of growth
hormone was measured by immunoreactivity and capability to stimulate growth of hormone
dependent NB211 cell line.
Enormous progress in biotechnology and genetic engineering in recent years may help in
research on xenotransplantation and provide solution to the shortage of human allografts. The
xenotransplantology is focused on receiving of genetically modified pigs lacking gene(s)
involved in graft rejection by recipients. Xenotransplantation comprises of several important
steps from interdisciplinary areas. The first step involves preparation of competitive,
inactivating and regulatory gene constructs. These gene constructs should allow (i) knock-out
of 1,3 galactosyltransferase (1,3GT) gene, (ii) expression of complement proteins and (iii)
regulate expression of proteins. Our transgenic pigs obtained by injection of gene construct to
the fertilized oocytes can be cloned after confirmation of transgene stability and expression.
Transgenic pigs could provide an unlimited source of cells and organs for persons suffering
from organ failure, diabetes or degenerative disorders. This made xenotransplantation very
attractive for biotechnology of reproduction.
Poster presentation
CRYOPRESERVATION OF EUROPEAN BISON (BISON BONASUS)
EPIDIDYMAL SPERMATOZOA: A PRELIMINARY RESULTS
Wiesław Demianowicz¹, Zygmunt GiŜejewski¹, Heriberto Rodriguez-Martinez ²,
Radosław Kowalski¹, Jan Glogowski¹
¹Institute of Animal Reproduction and Food Research of Polish Academy of
Science, 10-747 Olsztyn, Poland. E-mail: [email protected]
²Swedish Univ Agr Sci SLU, Dept Obstet&Gynaecol, Fac Vet Med, SE-75007
Uppsala, Sweden
European bison (Bison bonasus) is endangered and strictly protected species.
Assisted
reproductive techniques allow to increase heterozygosis for the maintenance of genetic
diversity and avoid inbreeding depression. Cryopreservation of spermatozoa from selected
individuals may be applied to establish genome resource banks. Furthermore it permits time
to acquire a better understanding of reproduction biology of this species.
Spermatozoa were collected post mortem from 3 European bison bulls, selected in Bialowieza
Primeval Forest (autumn 2003 and winter 2005). After evaluation of volume, motility and
concentration, the sperm from each bull were diluted with four extenders: citrate-fructose-egg
yolk (CYT); TRIS-egg yolk (TRIS); TRILADYL® (TRIL); BIOCIPHOS® (BIOC) and loaded
into 0,25 ml French straws. After 4 h of equilibration at 4º C, samples were placed on a 4-cmhigh styrofoam frame that was floating on liquid nitrogen. The frozen semen were stored at
-196ºC at least 2 months. Straw were thawed at 35º C for 30 seconds and samples were
evaluated for motility, viability (SYBR14/PI, Molecular Probes), morphology, acrosomal
status and integrity of sperm membrane (hypoosmotic swelling test- HOST).
The mean of concentration of spermatozoa in seminal fluid recovered from cauda epididymis
was 3.56±1.50x109/cm3. Motility of spermatozoa was different between animals (50, 20 and
40%). There were differences for motility after thawing between CYT vs. TRIS, TRIL and
BIOC extenders (6.7 vs. 20.0, 16.7 and 20.0 %). The percentage of live spermatozoa at
viability test was 11.5, 22.3, 21.8 and 18.5 respectively. Individually differences in motility
(11.3, 8.9, 27.5 %) and viability (18.6, 13.1, 25.3 %) of spermatozoa after freezing- thawing
were observed between bulls.
HOST-responsive cells preserved in BIOC-extender were numerically higher compare to
CYT, TRIS and TRIL (20.7 vs. 11.3, 13.7, 9.0%). All four extenders had no difference in
their effects on acrosome integrity and morphology. The main sperm abnormality observed
were cytoplasmatic droplets (33.2±7.2%).
The preliminary results of cryopreservation of epididymal spermatozoa of European bison
may offer good opportunities for the propagation of this threatened species.
SURVIVAL AND CELL CYCLE ANALYSIS OF VITRIFIED BOVINE
SOMATIC CELLS
Barbara Gajda, Lucyna Kątska-KsiąŜkiewicz, BoŜenna Ryńska, Michał Bochenek,
Zdzisław Smorąg
Animal Production, 32-083 Balice/Krakow, Poland.
The aim of experiment was to investigate the susceptibility of different types of bovine
somatic cells for vitrification and cytometric cycle analysis. The experiment was carried out
on cumulus and granulosa cells originating from ovarian oocytes at the germinal vesicle stage
(immature oocytes) or the metaphase II stage (in vitro matured oocytes) and fibroblast cells
originating from ear skin. The cells were cultured using routine treatment to confluency,
harvested, and passaged before being serum starved for 0, 8 or 12 days. Morphology and cell
concentration were evaluated before vitrification. The cell concentration of 1 to 10 x 106/ml
was used. Vitrification solution (EFS) consisted of 40% ethylene glycol, 18% ficoll and 0.3 M
sucrose dissolved in PBS medium. Before vitrification the cells were equilibrated for 5
minutes at room temperature in EFS solution and then were transferred to 0.25 ml plastic
straws. Immediately afterwards the straws were gradually immersed in liquid nitrogen.
Samples were stored in liquid nitrogen for 1 to 12 month. Following storage the straws were
thawed rapidly in water bath at 200C and diluted in 0.5 M sucrose. After five minutes cells
were centrifuged, washed with HEPES-buffered TCM 199 medium and placed in culture. The
effects of the vitrification treatment on cell survival were assessed during primary culture by
the ability of living cells, both to attach to slides and to proliferate up to the confluence stage.
After vitrification and culture the cells were cytometrically analysed. Cell cycle calculations
were performed after discrimination of cell pairs by the pulse processing method and
expressed as percentage of G0/G1, S and G2/M stages. The viability of vitrified somatic cells
depended on the concentration of the cells. The viability was higher (about 70 to 100% cells
prolif.) for cells vitrified at a concentration of 5 to 10 x 106/ml than (about 50 to 70% cells
prolif.) 1 to 5 x 106/ml (P<0.05 for cumulus and granulosa cells from immature oocytes, and
fibroblast). Higher susceptibility was observed for the vitrification of fibroblast cells starved
for 8 than 12 days (P<0.05). Vitrification did not decrease percentage (94.7) of cumulus cells
from immature oocytes non-starved in G0/G1 phase, of cumulus cells from in vitro matured
oocytes starved for 8 (80.7%) and 12 days (83.7%) and fibroblast cells starved for 0 (93.9%)
and 12 days (96.3%), but slightly decreased the percentage of fibroblast cells starved for 8
days (79.4%) compared to non-vitrified control (P<0.01). In conclusion, the proposed
vitrification method results in high survival rate of bovine cumulus and granulosa cells from
immature oocytes, cumulus cells from in vitro matured oocytes and fibroblasts. Cytometric
analysis of these cells confirmed cell cycle distribution compared to non-vitrified control
cycle.
CHARACTERISTICS OF THE VESTIGIAL MASCULINE UTERUS OF THE
EUROPEAN BISON /Bison bonasus L./ - PRELIMINARY RESULTS
Zygmunt GiŜejewski1, Jan Glogowski1, Wojciech Bielecki2, Wiesław Demianowicz1
¹Institute of Animal Reproduction and Food Research, Polish Academy of Sciences,
10-747 Olsztyn, E-mail: [email protected]
2
Faculty of Veterinary Medicine, Department of Clinical Sciences, Warsaw
Agricultural University, 02-776 Warsaw, Poland
Among native mammals, the masculine uterus is present in the European beaver and the
European bison, and its role remains unknown. In the present study reproductive organs were
collected from 13 male bison aged two to 15 years, culled for selection purposes. Among the
bison examined, 11 had bifid masculine uteruses of a different length (body + the longest
horn), varying from 17.1 to 53.0 cm, from which 0.5 to 38 ml fluid was collected by
squeezing. The consistency of uterine fluids was watery, or resembled fresh honey. The fluids
were light-yellow to light-brown in color. Histopathological examinations were performed on
the uteruses of five bison, fixed in 10% phosphate buffered formalin. Paraffin sections, 4µm
thick, were hematoxylin-eosin-, Van Gieson- and PAS-stained.
As shown in the macroscopic image, the uterus walls are composed of three layers, i.e.
mucous membrane, muscular coat and visceral peritoneum. The mucosa is covered by simple
cylindrical epithelium whose cells have oval nuclei and slightly acidophilic cytoplasm. The
framework of the mucous membrane is connective tissue with loosely arranged fibers. This
tissue contains irregular tubular structures, open towards the lumen of the organ. The
muscular coat is composed of three layers: internal and external, with cells arranged
longitudinally with respect to the longitudinal axis of the organ, and middle, with
transversally arranged muscle cells. Such a structure suggests the capability of uterine
contractions. The last layer is peritoneum, built of connective tissue, with matted fibers
providing support to simple squamous epithelium. Basophilic cytoplasm of epithelial cells of
tubular structures in the mucosa may indicate the presence of nucleic acids, i.e. secretory
capability of cells. Therefore, it can be stated that these are the glands producing secretion
found in the lumen of the masculine uterine. It seems that the epithelium lining the lumen of
the organ also produces secretion, contained in the fluid contents found in the lumen of the
uterus. In order to determine protein concentration and phosphatase activity in the uterine
fluids, they were - due to their consistency - suspended in physiologic saline, at a ratio of 1:9
(w/v). Taking into account the initial material, total protein content was determined at a level
of 94.1 mg/ml, alkaline phosphatase activity - 9 600 U/l and acid phosphatase activity - 890
U/l. Denaturing electrophoresis (SDS-PAGE) of uterine secretion showed low protein
heterogeneity, with the dominant fraction weighing approx. 70 kDa.
The secretory capability of the masculine uterus, observed in the European bison, is a new
scientific fact that has not been described in professional literature before. Macroscopic
analyses confirmed the presence of a canal connecting the lumen of the organ to the urethra in
all uteruses examined in the study, regardless of their size. This may indicate the contribution
of uterine fluids to ejaculate formation. The project was financially supported by the State
Committee for Scientific Research, PBZ-KBN-084/P06/2002/
IRON INDUCED LUMINESCENCE OF FRESH, EQUILIBRATED AND
FROZEN RAM SPERMATOZOA
Piotr Gogol, Mirosław Cegła
National Research Institute of Animal Production, Department of Biotechnology of
Animal Reproduction, Krakowska 1, 32-083 Balice, Poland.
Semen freezing can initiate the peroxidation of sperm cell membrane lipids. Some products of
lipid peroxidation are radiation inactivated and emit photons in the form of ultraweak
spontaneous luminescence. The intensity of this luminescence may therefore be indicative of
the peroxidation processes. In the case of spermatozoa, however, spontaneous luminescence is
very weak and thus difficult to measure. Recent studies have indicated that registration of
iron-induced luminescence can be alternatively used as a fairly simple method for measuring
sperm lipid peroxidation.
The aim of the present study was to evaluate the effect of equilibration and freezing on ironinduced luminescence of buck spermatozoa as an indicator of the oxidative damage to the
cell.
Buck semen was frozen using a modified method of Corteel (Kareta and Cegła, 1999).
Luminescence was measured directly after collection, after equilibration and following
freezing and thawing of semen.
Luminescence was measured at 20ºC using an AutoLumat LB953 (Berthold) luminometer
equipped with a cooled photomultiplier with a spectral response range from 370 to 620 nm.
Prior to measurement of luminescence, spermatozoa were separated from the seminal plasma
and diluent by two-fold centrifugation (700×g for 15 min) and resuspended in 0.9% NaCl to a
concentration of 200×106 cells/ml. To 500µl of the washed sperm suspension at a
concentration of 200×106 cells/ml, 10µl of 5mM luminol was added. Emission was induced
by adding (using automated injector system) 100µl 0.3mM FeSO4 solution (final
concentration 0.05mM). Immediately after injection light emission kinetics was measured
during 450 s.
Equilibration and freezing were found to have a significant effect on sperm luminescence.
The freezing process increased the intensity of luminescence, which indicates growing
oxidative damage to the spermatozoa.
Further on, attempts will be made to determine correlations between sperm luminescence and
sperm susceptibility to freezing.
This study was carried out as part of NRIAP statutory activity, project no. 3412.1.
IDENTIFICATION OF A NEW GENE ON THE MOUSE Y CHROMOSOME
Paweł Grzmil, Agnieszka Dziuba, Paweł Chomiak, Józefa Styrna
Department of Genetics and Evolution, Institute of Zoology, Jagiellonian University,
Ingardena 6, 30-060 Krakow, Poland, E-mail [email protected]
The only known active genes located on the long arm of the mouse Y chromosome belong to
the Ssty gene family and are present in more than 100 copies. They are supposed to be
involved in the last stage of spermatogenesis – spermiogenesis. Till now two genes of this
family are known Ssty1 and Ssty2, both expressed in the testis. But nothing more is known
about their exact function. The analysis of the mutants with partial deletion on the long arm of
the Y chromosome could throw some light on it. During the expression analysis of the Ssty1
gene we detected two different expression profiles. The first is characteristic for the entire
mRNA molecule, represented by 430 bp band in RT-PCR reaction, and its transcription was
detected when spermatids were present in the testis. The second was observed when the
primers for RT-PCR were located only in the last, third exon of the Ssty1, and was
represented by 342 bp band. This product was detectable when primary spermatocytes were
present in the testis, much earlier than it was described before. Exactly one year before, at the
conference in Gdansk, we presented our data concerning expression of the Ssty1and we posed
the question if the difference we observed was a result of the alternative splicing of this gene,
or maybe we found a new gene with very similar sequence to the third exon of Ssty1 located
on the long arm of the mouse Y chromosome. To answer this question we screened the RZPD
genomic library and we analyzed 52 genomic clones of the Y chromosome. Additionally we
searched the NCBI gene bank data base in order to find if there is a genomic sequence
corresponding to our predicted new gene. Here we present our results clearly demonstrating
that we found a new gene from the Ssty family with different expression profile comparing to
the Ssty1 and Ssty2. This new gene (Ssty3) has only one exon (like Ssty2) and its sequence is
highly similar to the sequence of the third exon of the Ssty1. It should be noted that this
sequence contains complete ORF coding for the hypothetical protein. Moreover our analysis
revealed that all three Ssty genes are expressed not only in germ cells but also in other cell
type. This finding can be very important in analysis of the function of these genes in
spermiogenesis.
This work was supported by the State Committee for Scientific Research (KBN) grant 3 P04C
033 23.
PRODUCTION OF F1 GENERATION OF TRANSGENIC PIGS SUITABLE
FOR XENOTRANSPLANTATION – EFFECTIVENES OF TRANSGEN
TRANSMISSION
Jacek Jura1, Zdzisław Smorąg1, Barbara Gajda1, Ryszard Słomski2, Daniel Lipiński2,
Robert Kalak2
1
National Research Institute of Animal Production, Division of Biotechnology of
Animal Reproduction, Balice, Poland, E-mail: [email protected]; 2Institute of
Human Genetics, Poznań, Poland
The aim of the experiment was to produce F1 generation of transgenic pigs suitable for
xenotransplantation and evaluation of transgene transmission to next generation. To produce
F0 generation of transgenic pigs a competitive gene construct coding the same substrate as the
endogenous enzyme of organ donor was introduced. The prepared gene construct contained
the human α1,2-fucosyltransferase gene that competes with α1, 3-galactosyltransferase for the
same substrate N-acetyl lactose amine. Insertion of human gene into pig genome should
conceal the epitope by reducing the affinity of anti-Gal antibodies. Decreased affinity of antiGal antibodies between human and pig can reduce species-specific immunological difference
and minimize the risk of xenograft rejection.
To produce F1 transgenic generation semen from transgenic F0 boar TG 1154 was used. 200
potentially transgenic pigs of F1 generation was produced. Molecular analysis of DNA
obtained from produced offspring showed that 87 (43,5%) of the produced piglets have
integrated gene inherited from transgenic boar TG 1154.
This work was supported by the State Committee for Scientific Research as a Solicited Project
number PBZ-KBN-048/PO5/2001/05 from 2002-2005.
SELECTION OF TRANSGENIC PIG EMBRYOS FOR
XENOTRANSPLANTATION PROJECT – THE USE OF GFP GENE
MARKER
Jacek Jura1, Ryszard Słomski2, Daniel Lipiński2, Barbara Gajda1, Zdzisław Smorąg1
1
National Research Institute of Animal Production, Division of Biotechnology of
Animal Reproduction, Balice, Poland, E-mail: [email protected]
2
Institute of Human Genetics, Poznań, Poland
Due to its potential, transgenesis of farm animals is an area of abiding interest to modern
biotechnology. A basic problem that needs to be resolved is the efficiency, which continues to
be disproportionately low compared to the financial outlays and labour. Improvement of
transgenesis efficiency has been the subject of many publications, which describe new
research approaches and enhancement techniques. The greatest challenge in transgenesis,
especially in xenotransplantation projects is the need to increase possibility to obtain
transgenic organ donors. Any other techniques, including cloning, are still less efficient than
standard microinjection. To overcome major problem for DNA microinjection – its
effectiveness – many attempts have been done by application of increasingly sophisticated
genetic constructs containing elements that should enhance transgene integration. The other
way to overcome mentioned problem is to introduce a procedure which will make possible to
select embryos with integrated desired gene.
In presented experiments to produce transgenic pig embryos for xenotransplantation project
gene construct CD 46 with GFP gene marker was used. The vector was injected into fertilized
pig eggs by standard microinjection.
After injection zygotes were placed into in vitro culture for five days to reach blastocyst stage
and were evaluated under fluorescence microscope. From 37 injected zygotes 12 developed to
blastocyst stage and five (41,7%) showed green bioluminescence. In compare, as a control
group 4 not microinjected zygotes were cultured to the blastocyst stage. They do not produce
any bioluminescence signal.
This work was supported by the State Committee for Scientific Research as a Solicited
Project number PBZ-KBN-048/PO5/2001/05 from 2002-2005.
OVULATION RATE AND PROLIFICACY IN EWES DIFFERING IN AGE,
BIRTH TYPE, AND PROPORTION OF PROLIFIC BREEDS IN GENOTYPE
Wiesław Kareta1, Kazimierz Korman2, Mirosław Cegła1
1
Department of Animal Reproduction Physiology, Balice n. Krakow, 2 Zootechnical
Experimental Station Kołuda Wielka, National Research Institute of Animal
Production, 30-960 Krakow, Poland, E-mail: mcegla@izoo. krakow.pl
One part of reproduction biotechnology is endoscopy for in vivo observation of the abdominal
cavity. This technique is indispensable for intrauterine insemination and has been applied to
determine the reproductive potential of small ruminants. The aim of the present study was to
determine the effect of age, birth type and proportion of prolific breeds in genotype on the rate
of ovulation estimated by the number of corpora lutea (CL) and on prolificacy determined
based on lambs born to hybrid ewes.
The observations were performed during December matings of sheep in 2000-2003 at the
Experimental Station Kołuda Wielka. A total of 483 hybrids between maternal breeds and
East Friesian milk sheep and prolific breeds of Finnsheep and Romanov sheep were
investigated. Teaser rams were used to detect oestrous ewes, which were then hand mated
twice at 12-h intervals. Ovaries of the ewes were examined laparoscopically within 4-9 days
of service. During the observations, the number of CL was counted on both ovaries in the age
groups of 8, 20, 32 and ≥44 months, according to birth type of ewes (single, twin, triplet +
quadruplet) and percentage of prolific breeds in genotype (0, 25, 37.5 and 50%). During
lambing, litter size was recorded and compared with the number of ovulations.
On the examined ovaries, 1 CL was found in 12.6% ewes, 2 CL in 56.9% ewes, 3 CL in
25.7% ewes and 4 CL in 4.8% ewes. Fertility of the analysed animals was 69.6%, with
prolificacy of 192.6%. On the ovaries of lambed ewes there was an average of 2.28 CL/ewe,
with an average of 1.93 lambs/ewe obtained after lambing. The difference between potential
prolificacy and the number of lambs born averaged 0.25 and ranged from 0.32 to 0.41
lambs/ewe according to age group, from 0.22 to 0.41 according to birth type, and from 0 to
0.52 according to the proportion of prolific blood. The number of CL and lambs born was
affected more by age at mating and proportion of prolific breed in the genotype than by birth
type of the ewes.
STEROID RECEPTORS IN DOMESTIC PIG AND WILD BOAR
CROSSBREED (PIG-BOAR) UTERUS DURING THE LUTEAL PHASE IN
ESTROUS CYCLE IN DIFFERENT SEASONS OF THE YEAR
Katarzyna Kozioł, Przemysław Gilun, Bartosz Jagusztyn, Marek Koziorowski
Department of Animal Physiology and Reproduction, Institute of Biotechnology,
University of Rzeszow Werynia near Kolbuszowa, Poland,
e-mail: [email protected]
Cyclical changes taking place in the uterus during the cycle depend on the presence of steroid
hormones in the destination cells of this organ. Estrogen (ERα), Progesterone (PR), Androgen
(AR) receptors were found in uterus tissues in many animal species and humans. During the
luteal phase of the estrous cycle many final changes take place in the uterus in order to create
a proper environment for conceiving and implantation of an embryo. Microtome section were
used for experiment. The tissues were taken from animals with controlled estral cycle: winter
(December), spring (March), summer (June) autumn (September). Immunohistochemical
reaction was induced with the use of specific antibodies reacting aganist ERα, PR, AR. The
presence of ERα, PR, AR was found in glandular epithelium of endometrium and
miometrium of the luteal phase of the estrus cycle independent of the season of the year. The
most intensive immunoreactivity for progesterone receptors was displayed by the miometrium
in all the seasons; immunocolouring for ERα, and AR were weaker. This points to the
importance of progesterone for blocking uterus contractions when there is the possibility of
embryos appearing in the uterus and later in the implantation process. The lack of differences
in immunoreactivity of ERα, PR, AR in uterus tissues in different seasons of the year
suggests that pig-boars can be pregnant any time of the year.
1
2
3
4
Fig.1 Localization of PR in miometrium of the luteal phase in estrous cycle in: 1-March, 2June, 3-September, 4-December (mag *400)
Research was supported by the State Committee for Scientifie Research as a Solicited Project
PBZ-KBN-084/P06/2002 from2003 to 2005 year
SEASONAL CHANGES IN INSULIN-LIKE GROWTH FACTOR – I (IGF-I)
CONCENTRATION IN PERIPHERAL BLOOD IN GILTS OF DOMESTIC
PIG AND WILD BOAR CROSSBREED (PIG-BOARS)
Marek Koziorowski, Przemysław Gilun, Bartosz Jagusztyn, Katarzyna Kozioł,
Ewelina Kowal
University of Rzeszów, Werynia near Kolbuszowa, Poland.
Insulin-like growth factor-I (IGF-I) is secreted by many peripheral tissues and is the mediator
of the anabolic and mitogenic activity of GH. Its secretion is controlled by the growth
hormone.
The aim of this research was to demonstrate the seasonal changes in IGF-I concentration in
peripheral blood. Domestic pig and wild boar crossbreed is characterized by the seasonally
changing metabolism status. Blood from 16 cycling gilts was collected in March, June,
September and December, within the period of 30 days, 3 times a day and measured with the
use of RIA method. Cycle phases and their duration were determined based on the previously
measured progesterone and estradiol levels. It was observed that the length of the cycle
changed together with the seasons of the year. In March the cycle lasted 24 days, in June 28,
in September 26 and in December 22 days.
450
***
400
***
***
350
***
***
300
ng/ml
***
250
200
150
100
50
VI Dec
VI Jun
VI Sep
VI Mar
V Dec
V Jun
V Sep
V Mar
IV Dec
IV Jun
IV Sep
IV Mar
III Dec
III Jun
III Sep
III Mar
II Dec
II Jun
II Sep
II Mar
I Dec
I Jun
I Sep
I Mar
0
Fig.1. Seasonal changes in IGF-I concentrations in blood plasma of pig-boar gilts in 4 different periods of the year
where I is the early luteal phase, II is the mid luteal phase, III is the late luteal phase, IV is the early luteolysis, V is the
full luteolysis and VI is the follicular phase; the statistically significant differences were marked with asterisks p≤0,001)
Although the significant influence of IGF-I on the regulation of reproductive functions was
widely claimed, the results of our research did not show significant differences in IGF-I
concentration in peripheral blood during the estrous cycle. It may mean, however, that the
IGF-I participates in reproductive regulations through its local concentration and circulation
in the vascular area of the uterus and ovaries.
The highest IGF-I level in peripheral blood, which was statistically significant, was observed
during the period of long daylight (June). This result is connected with the metabolism status
of the organism, which was confirmed in numerous papers on this hormone participationn in
general anabolic processes.
PBZ-KBN-084/P06/2002 from 2003 to 2005
SEASONAL CHANGES IN THYROID HORMONES CONCENTRATION IN
PERIPHERAL BLOOD IN GILTS OF DOMESTIC PIG AND WILD BOAR
CROSSBREED
Marek Koziorowski, Bartosz Jagusztyn, Przemysław Gilun, Katarzyna Kozioł
Department of Animal Physiology and Repoduction, Institute of Biotechnology,
University of Rzeszow, Werynia near Kolbuszowa, Poland,
Thyroid hormones – thyroxine (T4) and triiodothyronine (T3) are responsible for protein and
lipid transformations, growth speed, reproduction and regulation of lactation. In reproductive
processes in females, disorders in functioning of the thyroid gland are accompanied by sex
glands hypoplasia and follicular cysts. Hyperthyroidism causes limited secretion of LH. The
aim of research was defining the seasonal changes in thyroid hormones concentrations in
estrous cycle in domesticated pig and wild boar crossbred.
The analysis was conducted in four different periods of the year: spring equinox (March),
long daylight (June), autumn equinox (September), short daylight (December). Sixteen
crossbred gilts with controlled estrous cycle were used in the experiment. Blood for analysis
was collected within the period of 30 days, every eight hours from a cannulated jugular vein.
T3 and T4 concentrations in plasma was measured with the use of RIA method
75
2.5
B
B
70
B
B
65
B
B
60
2.0
55
50
A
45
nmol/l
nmol/l
1.5
A
A
A
A
A
40
35
30
1.0
25
20
0.5
15
10
5
0.0
Fig. 1 Seasonal changes in T3 concentration in plasma of
peripheral blood in gilts of domestic pig and wild boar
crossbred in 4 experimental times (I-early luteal phase, IImid luteal phase, III-late luteal phase, IV-early luteolysis, Vlate luteoyisis, VI-follicular phase).
VI Jun
VI Dec
VI Mar
VI Sep
V Jun
V Dec
V Mar
V Sep
IV Dec
IV Jun
IV Mar
IV Sep
III Jun
III Dec
III Mar
III Sep
II Jun
II Dec
II Mar
II Sep
I Jun
I Dec
I Mar
I Sep
VI Jun
VI Dec
VI Sep
V Dec
VI Mar
V Sep
V Mar
V Jun
IV Jun
IV Dec
IV Sep
III Dec
IV Mar
III Mar
III Jun
III Sep
II Jun
II Dec
II Sep
I Dec
II Mar
I Mar
I Jun
I Sep
0
Fig. 2 Seasonal changes in T4 concentration in plasma of
peripheral blood in gilts of domestic pig and wild boar
crossbreed in 4 experimental times (I-early luteal phase, IImid luteal phase, III-late luteal phase, IV-early luteolysis, Vlate luteoysis, VI-follicular phase; statistically significant
differences were marked different letters, p≤0,001).
Thyroid hormones (T3 and T4) concentration did not significantly change during the estrous
cycle. The highest thyroxin level was observed during the period of short daylight, and the
lowest during the period of spring equinox. Insignificant differences in T3 and T4
concentrations during the estrous cycle point to the fact that these hormones do not directly
participate in the cycle regulations. The highest thyroxin and triiodothyronine concentration in
winter points to a significant role of these hormones in regulations of energetic
transformations. The lowest T4 concentration and highest T3 concentration in March may
point to the organism adaptation to increasing metabolism due to longer daylight and higher
temperatures.
Research was supported by the State Committee for Scientific Research as a Solicited project
PBZ-KBN-084/P06/2002 from 2003 to 2005
SEASONAL CHANGES IN TOTAL ANTIOXIDANT ACTIVITY (TAA) IN
BLOOD PLASMA OF DOMESTIC PIG/WILD BOAR CROSSBREED (PIGBOAR) GILTS
Marek Koziorowski, Ewelina Kowal, Katarzyna Kozioł, Bartosz Jagusztyn,
Przemysław Gilun
Reactive oxygen species (ROS) are formed in the organism under the influence of
endogenous and exogenous factors as metabolism products. High reactivity of these species
causes damage to cells. The organism defense against ROS is essential. Small-particled
antioxidants play a significant role in defending the organism against ROS, especially when
blood plasma is poor in antioxidant enzymes. The changes in intensity of metabolic processes
depending on the amount of daylight in different periods of the year were used as a base for
research on total antioxidant activity in blood plasma in pig-boar gilts.
16 gilts of 80-100 kg were used in the experiments. Blood for analysis was collected within
the period of 30 days, 3 times a day, in the periods of spring equinox (March), long daylight
(June), autumn equinox (September) and short daylight (December). The mean of total
antioxidant activity was expressed in equivalents of Trolox.
The antioxidant activity of blood plasma undergoes seasonal changes. High activity in the
period of long daylight (June) may result from protesting organs from harmful effects of
increased ROS production. The cell membrane protection against organic free radicals,
specifically against peroxidation of membrane lipids is essential. Increased food and, at the
same time free radicals intake in the period of long daylight result in high antioxidant activity
in this period of time.
500
A
A
400
A
Trolox equivalents
A
AB
AB
AB
300
B
AB
B
B
B
B
B
B
B
200
100
VI Dec
VI Sep
VI Mar
VI Jun
V Dec
V Sep
V Mar
V Jun
IV Dec
IV Sep
IV Mar
IV Jun
III Dec
III Sep
III Mar
III Jun
II Dec
II Sep
II Mar
II Jun
I Dec
I Sep
I Mar
I Jun
0
Fig. 1 Seasonal changes in total antioxidant activity in blood plasma of pig-boar in 4 different periods of the year where I
is the early luteal phase, II is the mid luteal phase, III is the late luteal phase, IV is the early luteolysis, V is the full
luteolysis and VI is the follicular phase; the statistically significant differences were marked with different letters p≤0,001
PBZ-KBN-084/P06/2002 from 2003 to 2005
SEASONAL CHANGES IN Cu-Zn SOD CONCENTRATION IN RED-BLOOD
CELLS IN DOMESTIC PIG/WILD BOAR CROSSBREEDS (PIG-BOAR)
DURING ESTROUS CYCLE
Marek Koziorowski, Ewelina Kowal, Katarzyna Kozioł, Bartosz Jagusztyn,
Przemysław Gilun
A
A
A
A
A
A
B B
B
A
B
B
VI Dec
VI Jun
VI Mar
V Dec
V Jun
V Sep
V Mar
IV Jun
IV Sep
IV Mar
B
III Dec
III Jun
III Mar
II Dec
II Jun
II Sep
B
II Mar
I Dec
I Jun
B
B
B
C
C
IV Dec
B
III Sep
B
A
BC
VI Sep
A
I Sep
2 1 00
2 0 00
1 9 00
1 8 00
1 7 00
1 6 00
1 5 00
1 4 00
1 3 00
1 2 00
1 1 00
1 0 00
900
800
700
600
500
400
300
200
100
0
I Mar
U SOD/g Hb
Superoxide dismutase (SOD) is one of the main antioxidant enzymes in animals. Red blood
cells (RBC) Cu-ZnSOD is especially important in protecting erythrocytes from peroxide
products origniating from autoxidation hemoglobin. Seasonal changes in concentrations of
reactive oxygen species (ROS) caused by varying intensity levels of metabolism and seasonal
breeding of pig-boar gilts were the basis for determining the antioxidant state of RBC.
For the experiment we used 16 mature pig-boar gilts. Blood samples were taken every eight
hours for 30 days. The Cu-Zn SOD concentration was measured with Ransod equipment
from Randox Laboratories. Obtained results were displayed in units of SOD/gHb figure 1.
Fig.1 Seasonal changes in CU-Zn SOD concentration in red blood cells in pig-boar gilts in 4 different periods of the year
where I is the early luteal phase, II is the mid luteal phase, III is the late luteal phase, IV is the early luteolysis, V is the full
luteolysis and VI is the follicular phase; statistically significant differences were marked with different letters; p≤0,001
Achieved results show the seasonal changes in properties of antidoxidant blood. The
highest Cu-Zn SOD concentrations were found in autumn which was the best time for
creating optimal conditions for fertilization and embryo development. The role of CuZn SOD is to inactivate ROS and prevent processes which damage red blood-cells.
Research was supported by the State Committee for Scientific Research as a Solicited
Project PBZ-KBN-084/P06/2002 from 2003 to 2005
SEASONAL CHANGES IN LEPTIN CONCENTRATION DURING THE
ESTROUS CYCLE IN PERIPHERAL BLOOD PLASMA IN GILTS OF
DOMESTIC PIG AND WILD BOAR CROSSBREED
Marek Koziorowski, Katarzyna Kozioł, Agnieszka Sajdak, Bartosz Jagusztyn,
Przemysław Gilun
University of Rzeszów, Werynia near Kolbuszowa, Poland, E-mail: [email protected]
In wild mammals, depending on the time of the year, we observe changes in the reproductive
system activity levels and the amount of accumulated fatty tissue. The presence of leptin
receptors in the hypothalamus upbeats the LH and GnRH, which may point to leptin’s
significant role in the regulations of reproduction. Leptin is also known as a factor causing
decrease in appetite and increase in fatty tissue catabolism.
The aim of this research was to define leptin concentration in gilts of domestic pig and wild
boar crossbred ( a species which shows symptoms of heat and starts catabolising/burning fatty
tissue during the time of year when the daylight is becoming shorter) and domestic pig (a
poliestral species).
16 gilts of 80-100kg were used in the experiments. Blood for analysis was collected within
the period of 30 days, 3 times a day, in the periods of spring equinox (March), long daylight
(June), autumn equinox (September) and short daylight (December). Leptin concentration
was measured with the use of RIA method.
10
9
8
A
A
A
A
A
7
ng/ml
6
B
B
5
B
B
B
4
3
2
1
VI Dec
VI Jun
VI Sep
VI Mar
V Dec
V Jun
V Sep
V Mar
IV Dec
IV Jun
IV Sep
IV Mar
III Dec
III Jun
III Sep
III Mar
II Dec
II Jun
II Sep
II Mar
I Dec
I Jun
I Sep
I Mar
0
Fig. 1 Seasonal changes in leptin concentration in blood plasma of gilts of domestic pig and wild boar crossbred where I is the
early luteal phase, II is the mid luteal phase, III is the late luteal phase, IV is the early luteolysis phase, V is the full luteolysis
phase and VI is the follicular phase; statistically significant differences were marked with different letters; p≤0,001
The lowest concentration of leptin was observed in the period of the longest daylight, when
wild boars cease being sexually active and stop lactating. The highest concentration was
observed in the period of spring equinox when boars lactate and their fatty tissue burns
up/completely disappears.
These results suggested that leptin is a biological activator of reproductive processes as well
as lactopoesis, and its influence on the reproductive system is direct.
PBZ-KBN-084/P06/2002 from 2003 to 2005
SEASONAL CHANGES IN STEROID HORMONE CONCENTRATION
DURING ESTROUS CYCLE IN PERIPHERAL BLOOD IN GILTS OF
DOMESTIC PIG AND WILD BOAR CROSSBREED (PIG-BOAR)
1
Marek Koziorowski, 2Stanisława Stefańczyk-Krzymowska, 1Przemysław Gilun,
1
Anna Tabęcka- Łonczyńska, 1Katarzyna Kozioł, 1Bartosz Jagusztyn
1
Department of Animal Physiology of Reproduction, Institute of Biotechnology,
University of Rzeszow, Werynia near Kolbuszowa; 2Department of Local
Physiological Regulation, Institute of Animal Reproduction and Food Research,
Polish Academy of Science Tuwima 10, Olsztyn, Poland, E-mail: [email protected]
The aim of the research was to define sesonal changes in steroid hormone (E2, T, A4, and P4)
concentration during estrous cycle in peripheral blood in gilts of pig-boar crossbred. 16 cyclic
gilts (80-100 kg) were used for the experiments. Blood samples were collected three times a
day in: spring equinox (March), the long day light (June), autumn equinox (September), the
short day light (December). Hormone concentration was measured by RIA method.
50
A
27.5
25.0
A
22.5
40
A
A
Estradiol pg/m l
Progesterone ng/ml
20.0
30
20
17.5
15.0
A
A
A
A
12.5
10.0
B
B
B
7.5
B
B B
10
5.0
B
B
2.5
250
80
225
A
A
40
A A
A
A
A A
A
A
A
B
A
A
A
B
C
30
Androstendion pg/m l
A A
VI Jun
A
A
AB
A
175
AB
150
B
AB
125
B
B
100
75
B
20
B
B
B
C
10
VI Dec
VI Mar
VI Sep
V Jun
V Dec
V Mar
V Sep
IV Jun
IV Dec
IV Sep
IV Mar
III Jun
III Dec
III Sep
II Dec
III Mar
II Sep
I Dec
II Jun
II Mar
A
A
200
60
50
B
25
0
VI Dec
VI Jun
VI Sep
VI Mar
V Dec
V Jun
V Mar
V Sep
IV Dec
IV Jun
IV Mar
IV Sep
III Dec
III Sep
III Jun
II Dec
III Mar
II Sep
II Jun
II Mar
I Dec
I Jun
I Sep
VI Dec
VI Sep
VI Jun
V Dec
VI Mar
V Jun
V Sep
V Mar
IV Dec
IV Jun
IV Mar
IV Sep
III Jun
III Dec
III Sep
II Dec
III Mar
II Jun
II Mar
II Sep
I Jun
I Dec
I Mar
I Sep
0
I Mar
50
I Jun
I Mar
275
90
I Sep
VI Jun
100
70
Testosterone pg/m l
0.0
VI Dec
VI Sep
V Dec
VI Mar
V Mar
V Jun
V Sep
IV Dec
IV Mar
IV Jun
IV Sep
III Jun
III Dec
III Sep
II Dec
III Mar
II Mar
II Jun
II Sep
I Jun
I Dec
I Sep
I Mar
0
Fig. 1 Seasonal changes in progesterone, estradiol, testosterone and androstendione concentration in blood plasma of gilts of
domestic pig and wild boar crossbred where I is the early luteal phase, II is the mid luteal phase, III is the late luteal
phase, IV is the early luteolysis, V is the full luteolysis and VI is the follicular phase; significant differences were
marked with different letters; p≤0,001
The shortest cycle lasting 21-22 days was in December, in March it lasted 24 days, in June 28
days and in September 26 days.These results suggest that gilts of pig-boar crossbreed are
poliestral animals although they display sesonal changes in secretion of sex hormones. They
also suggests that the period from December to March is the best reproductive period for these
animals. Significantly higher level of estradiol in this time points to a high activity of
maturing follicles and, perhaps, their number. Finally, we have observed that testosterone
level is much higher in March than in December, when it decreases probably due to the
increase in the level of estradiol. The increased level of testosterone may suggest that during
this period the organism is readying itself for increased sexual activity.
PBZ-KBN-084/P06/2002 from 2003 to 2005.
BIODIVERSITY OF RED DEER POPULATIONS EXAMINED BY
AMPLICON-LENGTH POLYMORPHISM OF THE PREGNANCYASSOCIATED GLYCOPROTEIN GENES
Grzegorz Panasiewicz1, Adriana Cegiełka1, Jacek Rutkowski2 and BoŜena
Szafrańska1@*
1
Department of Animal Physiology, Faculty of Biology, Email:[email protected];
Department of Sheep Farming, Hunting and Goat Breeding, Faculty of Animal
Bioengineering, University of Warmia & Mazury in Olsztyn, 10-719 Olsztyn, Poland.
2
Numerous chorionic cDNAs of pregnancy-associated glycoprotein genes (PAG) have been
cloned mainly in domestic eutherians. In wild Artiodactyla taxons, several cDNAs of the PAG
genes were identified only in placental transcriptomes of the white-tailed deer (Odocoileus
virginianus, Cervidae family). The aim of this study was to identify length-polymorphism of the
PAG-like gene amplicons in four populations of the red deer (Rd; Cervus elaphus). Genomic
DNA (gDNA) templates were isolated from leukocytes harvested post mortem from hunted
female (f) and male (m) Rd individuals (N=106), eliminated from the various populations
located in Olsztyn region (7f/32m), Drygały region (28f/15m), or the Piska Forest: Strzałowo
(13m) and Popielno (6f/5m). The RdPAG-like gene amplicons were assembled by 40 PCR
cycles (94oC/60s, 60oC/120s and 72oC/120s). The amplification was performed with internal
sense and antisense primers specific for porcine PAG genes (pPAG) that created various
amplicons (corresponding to 5-9 exons with introns E-H of the porcine PAG2 gene structure).
Produced RdPAG-like amplicons were separated by electrophoresis, transferred on nylon
membranes and examined by cross-species Southern analysis. This heterogeneous
hybridization was performed with 32P-labelled pPAG probes produced on the basis of cDNA
of the pPAG10 gene - used also as positive control template. This autoradiography verified
the specificity of the RdPAG-like gene amplicons. Results revealed that applied PCRconditions (with use of RdgDNA templates and pPAG primers) amplified from one to ten of
the RdPAG-like amplicons (350-2800 bp), what allowed for the recognition of at least seven
distinct PAG-genotypes in examined Rd individuals. Length-polymorphism of the RdPAGlike amplicons was identified: 1) 650 bp; 2) 650 and 800 bp; 3) 1370, 1850 and 2450 bp; 4)
350, 650, 1250, 1370, 1500 and 1850 bp; 5) 350, 650, 1250, 1370, 1500, 1850, 2100, 2300
and 2800 bp; 6) 550, 650, 1000, 1200, 1500, 1850, 2100, 2300 and 2800 bp; or 7) 550, 650,
1000, 1200, 1500, 1850, 2100, 2300, 2350 and 2800 bp. Results indicated that applied PCR
and cross-species Southern hybridization was beneficial for the identification of the PAG-like
genotypes in various populations of the red deer. Optimal genetic management and genome
resource banking has the potential to decelerate the loss of small population diversity. This
method of the PAG-genotype identification can be advantageous also as genetic marker in other
members of the Cervidae family. Proposed PAG-marker should permit for genetic monitoring
of various populations and can allow for a proper conservation of wild animal biodiversity.
*Supported by PBZ-KBN-084/P06/02/3.4 and UWM528-0206.0805.
THE LIVE-DNA DIAGNOSTICS OF EARLY APOPTOTIC DEATH IN
PORCINE ADULT EAR SKIN-DERIVED FIBROBLASTS SELECTED TO
SOMATIC CELL NUCLEAR TRANSFER
Marcin Samiec, Maria Skrzyszowska
Department of Animal Reproduction Biotechnology, National Research Institute of
Animal Production, 32-083 Balice/Krakow, Poland, E-mail: [email protected]
One of the most important factors which determine the pre- and/or postimplantation
development of mammalian cloned embryos is structuro-functional quality of nuclear donor
cells. Therefore, system of early apoptosis diagnostics-mediated pre-selection would allow the
sorting of donor nuclei with high morphological and biochemical susceptibility to somatic cell
cloning. The purpose of our study was to examine the in vitro developmental potential of
porcine nuclear transfer-derived (NT) embryos reconstructed with gilt/sow ear skin-descended
fibroblast cells, which had been analyzed on apoptosis through the live-fluorescent labeling.
Frozen/thawed fibroblast cells, which had been in vitro cultured up to a total confluence state
after 2-6 passages, were used for analysis. To detect the early-apoptotic changes in the
fibroblast cells, single nuclear donor cell suspension was subjected to dyeing with live-DNA
green fluorochrome YO-PRO-1. The source of recipient cells were in vitro matured oocytes.
Maternal chromosomes were eliminated by chemically assisted microsurgical technique.
Fibroblast cell-ooplast couplets were simultaneously fused and activated. Reconstructed
embryos were in vitro cultured in NCSU-23/BSA/FBS medium for 6-7 days. The rates of
cleavage and development to morula/blastocyst stages were examined on Days 2 and 6/7,
respectively. After fluorescent analysis of adult skin fibroblast cells, it was shown that a
relatively high proportion of donor cells revealed ultrastructural proapoptotic changes. The
percentage of late-apoptotic cells with advanced morphological transformations ranges from
20 to 25%. A total of 236 enucleated oocytes were subjected to reconstruction and 135/236
(57.2%) were successfully fused with non-apoptotic nuclear donor cells. Out of 135 cultured
NT embryos, 88 (65.8%) were cleaved. The frequencies of cloned embryos, that reached the
morula and blastocyst stages, were 39/135 (28.9%) and 18/135 (13.3%), respectively. In
conclusion, a sufficient selection factor for detection of apoptosis in the cultured adult dermal
fibroblasts, which have been synchronized at G0/G1 phases of mitotic cell cycle by contact
inhibition of their migration and proliferative growth, are morphological criteria of the
classification to somatic cell cloning. Moreover, it was found that YO-PRO-1 fluorochrome
may be not able to detect the early phases of apoptosis, because only the morphologically
abnormal cells emitted the YO-PRO-1-derived fluorescence.
Project number PBZ-KBN-084/P06/2002/4.2 from 2003 to 2005 year.
ADVANTAGEOUS APPLICATION OF PORCINE PREGNANCYASSOCIATED GLYCOPROTEIN GENE FAMILY (pPAG) FOR DISCOVERY
OF THE PAG-LIKE AMPLICONS IN THE CHINCHILLA LANIGERA
GENOME
BoŜena Szafrańska 1@*, Grzegorz Panasiewicz1, Karol Bociek1, Beata Seremiak2, and
Małgorzata Sulik3
1
Department of Animal Physiology, Faculty of Biology, University of Warmia &
Mazury in Olsztyn, 10-719 Olsztyn, Poland, E-mail:[email protected];
2
Department of Animal Reproduction and 3Department of Fur-Animal Breeding,
Agricultural University of Szczecin, 71-460 Szczecin, Poland
The lack of genetic diversity increases homozygosity and inbreeding depression, thus genetic
monitoring permits for the selection of the best heterozygotic genitors. Placental PAG genes
(chorionic aspartic proteinases) have been identified by cDNA cloning in different eutherians,
including Artiodactyla (pigs), Rodentia (mouse) and other species (Perissodactyla and
Carnivora). Moreover, we identified diverse PAG-like gene amplicons with use of genomic
DNA (gDNA) templates isolated from hair roots or tail tissues in two taxons of the Rodentia
order: the European beaver and in the rat. It seems that the PAG gene family can be expected
also in other rodents with hemochorial placenta that is different than diffuse epitheliochorial
placenta of the pig. Surprisingly, the pregnancy length is similar in the Chinchilla lanigera
(111 days) and the pig (114 days). The objective of this study was to identify the PAG-like gene
family in genome of the Chinchilla lanigera (Chl). We used gDNA templates isolated from
tails of farm Chl males (n=47). The ChlPAG-like gene amplicons were produced by PCR
method used previously for other rodents. The amplicon syntheses were performed with internal
primers specific for pPAG gene family identified previously (pPAG 1-6, pPAG8 and pPAG10;
GenBank Acc. Nos.: L34360, L34361, AF315377, AF272734, AY188554, AF272735,
AY373029 and AY775784, respectively). These cDNAs were used as positive control
templates and the plasmids required for molecular probe productions. The ChlPAG-like
amplicons were electrophoresed, transferred on nylon membranes and hybridized with 32Plabelled pPAG probes (cross-species Southern) that confirmed amplicon specificity. Applied
gDNA amplification revealed from four to seven ChlPAG-like amplicons (500-1650 bp). The
profile comparisons of the ChlPAG-like gDNA amplicons indicated their lengthpolymorphism and distinct genotypes. The size of the ChlPAG-like amplicons in main
particular five genotype profiles were: 1) 615, 1050, 1400, 1650 bp; 2) 1050, 1200, 1400 and
1650 bp; 3) 500, 1050, 1400, 1600 and 1650 bp; 4) 570, 1050, 1200, 1400 and 1650 bp; or 5)
500, 615, 1050, 1200, 1400, 1600 and 1650 bp. This is the first report indicating the PAG-like
gene family in the Chinchilla lanigera genome. Presumably, careful monitoring of the ChlPAG
genotypes will allow for selection of the best genitors and will protect genetic biodiversity of
farm animals.
*Supported by PBZ-KBN-084/P06/02 and UWM528-0206.0805.
EFFECT OF SODIUM HYALURONIC ACID ADDED TO BOAR SEMEN
EXTENDER ON THE SURVIVAL TIME AND FERTILITY
Barbara Szczęśniak-Fabiańczyk1, Zdzisław Smorąg1, Waldemar Kowalski2
1
Department of Biotechnology of Animal Reproduction, Balice, 2Pig Research Station,
śerniki Wielkie, National Research Institute of Animal Production, 32-083 Balice,
There are different extenders for liquid semen we can use and the fertility is the cause for our
choice. Hyaluronic acid fulfils an important functions in fertility (protects the ova during
ovulation, assists the sperm binding to oocyte membrane). Sodium hyaluronic acid is useful in
cryopreservation, embryo transfer and in vitro fertilization. The aim of the experiment was to
determine the effect sodium hyaluronic acid supplementing one of the commercial available
extenders (EXTENDER X) on semen survival time and fertility of fresh porcine semen stored
for 7 days. The semen was divided into two groups and diluted with EXTENDER X and
EXTENDER X supplemented 0,5% solution of sodium hyaluronic acid (as 0.1% in 1.
experiment and as 1.0 % in 2. experiment concentration) and stored in 15-17ºC. The semen
motility was examined every day until it reached 30 % of normally motile sperm. The 0.1 %
addition of sodium hyaluronic acid to EXTENDER X increased sperm survival by 10.4 to
11.4 days, the 1.0% addition increased sperm survival by 14.9 to 17.2 days. Sows were
inseminated the semen which reached no less than 30% of normally motile sperm for 7 days
of storage. Sows inseminated with the EXTENDER X supplemented 0.1% of sodium
hyaluronic acid diluted semen (1. experiment) showed a increase in farrowing rate compared
with sows inseminated with EXTENDER X diluted semen (mean 72.4 % and 45.8%). They
bore more piglets, too (mean 9.4 and 9.0). Farrowing rate for sows inseminated with the
EXTENDER X supplemented 1.0% of sodium hyaluronic acid diluted semen (2. experiment)
was 51.6% and mean litter size was 9.1 of piglets. Semen diluted with EXTENDER X
survived shorter than 7 days and was not used for AI.
EFFECT OF GEOMAGNETIC DISTURBANCES CAUSED BY METAL
ELEMENTS ON SELECTED PARAMETERS OF RATS REPRODUCTION
Barbara Tombarkiewicz, Krzysztof Pawlak, Jerzy Niedziółka
Department of Animal Hygiene and Environmental Protection, Agricultural
University, Al. Mickiewicza 24/28, 30-059 Krakow, Poland, E-mail: [email protected]
For centuries, animal organisms have adapted to environmental conditions, including the
geomagnetic field (GMF), and often noticeably reacted to any field changes. In the era of
growing industrialization and "technological growth", static geomagnetic field is also
commonly disturbed by various metal constructions or interior design elements, e.g. metal
cages used in animal breeding and raising. The purpose of the study was to determine the
effect of magnetic disturbance caused by metal elements on selected parameters of
reproduction of laboratory rats. The experiment was carried out in two stages. In each stage,
the experimental material included 18 Wistar laboratory rats divided into 2 groups: one
experimental group (7♀ and 2♂) and one control group (7♀ and 2♂). All rats were
maintained in identical conditions, and the only factor distinguishing the experimental and the
control group was the presence or the lack of geomagnetic field. GMF was screened at the
dwelling place of the experimental rats (PGM vertical component value 0 nT), while the
control groups were staying far from field disturbance (vertical component value measured
with a BPM 2001 geomagnetometer amounted to ca. 35000 nT) (Mersmann, 1984).
At the first stage of the experiment after two-month adaptation to experimental conditions, the
rats were admitted to reproduction within each experimental group. Fertility, count of
newborn, count of those which survived, born litter’s mass, growth of litter mass, estimated
mass of 1 newborn and estimated growth of 1 animal were investigated, to find the influences
of GMF disturbances on rats’ reproduction. Weighing was carried out from 1 to 24 days of
life in 2-3 days intervals. The randomly chosen rats from F1 were used at the second stage of
experiment as a P1. They were divided into 2 groups like in the first stage. After reach
maturity the rats were admitted to reproduction. To find the influence of GMF on F2 were
carried out the same investigations (like in 1 stage). The results were statistically examined
with the t-Student test.
The conclusion that can be drawn from the experiment is that there are not statistically
significant differences between the experimental and control groups. On the other hand there
are some symptoms of GMF disturbances in rats’ reproduction. The can be especially noticed
in F2 . F2 from the experimental group is characterized by: - lower mass of newborn rats (5.5
g. in the experimental group and 6.16 g in control), - lower growth of 1 animal (30,75 g. in the
experimental group and 31.85g in control). Moreover the lower growth of female from the
experimental group P1 was noticed (142.57g. in the experimental group and 164.57g in
control). The rats from this group were under the influence of hypomagnetic field all their
lives- from conception till the end of experiment. In comparison the growth of female at the
first stage (P) were 81.8g and 80.7g respectively.
Studies supported by grants WHiBZ/2003 and BW-2230/ZHZiŚH
APOPTOSIS AND DEVELOPMENTAL COMPETENCE OF BOVINE
EMBRYOS DERIVED FROM OOCYTES MATURED IN TCM 199 MEDIUM
SUPPLEMENTED WITH FBS, fafBSA OR PVP40
Ewelina Warzych, Karolina Błajecka, Martyna Łabusińska, Dorota Lechniak
Department of Genetics and Animal Breeding, Agricultural University of Poznan,
60-637 Poznan, Poland, E-mail: [email protected]
It is well known that in vitro produced (IVP) embryos display lower developmental
competence when compared to their in vivo derived counterparts [Knijn et al. 2003]. The
composition of culture media significantly contributes to this phenomenon. During the past
decade a substantial effort has been made in order to improve embryo culture conditions,
whereas no important changes have been introduced into oocyte maturation (IVM) [Sutton et
al. 2003].
The objective of the present study was to investigate whether various protein (FBS, fafBSA)
or synthetic macromolecule (PVP40) supplements to oocyte maturation media affect the
cleavage rate, blastocyst yield, total number of blastomeres and the incidence of apoptosis in
resulting embryos. Cumulus-oocyte-complexes were matured in TCM199 optionally
supplemented with FBS, fafBSA or PVP40 for 24 hours in standard conditions for cattle
oocytes. After maturation oocytes were inseminated and cultured in vitro for 9 days as
previously described [Peippo et al. 2002]. Cleavage rate was assessed on day 2 post
insemination (pi) whereas blastocyst yield on day 9 pi. Detection of apoptotic cells (TUNEL)
and analysis of blastomeres number was performed in day 8 embryos.
In total, approx. 150 blastocysts were analyzed. The general influence of protein
supplementation on apoptotic index in blastocysts was found to be significant (p<0.05);
PVP40 addition was correlated with significantly higher level of apoptotic cells. No
significant differences were detected with regard to the total blastomere number in blastocysts
(FBS – 135.1, fafBSA – 124.8, PVP40 – 123.8). Also no significant variation in cleavage rate
among zygotes produced from oocytes matured in the three maturation media was noticed:
FBS – 70.5%, faf BSA – 72.1%, PVP40 – 71.1%. However, blastocyst rate on Day-9 of
culture was significantly lower (P<0.01) in TCM199+PVP40 (16%) in comparison to 22.4%
in TCM199+FBS and 22.1% in TCM199+BSA.
In conclusion, different protein supplementation during in vitro maturation of oocytes affects
further embryonic development, although these differences become evident at more advanced
developmental stages. Our study shows that embryos obtained from oocytes matured in
medium supplemented with PVP40 display lower developmental competence and elevated
rate of apoptotic cells, which may indicate reduced quality.
Research was supported by the State Committee
Solicited Project no PBZ-KBN-084 from 2003 to 2005 year.
for
Scientific
Research
as
THE PARTHENOGENETIC DEVELOPMENT OF RABBIT OOCYTES AS
AN EFFECT OF ELECTRIC FIELD
Agnieszka Wierzchoś
Animal Production, 32-083 Balice/Krakow, Poland,
The purpose of this study was to test the influence of electric field strength and duration as
well as the number of pulses applayed at a wide range of values and in several combinations
of the fusion, and parthenogenetic developmental rate of rabbit oocytes. There were analyzed
about 430 oocytes which developed to a blastocyst stage. Oocytes were exposed to electric
field of different parameters according to variants: variant I: 1 impulse 30 usek.
(1,0 kV/cm,1,5 kV/cm, 2,0 kV/cm, 2,0 kV/cm), variant II: 1 impulse 60 uesk., variant III: 3
impulses 30 usek., variant IV: 3 impulses 60 usek. We used also the extreme variant V: 3
impulses 60 usek.(3,0 kV/cm; 3,5 kV/cm; 4,0 kV/cm; 4,5 kV/cm).
Sexually matured female rabbits were superovulated with 100 j.m. PMSG. 72 h later they
received one injection of 100 j. m. HCG. 24 h after receiving HCG unfertilized ova were
recovered from oviducts of the does by flushing in phosphate-buffered saline. Oocytes were
placed separately between the electrodes and they were treated by electric field with
different parameters (number of pulses, strength and duration) Following electrical treatment
embryos were cultured in B2 medium supplemented with 10% of FCS to the blastocyst stage.
There were also analyzed oocytes which degenerate or which fragmented.
The analysis show that there wasn’t essential differences between different variants of our
work. Analysis of all groups of variants show slight but progressive degeneration of
oocytes. The positive correlation between number and duration of electric pulses and the
percentage of degenerated and fragmented oocytes was observed.
MORPHOLOGY AND ULTRASTRUCTURE OF RACCOON DOGS
(NYCTEREUTES PROCYONOIDES, GRAY) SPERMATOZOON
Katarzyna Zagórska-ŚwieŜy2, Maria Nowogrodzka-Zagórska1, Olga Szeleszczuk2,
Piotr Niedbała2, Janusz Bigaj3, Adam Miodoński1
1
Scanning Electron Microscope (SEM) Laboratory of Laryngological Clinic of
Collegium Medicum, Jagiellonian University; 2Department of Animal Reproduction
and Anatomy, 3Electron Microscopy Lab. of Agriculture University in Cracow, Al.
Mickiewicza 24/28 30-059 Cracow, Poland, E-mail: [email protected].
Cryoconservation process of raccoon dog semen caused less or more drastic decrease of
spermatozoa motility after thawing. Freezing and thawing of semen probably was responsible
for damaging of spermatozoon structure even after addition of cryoprotector.
We undertook investigations which aim was characterization of regular morphological
structure and ultrastructure of raccoon dogs spermatozoon in a fresh semen.
Manually collected samples of semen from ten 1-year old breeding raccoon dogs were used
for these analyses. Morphometric investigations of spermatozoa were provided on smears of
fresh semen stained with eosin and nigrosin and light microscope with micrometric
spectacles. During analyses, length and width of spermatozoa head were measured as well as
the length of tail. Photographic documentation indicate shape and it’s area (SEM) as well as
cross-section and internal structures of spermatozoa (TEM). Recorded SEM and TEM results
were obtained by a common methods used by microscope laboratories.
Raccoon dogs spermatozoa remind dogs’ spermatozoa. The surface of spermatozoa is smooth,
only midpiece because of mitochondria has different characteristic structure. The size of
spermatozoon head is: width – 4,9µm, length – 6,15 µm. The length of vibraculum tail is 57,9
µm. Approximate length of midpiece is 8 – 9 µm and width is: 0,6 – 0,8 µm.
This paper is supported by a grant No 2 PO6Z 046 26 from KBN – State Committee for
Scientific Research.
Session XI
Cancers of reproductive organs
Oral presentation
THE SEX STEROID RECEPTOR PHENOTYPE OF FEMALE BREAST
CANCER EXPLANTS INFLUENCE THE ACTION OF hGH AND IGF-I ON
THEIR PROLIFERATION AND APOPTOSIS
Tomasz Milewicz1, Ewa Ł. Gregoraszczuk2, Katarzyna Augustowska2, Ewa Stępień3,
Janusz Ryś4, Józef Krzysiek1
1
Dept. of Gynecological Endocrinology; Collegium Medicum; 2Lab of Reproductive
Toxicology, Institute of Zoology Jagiellonian University, Cracow; 3Lab of Molecular
Biology; John Paul II Hospital, Cracow, 4Dept of Pathology, Cracow Branch of
National Oncology Center; Cracow, Poland, E-mail:[email protected]
Aim:
The evaluation of sex steroid receptor phenotype influence on hGH and IGF-I action on
proliferation and apoptosis in female breast cancer explants.
Material & methods
The study was done on female breast cancer explants. The sex steroids receptors phenotype
was evaluated by means of immunohistochemistry. The explants were divided into
hormonally responsive [ER(+)PR(+)] and hormonally nonresponsive [ER(-)PR(-)]. Incubation
time with hGH and IGF-I was 48 hours. The explants were frozen until the moment of mRNA
expression evaluation. The proliferation was evaluated by means of Alamar Blue test.
Results:
Apoptosis was decreased by hGH and IGF-I in hormone responsive explants. Proliferation
was increased only by hGH and not affected by IGF-I in the same type of explants. Apoptosis
was not affected by hGH and IGF-I in hormone non responsive explants. In the same type of
explants IGF-I increased and hGH decreased the proliferation
Conclusion:
The action of hGH and IGF-I on proliferation and apoptosis differs according to the sex
steroid receptor phenotype of breast cancer explants.
LACK OF SYNERGY BETWEEN ESTROGEN AND PROGESTERONE ON
LOCAL IGF-I, IGFBP-2 AND IGFBP-3 SECRETION BY BOTH HORMONE
DEPENDENT AND HORMONE INDEPENDENT BREAST CANCER
EXPLANTS IN VITRO. INFLUENCE OF TAMOXIFENE AND
MIFEPRISTONE
Tomasz Milewicz1, Ewa Ł. Gregoraszczuk2, Krystyna Sztefko3, Katarzyna
Augustowska2, Józef Krzysiek1, Janusz Ryś4
1
Dept. of Gynecological Endocrinology, Collegium Medicum; 2Lab of Reproductive
Toxicology, Institute of Zoology, Jagiellonian University, Cracow, Poland; 3Lab of
Clinical Biochemistry; University Children Hospital, Cracow, Poland; 4Dept of
Pathology, Cracow Branch of National Oncology Center; Cracow, Poland,
The aim of the present study was to investigate direct effects of estrogen, progesterone vs.
estrogen+progestin on local IGF-I, IGFBP-3 and IGFBP-2 secretion. Explants obtained from
ER+/ PR+ and ER-/PR- tumors were incubated with E2, P4 or both. Tamoxifen was added to
E2; RU 486 to P4 both to E2 +P4 supplemented cultures. In hormone dependent and hormone
independent tissue treatment with estrogen+ progesterone, increased in the same manner as
E2 or P4 alone, increased IGF-I and IGFBP-2 secretion with concomitant decrease in IGFBP3. Tamoxifen decreased E2 and E2+P4 stimulated IGF-I secretion by hormone dependent
breast cancer explants. RU 486 decreased P4 and E2+P4 stimulated IGF-I secretion with
parallel stimulation of IGFBP-3 secretion by ER+/PR+ explants. In conclusion, presented data
suggest that there is no synergistic action of E2 and P4 on IGF/IGFBPs ratio and additionally
suggest a protective action of antiestrogen and antiprogestagen against excessive IGF-I
secretion.
ISOLATED TUMOR CELLS AND MICROMETASTASIS IN SENTINEL
LYMPH NODES IN SQUAMOUS UTERINE CERVICAL CANCER.
Kazimierz Pityński1, Marcin Opławski1, Krzysztof Okoń2, Antoni Basta1
1
Department of Gynecology and Obstetrics; 2Department of Pathomorphology,
Collegium Medicum, Jagiellonian University, Krakow, Poland
Presence of isolated tumor cells and micrometastasis in blood, bone marrow and lymph nodes
is one of the hottest problems in oncology. Optimal detection measures of neoplastic cells
outside tumor and their prognostic and therapeutic meanings are mainly discussed. The aim of
the study was the determination of occurrence of isolated tumor cells and micrometastasis in
sentinel lymph nodes in squamous uterine cervical cancer and connections of their presence
with typical prognostic factors. Study group consisted of 30 patients with squamous uterine
cervical cancer FIGO stage of Ia2-IIa, treated surgically in Department of Gynecology and
Obstetrics of Jagiellonian University. The presence of isolated tumor cells and
micrometastasis in sentinel nodes was detected immunohistochemically and by means of RT PCR, using cytoceratin 19 as tumor cell marker. Isolated tumor cells and micrometastasis in
negative sentinel node were present in immunhistochemical examination in 6(20%) and in
RT- PCR in 8(26,67%) of cases. Woman with micrometastasis had in standard
histopathologic investigation unfavorable prognostic factors. Conclusions. RT - PCR is
tenderer in relation to immunohistochemistry in detection of isolated tumor cells and
metastasis in lymph nodes. The occurrence of isolated tumor cells and micrometastasis in
sentinel lymph nodes joins with unfavorable prognostic factors of squamous uterine cervical
cancer
THE EXPRESSION OF VASCULAR GROWTH FACTORS A, C AND D
(VEGF - A, VEGF - C, VEGF - D) IN SQUAMOUS UTERINE CERVICAL
CANCER STAGE Ia2-IIa
1
Department of Gynecology and Obstetrics; 2Department of Pathomorphology,
Essential role of angiogenesis in progression of tumors was settled in many clinical and
experimental works. The investigations over lymphatic system in solid tumors were by long
time put aside due to the unacquaintance with factors regulative growth of lymphatics and
specific markers of these vessels. The end of XX brought the discovery the growth factors,
VEGF-C and D as well as the markers enabling the distinction in specimens of lymphatic
from blood vessels. Except VEGF C and D the importance for lymphangiogenesis is also
attributed to VEGF-A, which was connected first of all with angiogenesis. The aim of the
study was assessment whether significant differences in expression of VEGF-A, C and D in
squamous uterine cervical cancer and unchanged neoplastically cervix exist. Material and
methods. Study group consisted of 35 patients with squamous uterine cervical cancer FIGO
stage of Ia2-IIa, treated surgically in Department of Gynecology and Obstetrics of Jagellonian
University. The control group made up 30 women matched in relation to age, weigh and high
and without neoplastic disease. Expression of VEGF-A, C and D was marked
immunhistochemically in both groups and statistically analyzed. Results. The difference
between both groups analyzed was statistically significant. Conclusions. In squamous uterine
cervical cancer comes to change in expression of VEGF- A, C and D in comparison to
unchanged neoplastically cervix.
THE INVESTIGATION OVER LYMPHANGIOGENESIS IN SQUAMOUS
UTERINE CERVICAL CANCER.
1
Department of Gynecology and Obstetrics; 2 Department of Pathomorphology,
The lymphatic system is one of the main roads of spreading of squamous tumors. Recognition
of lymphatic vessels growth factors as well as their specific markers caused in last decade a
renaissance in investigation of that system in solid tumors. In comparison with blood vascular
system the mechanisms ruling lymphangiogenesis and metastasis within lymphatic system are
poorly known. The aim of the study was the determination whether lymphangiogenesis in
squamous uterine cervical cancer exists and if so the connection of it with different
histological features of the tumor. Material and methods. Study group consisted of 35 patients
with squamous uterine cervical cancer FIGO stage of Ia2-IIa, treated surgically in Department
of Gynecology and Obstetrics of Jagellonian University. The control group made up 30
women matched in relation to age, weigh and high and without neoplastic disease. The
number of LYVE-1 and Prox1 positive vessels within tumor, stroma-tumor border and stroma
were compared with the number of such vessels in the stroma of cervix-unchanged
neoplastically. Additionally the correlation between the numbers of lymphatic vessels within
tumor and its stroma and lymph node metastases, presence of inflammation, tumor grade and
necrosis was analyzed. Results. The presence of lymphatic vessels within the tumor and
significant differences between numbers of lymphatic vessels in tumor and its stroma and in
neoplastically unchanged cervix were ascertained. No dependence between the numbers of
lymphatic vessels and histological features of the tumor analyzed was found. Conclusion. In
squamous uterine cervical cancer comes to lymphangiogenesis within tumor and its stroma.
Poster presentation
(no abstracts were received)
Session XII
Reproduction of lower vertebrates
Oral presentation
SPERMATOZOA MOTILITY AND ADENYLATE ENERGY CHARGE OF
TWO TELEOST FISH (Cyprinus carpio AND Clarias gariepinus)
Anna Biegniewska1, Julian Świerczyński2, Frans Ollevier3, Edward F. Skorkowski1
1
Gdańsk University Biological Station, Laboratory of Comparative Biochemistry, 80680 Gdańsk-Sobieszewo, Poland, E-mail: [email protected],
2
Department of Biochemistry, Medical University of Gdańsk, Poland, 3Laboratory of
Aquatic Ecology, Katholieke Universiteit Leuven, Leuven, Belgium
The duration of motility of fish spermatozoa in the natural environment is limited to short
period from seconds to several minutes, but varies greatly between fish species. In
spermatozoa of externally fertilising fish sperm motility is one of the most important viability
parameters and is the major energy utilising process. It is well known from earlier studies that
when ATP decreased also decreased salmonid spermatozoa movement and that
phosphocreatine in rainbow trout spermatozoa can stimulate sperm motility. Some studies
suggest that the characteristics of progressive forward motility of the spermatozoa was related
to their fertilising capacity and the sperm motility was dependent on ATP content and
mitochondrial function. ATP provides the energy for spermatozoa movement, and the energy
is used by the dynein ATP-ase that is localised with the flagellar motile apparatus. It has been
shown recently that African catfish spermatozoa possess low level of ATP and low adenylate
energy charge as compared to carp spermatozoa. Fish spermatozoa motility and duration of
movement were measured during experiment by computer assisted sperm analysis (CASA).
Sperm movement decreased from the start of recording in both species. In catfish, however,
little movement was visible by 60 s postactivation at 0 hour or even earlier after exposure for
24 hours. The intensity and the duration of the movement were much more higher in carp than
in catfish spermatozoa. After 24 hours of incubation ATP concentration in catfish
spermatozoa dropped about 30%, AMP concentration growing about 7 times and finally
adenylate energy charge ratio follow down about 22%. In the same condition adenylate
energy charge ratio for carp spermatozoa is stable and kept value 0.96 for prolonged
incubation. Our results suggest that a relatively high rate of ATP catabolism contributes to the
low ATP concentration and low adenylate energy charge observed in catfish spermatozoa in
vitro.
INFLUENCE OF MELATONIN ON THE RELEASE OF DOPAMINE FROM
HYPOTHALAMIC AMINERGIC NUCLEI IN IMMATURE, MATURING
AND MATURE CARP FEMALE (Cyprinus carpio L.)
Ewa Drąg-Kozak, Ewa Łuszczek-Trojnar, Włodzimierz Popek
Department of Ichthyobiology and Fisheries, University of Agriculture, ul.
Spiczakowa 6, 30-199 Krakow, Poland, E-mail: [email protected]
Dopamine, a hormone produced and released in hypothalamic aminergic nuclei NRL and
NRP, influence on gonadotrophs of the pituitary gland, inhibiting the release of LH.
Dopamine antagonists are used in a stimulation of artificial spawning of carp to stop the
inhibiting action of dopamine. Looking for other ways to exclude dopamine action, we
focused our attention on melatonin – a hormone produced in the pineal gland. Research
carried out on mammals and birds indicate that melatonin is able to inhibit dopamine release
in retina and in the hypothalamus. The aim of our work was to indicate if melatonin influence
dopamine releasing in the hypothalamus of immature, maturing and mature carp female.
The experiment was carried out in carp spawning period in June. Twenty immature 2-years
old, 20 maturing 3-years old, and 20 mature 5-years old carp females were used. Ten fish of
each age group were made intraventricular (into the third brain ventricle) injections of
melatonin (1ug/1ul/1 kg of body weight). The rest of fish (control) were made similar
injections of salt solution. All operational treatments were made on fish under anesthesia.
Fifteen minutes after the injections all fish were decapitated. The fragments of hypothalami,
including NRL and NRP nuclei were weighted and radioimmunoassay (RIA) was used to
measure the concentration of dopamine. The mean concentration of dopamine in the
hypothalamus of the immature fish, which were made injections of melatonin 0.738 (±0.04)
ng/g of tissue, was significantly lower (p<0.01) than in control immature females 1.217
(±0.03) ng/g. The mean concentration of dopamine in the hypothalamus of the maturing fish
injected with melatonin equaled 1.041 (±0.04) ng/g, in control 1.407 (±0.04) ng/g. The mean
concentration of dopamine in the hypothalamus of the mature females injected with
melatonin 0.515 (±0.04) ng/g, was significantly higher (p<0.01) than in control fish 0.367
(±0.02) ng/g.
The results indicate that melatonin stimulates dopamine releasing from hypothalamic
aminergic nuclei in immature carp females, indirectly inhibiting processes of sexual
development. In mature carp female melatonin inhibits dopamine releasing, indirectly
stimulating reproduction processes during the spawning season.
FERTILIZING CAPACITY OF CRYOPRESERVED SPERM OF SIBERIAN
STURGEON AND STERLET AFTER SHORT-TIME EXPOSURE OF MILT
TO MERCURY AND CADMIUM
Jan Glogowski1, Grzegorz Dietrich1, Wiesław Demianowicz1, Radosław Kowalski1,
Roman Kujawa2, Adam Rzemieniecki3, Mariola Wojtczak1, Mariola Kotłowska1,
Eugeniusz Bogdan4, Andrzej Ciereszko1
1
10-747 Olsztyn, E-mail: [email protected];
2
Chair of Lake and River Fisheries, University of Warmia and Mazury, 10-719
Olsztyn; 3Department of General Zoology, Szczecin University, 71-412 Szczecin,
4
Fish Breeding Farm “Ryba”, Oleśnica, 64-800 ChodzieŜ, Poland.
Heavy metals are known to be cumulated in fish tissues, reaching concentrations of up to
20,000 fold higher than surrounding water environment. Moreover, heavy metals affect both
quality and quantity of the gametes as well as the endocrine system, disrupting the
gametogenesis.
The aim of the present work was examination of short-term exposure of Siberian sturgeon and
sterlet milt to mercury and cadmium on quality of cryopreserved spermatozoa.
Milt samples of Siberian sturgeon (n=4) and sterlet (n=5) were exposed (4h) to cadmium and
mercury ions at concentrations of 0; 1; 10 i 100 mg/l. After exposure samples were
cryopreserved using freezing solution containing Tris-saccharose-KCl medium (30 mM Tris,
23.4 mM saccharose, 0.25 mM KCl, pH 8.0) with 10% methanol. Samples, sucked into 250 µl
straws, were firstly frozen 4 cm above the surface of liquid nitrogen. After 3 min of cooling
the straws were transferred into liquid nitrogen and stored until fertilization. Just before
fertilization, straws were thawed in water bath (+400C) for 7s and adequate volume of diluted
milt (50,000 and 200,000 spermatozoa/egg for sterlet and Siberian sturgeon, respectively) was
added to 100 eggs, diluted with activation solution and left for 3 minutes.
Next, samples were washed with hatchery water, eggs were unstuck using milk and tanine
solution and finally were spread into incubation apparatus. In order to determine the
cryopreservation success, a hatching rate was determined after 7 days of incubation at +150C.
The percentage of hatched sterlet embryos in non-treated group (48.2±9.4%; mean±SD) was
not different from samples exposed to 1 mg/l Hg2+ or 1 and 10 mg/l Cd2+ (42.1±9.0%;
46.2±8.6% and 35.8±11.8%, respectively). Higher concentrations of these ions (10 and 100
mg/l Hg2+ and 100 mg/l Cd2+) caused significant alteration in hatching rates (3.8%, 3.3% and
2.3%; respectively). The percentage of hatched Siberian sturgeon embryos in non-treated
group (76.7±5,2%) was not different from samples exposed to 1 mg/l Hg2+ or 1, 10 and 100
mg/l Cd2+ (77.8±11.4%; 81.2±3.8%; 81.4±6.3% and 62.8±38.2%, respectively). Higher
concentrations of mercury ions (10 and 100 mg/l Hg2+) caused significant alteration in
hatching rates (8.1±10.2% and 0.4±0.9%; respectively). These results suggest that
spermatozoa of sturgeon fishes are relatively resistant to negative impact of heavy metals,
especially cadmium.
The project is supported by State Committee for Scientific Research (KBN) as a Solicited
Project , PBZ-KBN-084/P06/2002/5.8 in years 2003-2005.
THE EFFECTS OF TESTOSTERONE ON LH SECRETION IN RESPONSE
TO NALTREXONE (OPIOID RECEPTOR ANTAGONIST) IN COMMON
CARP (CYPRINUS CARPIO L.)
Mirosława Sokołowska-Mikołajczyk, Magdalena Socha. Piotr Epler
Department of Ichthyobiology and Fisheries, University of Agriculture, ul. Prof. T.
Spiczakowa 6, 30-199 Krakow-Mydlniki, Poland, E-mail: [email protected]
Endogenous opioid peptides affect the hypothalamo-pituitary-gonadal axis by the interaction
with hypothalamic secretion of the factors involved in the control of gonadotropin secretion or
by direct action at the level of the pituitary. Numerous data have shown that in mammals
opioids generally suppress LH and FSH secretion. In fish the influence of opioid peptides and
opiates on gonadotropin secretion is not that well established. There are however some data
showing that they affect in vivo LH release in goldfish and in common carp, both in vivo and
in vitro. Opioid peptides and opioids may modulate the release of the stimulatory (GnRH) and
inhibitory (DA) hypothalamic factors that control gonadotropin release. In the same time
opioids may act at the level of the pituitary by changing the sensitivity of the gonadotrophs
(number of the receptors?) to these main factors controlling LH secretion in fish. It was
shown that in common carp the effects of opioids on LH level (stimulation or inhibition of LH
secretion) depend on the sex of fish as well as the stage of their gonadal maturity, what
suggest that the feedback of gonadal steroids on LH release could be mediated by the
endogenous opioids.
The aim of the presented experiment was to examine the effect of testosterone and/or
naltrexone (opioid receptor antagonist) on GnRH analogue stimulated LH release in male carp
at the time of early gonad recrudescence (December).
Sexually mature male carp (Cyprinus carpio L.) were injected intraperitoneally with three
doses of testosterone (T), 5 mg per kg body weight (b.wt) per day. Control groups were
treated with a physiological solution. Twenty-four hours after the last T injection control and
experimental fish received 5 mg per kg b. wt of naltrexone (NALT) (time 0). Six hours later
all the fish were treated with salmon GnRH analogue (10 µg per kg b. wt). Blood samples
were taken from fish 24 hours after the last T injection (time 0) and then 6, 12 and 24 hours
later. LH levels in the plasma were determined by the specific ELISA method.
The statistical analysis of the results (non-parametric Man-Whitney test) showed (6 hours
after NALT administration) the difference in LH levels between the fish injected with NALT
alone and those which received T + NALT. Six hours later (six hours after sGnRH-A
injection) this difference was still observed, as well as the one between control and the
T+NALT injected group.
The results show that the injections of testosterone to recrudescing males change the
spontaneous as well as sGnRH stimulated LH secretion in response to naltrexone
administration. The results confirm our earlier data suggesting the involvement of sex steroids
in the modulatory role of the opioid peptides in LH secretion in fish.
TRANSFERRIN AND ANTIPROTEASES - MAJOR PROTEINS OF
COMMON CARP SEMINAL PLASMA
Mariola Wojtczak, Grzegorz J. Dietrich, Andrzej Ciereszko
Department of Semen Biology, Institute of Animal Reproduction and Food Research,
Polish Academy of Sciences, 10-747 Olsztyn, Poland, E-mail: [email protected]
Proteins are the main organic components of seminal plasma in teleost fishes, occurring in
very low concentrations of approximately 1-3 mg ml-l. Knowledge of fish seminal plasma
proteins is essential to better understand the physiology of the male reproductive system in
fish. During preliminary experiments using PAGE we identified three major proteins in
common carp seminal plasma, each occurring as two closely migrating bands.
The aim of the present work was to identify these major proteins in common carp Cyprinus
carpio L. seminal plasma. The two main fractions of proteins were observed after ion
exchange chromatography (IEC). Fraction I was predominant and contained the main seminal
plasma protein bands recognised as transferrin, slightly contaminated by protein of low
migration rates. Fraction II contained four protein bands identified as serine proteinase
inhibitors using zymogen techniques.
In order to purify transferrin from fraction I, a two-step procedure was used: hydrophobic
interaction chromatography followed by IEC. Two isoforms of 71 and 69 kDa transferrin
were isolated. N-terminal Edman sequencing revealed N-terminal blockage of two isoforms
of transferrin. Partial enzymatic cleavage of 71 kDa transferrin produced four peptide
fragments of molecular weights 60, 35, 24 and 21 kDa. However, partial proteolysis of 69
kDa transferrin produced only three peptides of molecular weights 35, 24 and 21 kDa. The
fragments of molecular weight 60 kDa had N-terminal blockage. Sequencing the 35 and 21
kDa fragments obtained after cleavage of 71 kDa transferrin and the 35 kDa fragment from 69
kDa transferrin produced 12 amino acids QDGKIEXXT ISHAEQQK, 24 amino acids
AVGDESKCKASSEEIYYGYDGAFRC
and
25
amino
acids
QDGKIEXXTISHAEQQXXDNLQIP, respectively. All obtained sequences showed high
homology with the sequence of common carp transferrin (residues 334-357). Moreover, the
sequence obtained from 21 kDa fragment from 69 kDa transferrin included the sequences –
YYGY- and –GAFR- (512-516 and 518-520 in common carp) which is present in almost all
presently identified transferrins of phylogenetically different animal groups and could
therefore be considered a conserved domain.
To our knowledge this is the first report to indicate the presence of transferrin in carp seminal
plasma and prove that transferrin is the major protein in this body fluid. Both transferrin and
serine proteinase inhibitors are major proteins of common carp seminal plasma and are
recognized as components of non-specific humoral defence mechanisms. This suggests that
transferrin and serine proteinase inhibitors are important parts of mechanisms for the
protection of spermatozoa.
Poster presentation
DIFFERENTIAL EFFECTS OF INHIBIN ON LH SECRETION IN MALE
AND FEMALE COMMON CARP: AN IN VITRO STUDY
Jarosław Chyb, Magdalena Socha, Mirosława Sokołowska-Mikołajczyk, Tomasz
Mikołajczyk, Piotr Epler
Department Ichthyobiology and Fisheries, University of Agriculture, ul. Prof.
Spiczakowa 6 30-199 Krakow, Poland, E-mail: [email protected]
Inhibins, which are proteins belonging to the TGFβ family, are found in the numerous tissues
in vertebrates, including gonads and the pituitary. They play an important role in the
regulation of synthesis and secretion of gonadotropins in vertebrates. In mammals these
factors influence mainly the synthesis and secretion of FSH, while in fish also luteinizing
hormone LH. The aim of this study was to evaluate the influence of recombinant human
inhibin on basal as well as sGnRH-stimulated secretion of LH from dispersed pituitary cells
of male and female common carp (Cyprinus carpio) during wintering (December) and during
spawning period (May).
For each experiment 6 to 8 male or female carps were used. Pituitaries were dispersed with
collagenase and then the obtained cells were placed onto microplates. After 60-hour period of
preincubation the dispersed cells were incubated with recombinant human inhibin A (NIBSC,
UK) at doses of 10 and 50 ng/ml and/or three different doses (10-9, 10-8 and 10-7 M) of
synthetic native sGnRH (Sigma-Aldrich, USA) for 24 hours. After incubation, media were
subjected to the analysis of LH levels using a specific ELISA method. LH levels were
expressed as mean + SEM. For each group n=6. Statistical analysis was performed using oneway ANOVA followed by Tukey’s posthoc-test.
In females during wintering recombinant human inhibin A at a dose of 50 ng/ml significantly
increased basal LH secretion Moreover, both doses of inhibin significantly elevated sGnRHstimulated release of this hormone. During preovulatory period inhibin at did not modify
basal LH secretion but inhibited LH secretion in response to 10-9M sGnRH. The secretion of
LH in male carp during wintering increased after incubation with inhibin at a dose of 50
ng/ml. At this stage inhibin increased also sGnRH-stimulated LH release. On the contrary,
inhibin failed to modify basal as well as sGnRH-stimulated LH secretion in males during the
spawning period.
The above results as well as the differences between inhibin effects on LH release in
wintering and prespawning period in both sexes indicate that inhibin influence on LH release
depends on the sex of fish and on the stage of gonad development. It suggests that probably
sexual steroids may be involved in this process.
FERTILIZING CAPACITY OF CRYOPRESERVED SPERM OF RAINBOW
TROUT AFTER SHORT-TIME EXPOSURE OF MILT TO MERCURY AND
CADMIUM
Grzegorz Dietrich1, Wiesław Demianowicz1, Radosław Kowalski1, Mariola
Wojtczak1, Stefan Dobosz2, Andrzej Ciereszko1, Jan Glogowski1
1
10-747 Olsztyn, E-mail: [email protected]
2
Department of Salmonid Research, Inland Fisheries Institute, Rutki, 83-330 śukowo,
Poland
Mercury and cadmium are known to affect sexual maturation of fish as well as
gametogenesis, spawning behavior, fertilization success and development of the offspring.
Furthermore, fishes may accumulate in tissues up to 20,000 fold more mercury or cadmium
ions comparing to concentrations of these heavy metals in surrounding water.
The aim of the present work was examination of short-term exposure rainbow trout milt to
mercury and cadmium on quality of cryopreserved spermatozoa.
Milt samples of rainbow trout sperm (n=5) were short-time (4h) exposed to cadmium and
mercury ions at concentrations of 0; 1; 10 and 100 mg/l. After exposure samples were
cryopreserved using freezing solution containing 0.6 M saccharose with 10% DMSO. Diluted
(1:3; milt- extender) samples were dropped (c. 0.05 ml pellets) into indents made in dry ice.
After 5 min, frozen pellets were placed in liquid nitrogen. Next, frozen pellets were thawed in
25 ml of activating solution (+25°C) and after 7s adequate volume of sperm suspension
(2,000,000 spermatozoa/egg) was added to 200 eggs (in replicates for each experimental
sample) and left for 3 minutes.
Consequently, samples were washed with hatchery water and washed eggs were incubated in
California-style trays. In order to determine the quality of the cryopreserved spermatozoa, a
hatching rate was determined 300 degree-days post fertilization.
The percentage of hatched rainbow trout embryos in non-treated group (57.8±6.9%;
mean±SD) was not different from samples exposed to 1 mg/l Hg2+ and 1 Cd2+ (47.9±17.0%
and 53.4±9.3%, respectively). Higher concentrations of these ions (10 and 100 mg/l Hg2+ and
10 and 100 mg/l Cd2+) caused significant decrease in hatching rates (6.0%, 0.04% and 24.4%,
0.1%; respectively). These results suggest that spermatozoa of rainbow trout are relatively
resistant to negative impact of heavy metals.
The project is supported by State Committee for Scientific Research (KBN) as a Solicited
Project , PBZ-KBN-084/P06/2002/5.8 in years 2003-2005.
OVARIAN PUBERTY OF THE NASE CHONDROSTOMA NASUS (L.)
(PISCES, CYPRINDIAE) IN THE SPRING – AUTUMN SEASON
1
Bartosz Jagusztyn, 2Alicja Boroń, 2Dorota Juchno, 1Marek Koziorowski
1
Department of Physiology and Animal Reproduction, Institute of Biotechnology of
Rzeszów University, 36-100 Kolbuszowa, E-mail: [email protected]
2
Department of Zoology, Faculty of Biology of the Warmian-Mazurian University, 10957 Olsztyn – Kortowo, Poland.
The nase is a rheophile fish living in submontane rivers. The spawning season of the species
takes place in early spring, soon after snowfall waters have flown. Over the past decades we
have observed a systematical reduction in their population in Poland. The aim of the research
was trace the seasonal and ontogenetic changeability of stages in ovarian puberty of the nase
within the spring-autumn period.
Females in full puberty, five per sample, were caught from the Wisłok River, at
month intervals, from April to October 2003. The temperature was measured at the place of
taking the samples. Ovaries were preserved in buffered formalin, while the histological
section were dyed with hematoxylin and eosin. The age of the females was determined on the
basis of annual scale growth. The number of particular growth stage in oocytes was
established on histological specimen in five different fields of vision. The puberty stages of
gonads were accepted by Sakun and Buckaja (1968), while the growth stages of oocytes,
marked with letters B to E, by Mejen (1927) and Pimpicka (1989).
The spawning season of the nase took place in the Wisłok River in April, at temperature of 910 ºC, and it lasted 2-3 days. The females were at the age of 9-11 (on average, 10 years old).
The process of vitellogenesis (accumulating yolk) began in July (gonad puberty – stage 3),
during the period of the best possible food access, and probably finished in October (stage 4),
before the winter lack of food. In April the ovaries achieved the 5th stage of puberty, which
meant the females’ readiness to lay eggs, while as early as in May they were in stage 6
(postspawning), where previtellogenic oocytes B (58,2 %) and empty follicular shields were
primarily observed.
The presence of oocytes B ranged from 10,4 % in August to 58,2 % in May, while that of
oocytes C from 5,2 % in April to 60,3 % in June. Oocytes in stage D ranged from 1,9 % in
April to 54,2 % of their total number in August. Stage E reached the minimum (40,6 %) in
September, and the maximum (70,0 %) in October. The achieved results concerning the
spawning time as well as the stages of ovary puberty of the nase in the spring-autumn cycle
were discussed and compared with those from other papers.
SPERM MOTILITY PARAMETERS OF HOMO (XX, YY) AND
HETEROGAMETIC (XY) RAINBOW TROUT (ONCORHYNCHUS MYKISS
WALBAUM)
Radosław Kowalski1, Wiesław Demianowicz1, Beata Sarosiek1, Stefan Dobosz2, Jan
Glogowski1
1
10-747 Olsztyn, Poland, E-mail:[email protected]
2
Department of Salmonid Research, Inland Fisheries Institute, Rutki, 83-330 śukowo,
Poland
Specificity of fish reproduction and their phenotypic plasticity give us broad opportunity to
perform genetic and hormonal manipulation such as androgenesis or sex reversing.
Gynogenesis combinated with sex reversion allow us to obtain homogametic XX males and
androgenesis allow us to obtain homogametic YY males. The purpose of this study was to
determine and compare the sperm motility parameters measured in milt originated from
androgenetic (YY), control (XY) rainbow trout males and sex reversed female (XX).
Sperm samples of rainbow trout (n=14 in each group) were collected both, from spermatic
duct and from homogenized testis. In sex reversed group without developed spermatic duct
only testicular sperm were collected. Sample of sperm were diluted in appropriate ratio
(dependent of sperm concentrations) that vary from 1:200 to 1:400. Billard solution with
ovary fluid (ratio 1:1) was used as an activating medium. Shortly before activation, testicular
sperm was prediluted (ratio 1:2) with frozen/thawed rainbow trout seminal plasma as an
immobilizing solution. Sperm motility parameters were estimated using the Hobson sperm
tracker and associated software (Computer Assisted Sperm Ananlysis).
Sperm motility parameters were higher in the sperm obtained from spermatic duct of
androgenetic and control group and testis of sex reversed female. Average motility was
estimated around 70% in these samples. This parameter was lower in testicular sperm from
both, control (48%) and androgenetic (33%) males. Also curvilinear velocity (VCL) and
average path velocity (VAP) was higher in the sperm from spermatic duct of androgenetic and
control group and testis of sex reversed female. Straight line velocity (VSL) was around
40µm/s in sample from spermatic duct and around 30 µm/s in testicular sperm. Beat crossfrequency (BCF) value was similar in all samples, average 5 Hz. These results imply that
motility of the sperm obtained from testis of sex reversed females is comparable to the
motility of the sperm obtained from the spermatic duct of the androgenetic and control males.
The project was supported by State Committee for Scientific Research (KBN) grant nr 2 P06D
023 27
THE INFLUENCE OF HEAVY METALS (MERCURY AND CADMIUM) ON
THE ACTIVITIES OF SIBERIAN STURGEON (ACIPENSER RUTHENUS)
SPERMATOZOA ENZYMES
Beata Sarosiek1, Radosław Kowalski1, Jan Glogowski1
1
Department of Molecular Andrology Institute of Animal Reproduction and Food
Research, Polish Academy of Science, ul. Bydgoska 1/8 10-243Olsztyn, Poland,
One of the most dangerous pollutants that can be found in waters is mercury and cadmium.
These metals causes multiple negative physiological effects in fish e.g. hemolysis of
erythrocytes, but first of all affects many reproductive processes. The aim of this work was to
evaluate the influence of mercury and cadmium on the activities of siberian sturgeon
spermatozoa enzymes: lactic dehydrogenase (LDH), arylsulfatase, β-N-acetylglucosaminidase
and acid phosphatase.
The enzymatic activity were measured in supernatants obtained after semen exposition on
metals 0, 4 and 24 h, centrifugation, extraction of spermatozoa (in 0.7% NaCl) and the next
centrifugation. The 10 mg Hg2+/l decreased the LDH activity in seminal plasma by 100%, the
β-N-acetylglucosaminidase activity was decreased by 95% and arylsulfatase by 22%. The
analyzed enzymes were not affected by addition of 1 mg/l Hg2+. The same density of
cadmium did not inhibit the enzymes either (except LDH: 6% inhibition after 4 h and 26%
after 24 h). 10 and 100 mg/l addition of cadmium ions caused of inhibition of the LDH, acid
phosphatase and β-N-acetylglucosaminidase activities, and its depended on the metal density
and the enzymes exposition period on this metal.
We also checked the enzymes activities in supernatants (obtained after centrifugation of
freezing-thawing semen) exposed on heavy metals. 1 mg Hg2+/l inhibited the LDH activity by
15%, arylsulfatase by 10% and acid phosphatase by 22%, after 24 h of sperm-Hg2+ contact. 10
mg Hg2+/l caused 100% inhibition of LDH, 77% of arylsulfatase and 57% of acid phosphatase
activities. The Cd ions also decreased the enzymes activities. 10 mg Cd2+/l caused 51%
inhibition of LDH, 22% of arylsulfatase and 37% of acid phosphatase activities, after 24 h.
The 100 mg Cd2+/l density decreased the enzymes activity properly of 100, 65, 81%.
Our results showed that the heavy metals (mercury and cadmium) possessed destructive
influence on sperm enzymes activities. The negative effect of these metals depended on its
density and exposition period.
The project is supported by State Committee for Scientific Research (KBN) as
a Solicited Project , PBZ-KBN-084/P06/2002/5.8 in years 2003-2005.
ISOLATION AND CHARACTERIZATION OF α-1-ANTIPROTEINASE
FROM COMMON CARP SEMINAL PLASMA
Mariola Wojtczak1, Jan Glogowski2, Andrzej Ciereszko1
1
Department of Semen Biology,2 Department of Andrology, Institute of Animal
Reproduction and Food Research, Polish Academy of Sciences, 10-747 Olsztyn,
Seminal plasma of teleost fishes contains serine proteinase inhibitors. We previously
identified three serine proteinase inhibitors in common carp seminal plasma: inhibitors of low
(inhibitor I), moderate (inhibitor II) and fast migration rate (inhibitor III).
The objective of this study was to isolate and characterize two isoforms (IIA and IIB) of
serine proteinase inhibitor with close electrophoretic migration rates. These inhibitors were
purified by sequential application of hydrophobic interaction chromatography, ion exchange
chromatography and reverse phase high performance chromatography or preparative
electrophoresis. Using this four-step procedure we obtained homogenous inhibitors IIA and
IIB. The molecular weight of the isolated inhibitors IIA and IIB, estimated using SDS-PAGE,
were determined to be 58.5 kDa and 57.5 kDa, respectively. N-terminal Edman sequencing
revealed N-terminal blockage of inhibitor IIB and allowed the identification of only five
amino acids HEGHD for inhibitor IIA. Purified inhibitors IIA and IIB formed SDS-stable
complexes with cod and bovine trypsin, chymotrypsin and elastase, a unique feature of
serpins (serine proteinase inhibitors). Molecular weights of complexes appeared to be 2-3 kDa
lower than the sum of the individual components. After complex formation of both isoforms
with trypsin, the following 33 amino acids of released peptide (P1’- P33’)
SLPDTVILNRPFLVLIVEDTTKSILFMGKITNP were identified for isoform IIB. The same
first 10 amino acids SLPDTVILNR were obtained for isoform IIA. These small peptide
fragments represent the highly conserved C-terminal sequence of the sepins. The amino acid
sequence within the reactive centre of inhibitors IIA and IIB showed a high degree of
homology with rainbow trout and mammalian α1-antiproteinase, including 100% identity
with α1-antitrypsin homolog precursor from carp perimeningeal fluid. These results suggest
that inhibitor II is similar to α1-antiproteinase. Both isoforms are glycoproteins. The
glycoprotein character of these inhibitors was determined using Pro-Q Emerald 300
Glycoprotein Gel Stain Kit. After deglycosylation with N-glycosidase F the molecular weight
of inhibitors IIA and IIB decreased to about 52.5 kDa and 50.4 kDa, respectively. Time
dependent deglycosylation analysed by SDS-PAGE revealed the presence of one intermediate
of molecular weight 54.8 and 54.1 kDa for inhibitors IIA and IIB, respectively. The sugar
moiety of these inhibitors contains at least two N-linked oligosaccharide chains.
Inhibitors IIA and IIB are one of the main proteins of seminal plasma and their physiological
role may be related to protection of spermatozoa from proteolytic attract. The protective role
of α1-antitrypsin against spermatozoa may also include its antiapoptotic and bacteriostatic
actions.
Session XIII
Early pregnancy
Oral presentation
THE ACTIVITY OF NITRIC OXIDE SYNTHASE IN THE PORCINE
UTERUS DURING EARLY PREGNANCY
Aneta Andronowska, Marcin Chruściel, Teresa Doboszyńska
Nitric oxide plays a key role as a regulator of various female reproductive processes such as
ovulation, endometrial receptivity, implantation and myometrial relaxation. NO is produced
from L-arginine by enzyme nitric oxide synthases (NOSs), the family of enzymes in which
three izoforms have been identified: nNOS, iNOS and eNOS. The aim of the present study
was to determine the localization and hormonal regulation of NOS activity in the porcine
uterus during the early pregnancy.
Uteri from pregnant pigs at days 5, 10, 15, 20, 25, 30 of pregnancy were collected
immediately after slaughter. Tissues for localization of NOS izoforms were stained
immunohistochemically using monoclonal antibody against eNOS and iNOS. About 100 mg
of endometrial (all studied days of pregnancy) and placental (days 20, 25, 30 of pregnancy)
strips were cultured at 37°C for 24 hours in 2 ml Dulbecos Modified Eagles Medium
containing one of following: 1) 0,05, 0,2, 0,5 ng/ml estradiol (E2); or 10, 20, 40 ng/ml
progesterone (P4); or E2 + P4 (in combinations). After 24-h of culture tissues were analyzed
for eNOS and iNOS expression, whereas media were used for nitrate and nitrite concentration
(stable oxidation products of NO) by modified Griess method. Additionally in endometrial
and placental tissues obtained immediately after slaughter expression of NOS and nitrate and
nitrite level were estimated.
Light microscopic observations revealed that eNOS is present in the luminal and glandular
epithelium, vascular endothelium and myometrium, whereas slight iNOS staining was
observed mainly in vascular endothelium and luminal epithelium. Comparison of nitrate and
nitrite concentrations in the pregnant endometrium in the implantation and interimplantation
region showed significant increase of NO production in the implantation site. In the
endometrium the dose-dependent effect of E2 on nitrate and nitrite concentration was found
on days 25 and 30. The lack of E2 influence on nitric oxide synthase activity on days 15-20
was probably caused by endogenous estrogen production by trophoblast. P4 stimulate NO
production on days 5 – 25 of pregnancy. In placenta obtained on days 25 and 30 both E2 and
P4 affected on NO production.
NO synthesis, stimulated by steroid hormones, may play an important role in placental
angiogenesis for supporting the rapid fetal growth during early pregnancy.
Supported by KBN grant 3P06D 02123.
PLEIOTROPIC ACTIVITY OF IFN-TAU DURING PERI-IMPLANTATION
PERIOD OF PREGNANCY IN RUMINANTS
Anna Chełmońska-Soyta
Department of Veterinary Prevention and Immunology, Faculty of Veterinary
Medicine, Agriculture University of Wroclaw, 50-375 Wroclaw, Poland,
IFN-tau is a signaling protein secreted by the bovine conceptus during the peri-implantation
period and responsible for pregnancy recognition. Its pleiotropic actions can be explained by
widespread presence of interferon receptors on many types of the cells and the ability to
activate multiple signaling actions mainly involved STAT (Signal Transducers and Activators
of Transcription) family of proteins. IFN-tau acts in paracrine manner on the endometrial
luminal epithelium (LE), superficial ductal glandular epithelium(GE) and stromal cells. On
the other hand, IFN-tau exerts pronounced immunomodulatory effect on mononuclear
leukocytes in vitro. Pleiotropic effects of IFN- tau promote the mechanisms of recognition
and establishing of pregnancy which considerably interfere with differentiation and secretion
activity, proliferation and apoptosis of the target cells. The main role of IFN-tau in sheep is to
block transcription of the estrogen receptor gene and, therefore, the oxytocin receptor gene to
prevent uterine release of luteolytic pulses of PGF2α. In cattle it has probably direct influence
on COX-2 gene expression and TNF-α induced PGF2α synthesis. IFN-tau induces in
endometrial cells genes containing ISRE elements and increases expressions of proteins and
growth factors: 2’,5’ oligoadenylate synthetase, beta2-microglobulin, interferon regulatory
factor (IRF)-1 and IRF -2, ubiquitin cross reactive protein(UCRP) and Mx protein, cytokines:
GCP-2, MCP-1,2, GM-CSF and it also stimulates MHC I antigens expression. In bovine
mononuclear leucocytes stimulated with ConA, it induces GM-CSF production and according
to our observations IFN-tau increases of MHC I antigens expression during allogenic
response. It also influences the process of differentiation of luminal epithelial cells and
lymphocytes of endometrial LE. After mitogenic and allogenic stimulation IFN-tau changes
the percentage of lymphocytes subpopulations in ovine, cattle, murine and human species.
Based on our own experiments we showed that this cytokine diminished the population of
activated T gamma delta lymphocytes and increased the population of B cells. IFN-tau also
directly promotes IVFM/IVF derived embryos development. Antiproliferatve activity of IFNtau is accomplish by inducing of apoptosis and inhibition of cell cycle progression. It exerts
weak antiproliferative effect on ovine endometrial epithelial cells but induces of apoptosis in
30% of these cells. On the other hand it is observed pronounced, dose dependent
antiproliferative activity of this cytokine towards ruminant lymphocytes stimulated with
mitogens and allogenic antigen. We showed increased number of apoptotic cells and
accumulation of resting and PHA and PWM activated bovine PMN in G0/G1 phase of cell
cycle stimulated with IFN-tau.
This work was supported by State Committee for Scientific Research grant no. 3 PO6 D
03423.
LEPTIN AND ITS ROLE IN IMPLANTATION PROCESS
Gabriela Siawrys1, Tadeusz Kamiński1, Nina Smolińska1, Alina Gajewska2, Stanisław
Okrasa1, Kazimierz Kochman2 , Jadwiga Przała1,3
1
Department of Animal Physiology, University of Warmia and Mazury in Olsztyn, 10718 Olsztyn-Kortowo 5, Poland, 2 The Kielanowski Institute of Animal Physiology and
Nutrition near Warsaw, Poland, 3E-mail: [email protected]
Recent evidences indicate that leptin, the product of the ob gene, is involved in the regulation
of reproductive functions through its effect on the hypothalamic–pituitary–gonadal axis.
Moreover, a key role for leptin during pregnancy has been suggested. In human and rat serum
leptin concentration significantly increased during pregnancy and decreased at the beginning
of lactation period. Embryonic implantation is a crucial event in the establishment of
pregnancy. During this period expression of multiple genes for variety of factors critical for
embryo growth and development occurs and it is likely that leptin is one of these factors.
Study with ob/ob mice has been shown that exogenous leptin supplementation to the mother
was required at least for 6,5 days post coitum to retain pregnancy and go to term. Data of
leptin gene expression during implantation in pigs, when approximately 30-40% of embryos
is lost, are limited. Thus, studies on the expression of this gene in this critical period may
significantly contribute to the improvement of reproductiveness in pigs.
The objective of this study was to detect and comparison of leptin gene expression level
between mid luteal phase (10-12 day) and early pregnancy (14-16 day) in the discrete areas of
the hypothalamus [medial basal hypothalamus (MBH); preoptic area (POA), stalk median
eminence (SME)], anterior pituitary (AP), neural pituitary (NP), ovary (corpus luteum,
stroma) and uterus (endometrium, myometrium) in the pig.
The results of semiquantitative RT-PCR analysis showed that during early pregnancy (14-16
day) leptin mRNA expression was decreased in the MBH, POA, SME, NP, endometrium and
corpus luteum when compared to cyclic animals (10-12 day). No significant differences in
leptin gene activity were observed within AP, myometrium and stroma of ovary between
cyclic and pregnant pigs.
Conclusion. The finding of differences in leptin gene expression level between mid luteal
phase (10-12 day) and early pregnancy (14-16 day) in the discrete areas of the hypothalamus,
pituitary, ovary and uterus of the pig (reduction of expression during pregnancy) suggests that
leptin might affect regulation of the hypothalamic-pituitary-gonadal axis and be involved in
the implantation process.
This research was supported by the State Committee for Scientific Research (project nr PBZ
KBN-084/P06/2002 and No 0206.0805).
GENE EXPRESSION ANALYSIS AS A TOOL FOR QUALITY ASSESSMENT
OF BOVINE EMBRYOS
Ewelina Warzych, Dorota Lechniak
Agricultural University of Poznań, Department of Genetics and Animal Breeding,
ul. Wolynska 33,60-637 Poznań, Poland, E-mail: [email protected]
In vitro fertilization and embryo culture (IVP) became an alternative source of embryos for
cattle reproduction and research. IVP embryos however, display reduced quality and
developmental potential than their in vivo derived counterparts. Several factors supporting this
hypothesis have been identified e.g. reduced implantation and pregnancy rates, impaired
morphology (lower cell number, impaired compaction, early cavitation), increased incidence
of chromosomal abnormalities, altered pattern of expression of developmentally important
genes. Moreover, it is calculated that about 30% of bovine and sheep embryos derived from in
vitro system give rise to big size fetuses (large offspring syndrome – LOS). It has been shown
that preimplantation embryos display capacity to cope with sub-optimal culture conditions
and can compensate, at least partially, by adjusting their developmental program.
The quality of an embryo can be defined as its ability/potential to reach the advanced
developmental stages (e.g. blastocyst) able to implant and develop into a viable offspring.
Therefore, criteria used to characterize embryonic quality in vitro need to be verified by
embryo transfer to a donor and by monitoring the resulting pregnancy. Analysis of gene
expression in embryos concerns mainly the transcription level and characterizes an existing
pool of mRNAs. The current status of expression studies in bovine embryos displays
approximately 100 genes analyzed with regard to qualitative and quantitative aspects. These
are mainly genes involved in various biological processes, which have crucial role in embryo
development. Due to methodological limitations it is still impossible to successfully analyze a
single cell derived from morula or blastocyst and therefore the analysis is based on an entire
embryo or a group of embryos.
In this presentation we will discuss the most important aspects of gene expression
investigation in mammalian embryos in relation to their quality and developmental potential,
like:
1. qualitative and quantitative comparison of transcript pattern in in vivo and in vitro
derived embryos,
2. influence of in vitro culture conditions on gene expression pattern,
3. alternation in mRNA expression of genes controlling main biological functions in
embryos (first cleavage, genome activation, compaction and blastocyst formation
etc.),
4. expression pattern of imprinted genes in in vitro culture conditions,
5. gene expression in cloned embryos (e.g. by nuclear transfer),
6. diet composition and embryo quality in vivo.
Research was supported by the State Committee
Solicited Project no PBZ-KBN-084 from 2003 to 2005 year.
for
Scientific
Research
as
VASCULO- AND ANGIOGENESIS IN THE FETOMATERNAL UNIT
Marek Zygmunt
Department of Obstetrics and Gynecology, University of Giessen,
Klinikstrasse 32 35385 Giessen, Germany
An adequate nutrient and substrate supply is essential for normal intrauterine development of
the fetus. Disturbances in uterine blood supply are associated with higher perinatal morbidity
and mortality caused by preterm delivery, pre-eclampsia or intrauterine growth restriction.
Since the uterus and the surrounding tissue demand an increased blood supply during
pregnancy, its vasculature undergoes three main adaptative changes: vasodilatation; increased
permeability; development and maturation of new vessels (vasculo- and angiogenesis). In
general, the angiogenic process is initiated by growth factors such as bFGF, VEGF, or PlGF
which are highly abundant in the endometrium, decidua and placenta. In order to identify
respective angiogenic factors of trophoblast origin within the feto-maternal interface, we
focused our experiments on human chorionic gonadotropin (hCG), a major peptide hormone
that is responsible for numerous pregnancy related and pregnancy-maintaining processes.
We demonstrated that hCG has a direct angiogenic function and that hCG/LH receptorexpressing uterine endothelial cells could respond to physiological doses hCG with increased
capillary formation in vitro. In subsequent experiments, we showed that phorbol ester (kinase
C activator), but not cAMP or forskolin (kinase A) mimicked hCG actions in the angiogenesis
model and PKC inhibitory peptide abolished hCG-induced sprout formation, indicating that
protein kinase C (PKC) could be part of the signal transduction pathway utilized during tube
formation. Possible subsequent events of signal transduction may involve activation of
extracellular signal-regulated kinases (ERK-1 and ERK-2), mitogen-activated protein kinases
(MAP) as well as myosin light chain kinase (MLCK). Because of striking similarities
between, on the one hand, tumor invasion and tumor vascularization and, on the other,
blastocyst implantation and placental development and the fact that hCG is secreted by
a broad spectrum of genital and non-genital solid tumors, we also investigated the role of hCG
in tumor-induced angiogenesis and development. The observation that the angiogenic activity
of hCG secreting tumor cells could be inhibited by anti-hCG antibodies not only underlines
the specificity of this novel function of hCG, but may also serve as a potential approach for
antitumor treatment.
Poster presentation
NON-GENOMIC EFFECT OF STEROIDS ON OXYTOCIN-STIMULATED
INTRACELLULAR MOBILIZATION OF CALCIUM AND ON
PROSTAGLANDIN F2α AND E2 SECRETION FROM BOVINE
ENDOMETRIAL CELLS
Magdalena Duras, Jan Kotwica
Progesterone (P4) decreases the ability of OT-stimulated PGF2α secretion from the bovine
endometrial cells and this effect is partly via non-genomic way. Therefore we studied (a) the
effect of P4, its precursor (pregnenolone; P5) and metabolite (17β-hydroxyprogesterone;
17OHP4) on OT-stimulated PGF2α and PGE2 secretion by bovine endometrium and (b) the
effect of these steroids on intracellular Ca2+ mobilization ([Ca2+]).
Uteri were collected from nonpregnant cows and heifers on days 14-18 of the estrous cycle. In
Exp. 1, endometrial slices (30-35 mg) were preincubated in medium M199 with 0.1% BSA
(38°C; atmosphere of air and 5%CO2). After 24 h medium was replaced by fresh and cells
were incubated for 6h with: P4, P5, 17OHP4 at the dose of 10-5M, OT (10-7M) and OT with
each of these steroids. In Exp. 2, dispersed endometrial cells (105/ml) were incubated as above
in DMEM/Ham’s-F12 medium with 5% FCS for 72-96 h. The last 24 h the cells were
incubated in DMEM/Ham’s-F12 with 0.1% BSA and then for 4 h with: (a) arachidonic acid
(10-5M) as a positive control, (b) OT (10-7M), (c) P4 (10-5M), (d) P5 (10-5M), (e) 17OHP4 (105
M) alone and with OT. The concentration of PGFM and PGE2 was determined in medium by
EIA. In Exp. 3 [Ca2+] mobilization was monitored using the cell permeable form of the
fluorescent Ca2+ indicator Fura-2AM (5 µg/ml). The endometrial cells were incubated with
P4, P5 i 17OHP4 (10-5M each) for 30 min and [Ca2+] mobilization was measured every 5 s for
15 s before OT treatment and for 60 s thereafter. Concentrations of [Ca2+] were analyzed by
means of computer software (Micro Image 4.0; Olympus Optical Co.). Each experiment was
done for 4-7 animals, in triplicate for each treatment.
OT stimulated (P<0.01) PGF2α secretion from endometrial slices and both PGF2α and PGE2
from dispersed cells on days 14-18 of the estrous cycle. All used steroids inhibited (P<0.01)
OT-stimulated PGF2α, but not PGE2 production/secretion. P4, P5 and 17OHP4 decreased
(P<0.05) total amount of Ca2+ concentrations in cells compared to control. OT induced rapid
increase of [Ca2+] mobilization within 15 s, while in the cells pretreated with steroids this
effect did not appear before 45 s after OT treatment.
Studied steroids are able to suppress the ability of OT to stimulate endometrial secretion of
PGF2α and decrease [Ca2+] mobilization in response to OT via non-genomic way. This effect
of steroids can directly modulate function of endometrial cells and supposedly other cells.
This phenomenon might be important in evoking a local effect at the site of hormone
secretion or treatment (eg. injection or use of ointment with steroids as an active substance).
This work was supported by grant from KBN (5P06D 025 22 )
EFFECT OF OXYTOCIN ON EXPRESSION OF ENZYMES OF
CYCLOOXYGENASE PATHWAY IN PORCINE MYOMETRIAL CELLS
Anita Franczak1, Izabela Wocławek-Potocka2, Agnieszka Oponowicz1,
Beata Kurowicka1, Genowefa Kotwica1
1
Mazury, 10-719 Olsztyn, 2Department of Reproductive Immunology, Institute of
Animal Reproduction and Food Research, Polish Academy of Science,
Our previous study indicated that oxytocin receptors (OTR) are present in the porcine
myometrium and that oxytocin (OT) stimulated PGF2α and PGE2 secretion by myometrial
slices isolated from uterus of sows at luteolysis and early pregnancy. Increase of accumulation
of PGF2α in porcine myometrium was observed at luteolysis. Thus, it cannot be excluded that
myometrial tissue is responsible for localized synthesis of prostaglandins and that OT may
regulate this process. The present study was undertaken to ascertain whether OT influences
myometrial expression of enzymes in the cyclooxygenase pathway: cyclooxygenase 2
(COX2), synthase PGF2α (PGFS) and synthase PGE2 (PGES). Myometrial tissue was isolated
from cyclic (n=4; Days 14-16 of estrous cycle) and early pregnant (n=4; Days 14-16) sows.
Slices of myometrium were incubated for 6h in the presence of arachidonic acid (AA;
2mg/ml; positive control), OT (10-7 M) or without treatments (control). Homogenates of the
slices were prepared for Western blotting analysis. The intensities of COX2, PGFS and PGES
protein expression were estimated by measuring the optical density in a defined area using a
Kodak system (Kodak 1D Image Analysis Software). The expression of COX2, PGFS and
PGES proteins was evident in myometrial explants of cyclic as well as early pregnant sows
and AA increased (P<0.05) this expression. Oxytocin stimulated (P<0.05) expression of
COX2 and PGFS in cyclic and early pregnant myometrial slices. Expression of PGES was
higher (P<0.01) in myometrial explants at early pregnancy compared to those at luteolysis and
OT did not significantly change this expression. In conclusion: these results demonstrate
presence of an active cyclooxygenase pathway in porcine myometrial tissue at luteolysis and
early pregnancy. Thus, myometrium may be a source of the prostaglandins. Oxytocin is
involved in regulation of an activity of this pathway.
This work was supported by the State Committee for Scientific Research as a Solicited Project
PBZ-KBN-084/Po6/2002 from 2003 to 2005 and by grant 0206. 206 (University of Warmia
and Mazury).
CONNEXIN43 GAP JUNCTIONS IN PREPUBERTAL PORCINE
MYOMETRIUM
Marta Kojs, Marek Romek, Janusz Karasinski
Department of Cytology and Histology, Institute of Zoology, Jagiellonian University,
Connexin43 is the most abundant connexin expressed in myometrial smooth muscle cells.
Connexin43 forms transmembrane channels, the gap junctions, which allow exchange of
small signalling molecules and mediate flow of action potentials between adjacent cells. In
the pregnant uterus the number and size of connexin43 gap junction plaques increase
dramatically at term and coincide with development of synchronized uterine contractions
typical of labor. However, in the non-pregnant uterus the pattern of myometrial gap junctions
expression and their functional significance are still unclear.
The purpose of this study was to analyse the distribution of Cx43 protein and gap junction
plaques in the longitudinal and circular layers of prepubertal porcine myometrium by confocal
microscopy, Western blotting and transmission electron microscopy. Uterine tissue was
obtained immediately after slaughter of the animals and examination of ovarian morphology.
The tissue was frozen for cryosectioning or fixed for standard thin-section electron
microscopy. Cryosections were immunofluorescently labelled using antibodies against
connexin43 and desmin and examined by confocal microscopy. Cryosections were also used
for protein electrophoresis followed by Western immunoblotting. Electron microscopy was
applied to investigate the mean diameter of gap junction plaques and permit unequivocal
identification of gap junctions by their pentalaminar structure.
Immunofluorescence confocal microscopy showed prominent labelling for the gap junction
protein connexin43 in the circular layer and only week labelling in the longitudinal layer. In
the circular muscle high level of connexin43 label occurred in the region bordering the
endometrium, while the amount of labelling decreased progressively towards the longitudinal
muscle. Both layers of the myometrium were strongly positive for smooth muscle cell marker
desmin. Western immunoblotting showed a corresponding abundance of connexin43 protein
in the circular layer but not the longitudinal layer of the myometrium. These results indicate
that the differential expression of connexin43 gap junctions in the longitudinal and circular
porcine myometrium is maintained even before cyclic ovarian activity.
This work was supported by KBN grant 3 P06K 030 24.
OXYTOCIN RECEPTOR GENE EXPRESSION IN PORCINE
ENDOMETRIUM AND MYOMETRIUM DURING LUTEOLYSIS AND
EARLY PREGNANCY
Agnieszka Oponowicz, Anita Franczak, Beata Kurowicka, Genowefa Kotwica1
Mazury, 10-719 Olsztyn, Poland, 1E-mail: [email protected]
Oxytocin (OT) and oxytocin receptors (OTR) are involved in the regulation of uterine
functions in sows. The results of in vivo and in vitro studies have shown that OT stimulated
secretion of PGF2α from endometrial cells at luteolysis and PGE2 at early pregnancy. The
increase of myometrial contractions was observed in response to OT in cyclic but not in early
pregnant sows. Concentration of OTR in endometrium (E) and myometrium (M) changes
dependent on days of the oestrous cycle and pregnancy. The biological effect of OT in uterine
tissues is controlled on many levels of intracellular pathway but on the top of this
relationships is the expression of OTR gene. Thus, the fluctuations in the sensitivity to OT in
both uterine tissues during the different stages of the oestrous cycle and pregnancy suggest
that OT plays a role in regulation of these reproductive periods in sows. The aim of this study
was to compare the expression of OTR gene in E and M of sows during luteolysis and early
pregnancy. Uterine tissues were obtained from cyclic (n=4; Days 14-16) and early pregnant
(n=4; Days 14-16) sows. Total RNA was isolated from 30 mg-weighted slices of E and M
using RNeasy Mini Kit, Qiagen. Purity of RNA was determined spectrophotometrically. Total
RNA was reverse-transcribed to cDNA using Omniscript RT Kit (Qiagen). Samples were
incubated at 37°C for 1 h and then at 95°C for 5 min. PCR was prepared using HotStarTaq
Master Mix Kit, Qiagen. Conditions for amplification of OTR and GAPDH genes were
performed based on those previously described and after optimalization of cycles number.
The reaction was carried out for 35 and 29 cycles for OTR and GAPDH, respectively.
Temperature for annealing of the primers was 65°C. Amplification products were analyzed by
electophoresis in 1,5% agarose gel and next by densytometric analysis. Homology of the PCR
products to the sequence for OTR cDNA was 98%. The results were presented as a ratio of
optical density of OTR to GAPDH (mean ± S.E.M). Expression of the OTR gene was
revealed in E and M of porcine uterus from the oestrous cycle and early pregnancy. The
differences in expression of the OTR gene in E between early pregnant and cyclic gilts was
not signifficant, because of large individual differences. A tendency (P<0.07) to a lower
expression of the OTR gene in early pregnant compared to cyclic myometrial cells was
observed. The results shown that biological effect of OT in uterine tissues may be regulated at
the level of expression of OTR gene.
Project PBZ–KBN–084/P06/2002 from 2003 to 2005 year.
BINDING OF SEMI-PURIFIED PORCINE AND BOVINE SECRETORY
CHORIONIC LIGANDS BY GONADAL AND EXTRAGONADAL
GONADOTROPHIN RECEPTORS
Grzegorz Panasiewicz, Marta Majewska, Adriana Cegiełka, Aleksandra
Romanowska, Jolanta Dajnowiec, Janina Bukowska and BoŜena Szafrańska@*
Department of Animal Physiology, University of Warmia & Mazury, Faculty of
Biology, 10-719 Olsztyn-Kortowo, Poland, E-mail: [email protected]
Proteomic studies revealed that some purified pregnancy-associated glycoproteins (PAG) are
useful diagnostic prenatal markers of embryonic mortality in domestic and wild ruminants.
However, a function or receptors of the PAG family remain still unclear. The aim of this study
was to compare radiocompetition effectiveness of semi-purified porcine and bovine secretory
chorionic proteins (including PAGs), as ligands of gonadal and extragonadal gonadotrophin
receptors of cyclic pigs and cows. Various trophectoderm (TRD) explants were harvested in
pregnant pigs (n=8; 21-61 dpc - day post coitum) or cows (n=6; 40-110 dpc), then TRD explants
were long-term cultured. De novo produced TRD proteins (+PAG) were isolated from media.
Porcine TRD (21 dpc) and bovine (40-110 dpc) cotyledonary (CT) or interCT-TRD proteins
were fractionated by Amicon cartridges (MWCO 10 kDa). According to increased mass of
porcine explants during advanced pregnancy (30-61 dpc) and subsequent bigger volumes of
used culture media, released TRD proteins were recovered by precipitation with 20%, 40%
and 75% saturation of (NH4)2SO4 and dialysis (MWCO 12-14 kDa). Western/PAGE blotting
with well-defined anti-PAG sera monitored the PAG proteins during isolations. The semipurified and fractionated TRD proteins (>10 kDa) were applied as secretory ligands (0.78-25
µg/ml) for radioreceptor assay (RRA), parralel to pLH and hCG (0.39-50 ng/ml) - used as
positive control RRA ligands. Gonadal and extragonadal receptors of cyclic pigs and cows
(cRc) were isolated from late luteal-phase corpora lutea (cCLRc) and uterine myometrium
(cMYORc). The competition was accomplished with diluted cCLRc (1:30) or cMYORc (1:1)
and iodinated hCG (125I-hCG), as the tracer. Both control ligands (pLH and hCG), displaced
125
I-hCG from porcine cCLRc (up to 15 %/B0) or cMYORc (40 %/B0) and bovine cCLRc (only
82 %/B0). This is the first study reporting that various fractions of semi-purified porcine TRD
(+PAG) ligands competed with 125I-hCG for binding by cCLRc and cMYORc in a
concentration-dependent and pregnancy stage-dependent manner. The highest 125I-hCG
competition with cCLRc (15%/B0) or cMYORc (25%/B0) was detected for porcine TRD
protein ligands (31-38 dpc) precipitated by 20% saturation of (NH4)2SO4. The semi-purified
porcine TRD ligands from more advanced pregnancy (40-61 dpc) were less effective in the tracer
competition with cCLRc (73%/B0) and cMYORc (80%/B0). The competition of bovine TRD
proteins with bovine cCLRc was also low (71%/B0). Thus, RRA suggest that the PAG protein
family is very promiscuous and may play more important role during post-implantation than
during advanced placentation periods.
*
Supported by PBZ-KBN-084/P06/02/3.4 and UWM528-0206.0805.
LUTEINIZING HORMONE ATTENUATES THE VASCULAR RESPONSE
TO NOREPINEPHRINE
Janina Skipor, Andrzej Kowalik
Vascular smooth muscle and endothelial cells are a target for the action of LH, since
functional LH/hCG receptors in female genital tract has been demonstrated. There are
evidence for direct and indirect action of LH in these vessels, however the exact mechanism
involved in vasoactive action of LH/hCG is not well known. In vitro studies of human uterine
arteries showed that hCG increases release of vasodilative and decrease release of
vasoconstrictive eicosanoids.
The present study examines direct effect of LH on the uterine artery from pigs in an in vitro
preparation. We have focused on LH effect on vessels response to norepinephrine (NE).
Third order arteries (200 µm) from the tree of uterine artery were isolated during early luteal
phase of the estrous cycle. They were cleaned of loose connective tissue, cut into rings (length
3 mm) free of side branches and prepared for isometric tension recording. Briefly, the method
consists of passing two fine (80 µm), stainless steel triangles through the lumen of the
vascular ring. One triangle was fixed to the stationary rod, while the other was hooked to
isometric force transducer (F30, HUGO Sachs). The arteries were fixed in 6 ml organ bath at
37°C, containing modified Krebs-Henseleit solution with the following composition
(millimolar): NaCl, 115; KCl, 4,6; KH2PO4 1,2; CaCl2, 2,5; NaHCO3, 25; glucose, 11,1. The
pH was maintained at 7.4 by constant bubbling with 95% O2-5% CO2. After 60 min, rings
were stretched incrementally to their optimal passive tone, as determined previously by their
contractile response to 60 mM KCL. After 30 min equilibration, one part of vessels were
treated with 10 ng/ml of LH in PBS (experimental), while a second part of vessels were
treated with 10 ng/ml of BSA in PBS (control). After 30 min of equilibration, NE was given
to each organ bath in cumulative concentration manner ranged between 1 x 10-8 mol/l to 3 x
10-4 mol/l.
Norepinephrine caused a dose-dependent contraction of all experimental and control arteries.
Addition of LH caused a rightward shift of the dose-response curve to NE. The
corresponding EC50 values were 2,17 (± 0.39) µmol/l in PBS pretreated vessels and 3,35 (±
0.41) µmol/l in LH pretreated vessels (P<0.05).
The results of present study demonstrated that LH attenuates the vascular response to NE in
third order branches of uterine artery. Therefore, it can be suggested that and additional
mechanism is engaged in direct action of LH on blood flow in uterine arteries in pigs.
IMMUNOLOCALIZATION OF ANDROGEN RECEPTOR (AR) AND
CYTOCHROME P450 AROMATASE IN PORCINE EMBRYO TISSUES
Maria Słomczyńska1, Małgorzata Duda1, Małgorzata Burek1, Jerzy Galas1, Marek
Koziorowski2
1
Lab of Endocrinology & Tissue Culture, Institute of Zoology, Jagiellonian
University, 30-060 Krakow, Poland, E-mail: [email protected]
2
Department of Physiology and Reproduction of Animal, University of Rzeszów,
Poland
Androgen, plays a number of important physiological roles in normal female development. It
may act on target gene trough androgen receptor (AR) or serve as a substrate for cytochrome
P450 aromatase (P450 arom), which is responsible for estrogen synthesis. The AR protein
belongs together with estradiol, progesterone and mineralocorticoid receptor to the big family
of steroid receptors which, after interaction with ligands serve as transcription factors. AR has
been considered as the critical mediator for the action of sex steroids on the female
differentiation and development. Relatively little is known about steroid hormones production
and their receptors localization during pregnancy. Secretions of porcine conceptus and the
uterine endometrium provide an optimal and essential environment for proper conceptus
development. It is well known that elongating trophoblast secretes estrogens and a certain
amount of androgens. However, no estrogen receptor protein was detected in that tissue and
there is no data concerning AR. In the fetal testis, unlike in the quiescent fetal ovary, hormone
production is very active. Testosterone with anti-Mülerian hormone, play a key role in the
induction and regulation of male sexual differentiation.
The purpose of this work was to compare the presence of AR and P450 arom at various
developmental stages of the gonads as well as other fetal organs: heart, lung, kidney, intestine
and spleen. Porcine fetuses were recovered at 18, 32, 50, 71 and 90 days of gestation. The
sections were exposed to polyclonal antibodies against AR and aromatase (gift from dr
Osawa) and color reaction was developed using DAB system. The intensity of
immunoreaction was analyzed in each section. The most prominent differences in the
immunostaining were observed in AR and aromatase within fetal testes. AR immunostaining
was also detected in embryonic kidney and lung. Serial sections used for immunostaining
were also stained using Goldner procedure.
Supported by a Solicited Project PBZ-KBN-084/PO6/2002 realized from 2003-2005
LOCAL TRANSFER OF PROSTAGLANDIN E2 INTO THE OVARY AND
OVIDUCT AND ITS RETOGRADE TRANSFER INTO THE UTERUS IN
EARLY PREGNANT SOWS
Stanisława Stefańczyk-Krzymowska, Jolanta Chłopek, Waldemar Grzegorzewski,
Michał Radomski
Food Research of The Polish Academy of Sciences, 10-747 Olsztyn, Poland.
This study was designed to establish whether PGE2 can reach the ovary and oviduct by local
pathway and what is the contribution of lymphatic vessels to this transfer, and whether PGE2
can permeate from venous and lymphatic vessels of the mesometium to arterial blood and be
delivered to the uterine horn during maternal recognition of pregnancy in gilts.
The reproductive tract was excised from gilts (n=10) on 14 day after mating. The uterine horn
was isolated with the ovary and broad ligament and perfused with warmed and oxygenated
autologous blood. A total dose of 5.5 x 107 d.p.m. (49 ng) of 3H-PGE2 was infused into small
branches of the uterine vein on the broad ligament or into lymphatic vessels. Frequent blood
samples were collected from the branch of the uterine artery and from venous effluent. Tissue
samples were collected from the uterine horn, from the ovary and from the broad ligament.
Concentration of 3H-PGE2 was significantly higher in the ovary (P < 0.001), oviduct (P <
0.01), endometrium (P < 0.01), myometrium (P < 0.001) and mesometrium (P < 0.001) after
infusion of 3H-PGE2 into lymphatic vessels than into the branches of the uterine vein. In
contrary, concentration of 3H-PGE2 was significantly higher in arterial blood supplying the
uterine horn (P < 0.01) and in venous effluent (P < 0.001) after infusion of 3H-PGE2 into the
branches of uterine vein than into lymphatic vessels. These results demonstrated local transfer
of 3H-PGE2 into the ovary, oviduct and uterine horn from lymphatic and venous vessels of the
mesometrium. However, the efficiency of this transfer was considerably higher after infusion
into lymphatic vessels than into branches of the uterine vein. We conclude that the lymphatic
pathway is a fundamental mechanism in the local transfer of PGE2 from the uterus to ovary
and oviduct during early pregnancy in the pig.
The study was supported by the State Committee for Scientific Research as Solicited Project
EXPRESSION PATTERN OF MICROSOMAL PROSTAGLANDIN E2
SYNTHASE-1 IN THE PORCINE CORPUS LUTEUM AND TROPHOBLAST
Agnieszka Wacławik, Monika M. Kaczmarek, Agnieszka Blitek and Adam J. Zięcik
Maintenance and regression of corpus luteum (CL) is regulated by complex interactions
between luteolytic and luteotrophic mediators. Prostaglandin E2 (PGE2) has been considered
to play important role as a luteoprotective factor during maternal recognition of pregnancy in
the pig. Recently, we have identified expression of microsomal prostaglandin E2 synthase-1
(PGES) in endometrium during the estrous cycle and pregnancy and in trophoblast in the pig
(Waclawik et al., 2004 and 2005). Uterine PGE2 reach the corpus luteum by local and/or
systemic mechanisms. It has been suggested that conceptus/trophoblast and CL also possess
capacity to produce prostaglandins in many species. We have hereby investigated the
functional changes of PGES expression in the porcine corpus luteum and
conceptus/trophoblast.
In present studies, samples were collected from gilts on Days 1-21 of the estrous cycle (n=29)
and on Days 10-25 of pregnancy (n=29). Luteal PGES protein expression was analyzed
throughout the estrous cycle and early pregnancy by using Western blotting. Furthermore,
expression of PGES in conceptus/trophoblast was analyzed by Western blotting and real-time
RT-PCR.
Quantification of mPGES-1 mRNA in conceptus/trophoblast revealed up-regulation on Days
10-12 and 25 (mean±SEM, 314.3+/-51.4, 219.3±40.4; respectively; p<0.05 vs all other
examined stages of pregnancy). The lowest expression of mPGES-1 mRNA was on Days 1417 (12.5±1.6). Correspondingly, mPGES-1 protein levels were high on Days 10-12 and 22-25
of pregnancy. In contrast to endometrium, luteal PGES protein was expressed at constant
levels both during the estrous cycle and early pregnancy.
Summarizing, this is the first report showing identification of PGE2 synthase in the porcine
CL. Although autocrine and paracrine actions of luteal PGE2 in autoregulation of CL function
can not be excluded, our results suggest that rather synthesis of PGE2 in trophoblast and
endometrium than in CL could be involved in the process of maternal recognition of
pregnancy in pigs.
This work was partially supported by the State Committee for Scientific Research in Poland
as a Project 2 P06D 041 26.
PRECURSORS AND ENZYMES RESPONSIBLE FOR PROSTAGLANDIN
(PG) F2αα SYNTHESIS IN THE BOVINE ENDOMETRIUM
Izabela Wocławek-Potocka, Anna Korzekwa, Dariusz J. SkarŜyński
Division of Reproductive Endocrinology and Pathophysiology, Institute of Animal
Reproduction and Food Research, PAS, Tuwima-St 10, Olsztyn 10-747, Poland,
The proper balance of oppositely acting prostaglandins (PG)s and other arachidonic acid (AA)
metabolites regulates many regulatory mechanizms in the bovine reproductive organs.
Arachidonic acid is converted by cyclooxygenase-2 to PGH2, which under the influence of three
synthases: PGD2 synthase (PGDS), PGES and PGFS is converted to respective PGs. Moreover, 9keto-reductase PG (9k-PGR)-dependent PGE2 conversion to luteolytic PGF2α, has been suggested. In
ruminants corpus luteum (CL) development as well as pregnancy establishment and recognition
depend on higher PGE2 to PGF2α concentration. On the other hand, higher ratio PGF2α to PGE2 is
required, in case of the lack of pregnancy, when coming back to cyclicity is needed. Taking into
consideration the great importance of the proper PGE2 and PGF2α concentrations, the aim of this
study was to examine which precursors and enzymes are responsible for PGF2α synthesis in the
bovine endometrium during early and luteal stages of the estrous cycle. In Exp. 1 epithelial cells
enzymatically isolated from the uteri (21-1 and 8-12 day of the estrous cycle – early and mid luteal
stages of the cycle) were incubated in suspension for 6 hours with PGF2α precursors: PGE2, PGD2,
AA and oxytocin as a positive control (OT; all factors: 10-7 M). In the incubation medium
PGF2α. concentration was measured by EIA. During early luteal phase PGE2 and AA were the main
precursors for PGF2α synthesis in epithelial cells, whereas during mid-luteal phase PGD2 was the
main precursor for PGF2α synthesis in the cells. In Exp. 2 epithelial cells enzymatically isolated
from the uteri (21-1 and 8-12 day of the estrous cycle - early and mid luteal stages of the cycle) were
incubated in suspension for 6 hours with PGF2α precursors: PGE2, PGD2, AA and oxytocin as a
positive control (OT; all factors: 10-7 M). In the cells gene expression for PGFS and 9k-PGR was
measured by RT-PCR. During early luteal phase PGF2α synthesis depended on 9k-PG activation
(P<0.05), whereas during mid-luteal phase PGF2α synthesis depended on PGFS activation
(P<0.05).
Summarizing: (1) We found higher 9k-PGR and PGFS gene expression at the early luteal phase of
the estrous cycle in comparison to mid-luteal phase, which can lead to higher luteolytic PGF2α
synthesis from its basic precursors - PGE2 and PGG/H. (2) At the mid luteal phase of the estrous
cycle PGF2α synthesis depended on 9k-PG activation. (3) At the mid luteal phase of the estrous
cycle PGF2α synthesis depended on PGFS activation.
Session XIV
Avian reproduction
Oral presentation
EFFECT OF EGZO- AND ENDOGENOUS EMBRYONIC CELLS ON
SELECTED REPRODUCTIVE TRAITS OF BIRDS*
Marek Bednarczyk1, Paweł Łakota1, Przemysław Czekalski2, Mirosław Lisowski2,
Magdalena Wawrzyńska1
1
Department of Animal Biotechnology, University of Technology and Agriculture,
Bydgoszcz, Poland, E-mail: [email protected]; 2National Research
Institute of Animal Production, Krakow, Poland
The investigation was conducted on the cocks and hens of ZielononóŜka kuropatwiana breed Zk, White leghorn – WI, and chimeric - Ch chickens, raised and housed in the same
environmental conditions. The chicken somatic and germline chimeras were obtained by the
transfer of donor (Zk) blastoderm cells (BCs) into recipient stage X embryos (Wl), according
to the method proposed be Petitte et al., (1990), with some modifications (Bednarczyk et al.,
2000). Slight number of BCs (1 200) injected into recipient embryo can proliferate, and
colonize different tissues/organs of treated embryo, and first of all can significantly change
the mono- and polygenous physiological and productive traits of recipient. The statistically
significant influence of chimerism on the following reproductive and egg quality traits:
spermatozoa sperm concentration, eggs number laid, eggs weight, yolk weight, albumen
weight, percent of albumen, shell weight, percent of shell, shell colour, albumen height,
Haugh unite, and shell density, was demonstrated. The all chimeric hens laid eggs but 25% of
them produced no offspring. Three cock chimeras produced the low spermatozoa sperm
concentration (from 653.000 to 772.000 /ml), and two chimeras did not produce sperm.
Among the mentioned cocks, some of them had underdeveloped testis and/or another showed
in blood the chromosome W. On the other hand, three chimeras produced the high number of
spermatozoa (from 2. 289.000 to 2. 681.000 / ml) and two of them characterised the highest
spermatozoa production in comparison with the all investigated Wl, Zk and Ch cocks.
*
Grant sponsor: State Committee for Scientific Research (KBN) Project No. PBZ-KBN084/P06/2002/4.3
IN VITRO FERTILISATION IN BIRDS
Anna Hrabia1, Kiyoshi Shimada2, Soichi Takagi2, Janusz Rząsa1
1
30-059 Krakόw, Poland, E-mail: [email protected], 2Laboratory of Animal
Physiology, Graduate School of Bioagricultural Sciences, Nagoya University,
Nagoya, Aichi 464-8601, Japan.
Naturally, fertilisation in birds takes place in the infundibulum, soon after ovulation. This
must occur before the outer layer of vitelline membrane is laid down (Bakst & Howarth 1977;
Howarth 1984). Birds exhibit physiological polyspermy, it means that many sperm enter the
germinal disc of an oocyte, where undergo decondensation and form male pronuclei, although
only one successfully fertilises the oocyte. The supernumerary pronuclei degenerate at the
very early stages of embryo development (Okamura et al. 1978; Perry 1987).
Information about in vitro fertilisation (IVF) is very limited in birds due at least in part to the
difficulties in obtaining of sufficient number of newly ovulated oocytes and specific embryo
development.
For the first time Howarth (1971) showed by haematoxylin staining of nuclei that the ovum
immediately after ovulation could be fertilised in vitro when the germinal disc of the ovum
was introduced to millions of spermatozoa in medium. More recently, Nakanishi et al. (1990)
provided direct evidence of IVF in chicken by showing male and female pronuclei formation.
Furthermore, Nakanishi et al. (1991) demonstrated the fertility competency of multipleovulated oocytes in the chicken after IVF. Viable chicks by implanting in vitro fertilised ova
into the oviduct of recipient hens followed by incubation of the eggs were produced by
Tanaka et al. (1994). Recently, Olszańska et al. (2002) investigated development of quail
embryos after IVF using in vitro ovulated oocytes. They demonstrated for the first time that
cytoplasmic segmentation can occur in the absence of nuclear divisions in the germinal disc
of the quail and showed the existence and significance of ooplasmic maternal information in
birds.
Only one study demonstrating in vitro fertilisation by intracytoplasmic sperm injection (ICSI)
has been published in birds (Hrabia et al., 2003). The authors showed that intracytoplasmic
injection of a single spermatozoon into quail oocyte can activate the oocyte and lead to
fertilisation. Oocytes prematurely ovulated are capable of fertilising with mature sperm as are
those spontaneously ovulated. In addition, the results suggest that testicular round spermatids
may not posses sufficient oocyte-activating potency but that the elongated spermatids and
immature spermatozoa are competent to participate in fertilisation and early embryonic
development in quail. The method of microfertilisation in birds is advantageous to study the
mechanism of fertilisation and the role of gametes in this process. The use of sperm as a
vector to carry foreign DNA to the oocyte may help for production of transgenic birds.
MELATONIN, ITS SYNTHESIS AND ROLE DURING EARLY AVIAN
DEVELOPMENT
BoŜenna Olszańska1, Rusłan Obłap1, Paweł Majewski2, Bogdan Lewczuk3, Urszula
Stępińska1, Anna Barańska2, Krystyna Skwarło-Sońta2
1
Institute of Genetics and Animal Breeding PAS, Jastrzębiec n. Warsaw, E-mail:
[email protected]; 2Lab. of Vertebrate Physiology, Institute of Zoologyi,
University of Warsaw, 3Department of Histology and Embryology, Faculty of
Veterinary Medicine, University of Warmia & Mazury, Olsztyn, Poland.
Melatonin (MEL) is a multifunctional neurohormone regulating circadian and seasonal
physiological processes in vertebrates, including those of reproduction and behaviour. It is
also known as a potent free radical scavenger protecting cells from oxidative stress. MEL is
synthesised mainly, but not uniquely, in the pineal gland from where it is secreted into blood
system. Its synthesis in the pineal gland is regulated by internal clock and by light. MEL
effect on the metabolic processes in cells is mediated by membrane receptors located in the
brain structures and many peripheral tissues. In birds there are 3 MEL receptors: mel-1a, mel1b and mel-1c, and in mammals: mt1 and MT2 (corresponding to mel-1 and b). The key
enzyme in MEL synthesis is arylalkylamine N-acetyl-transferase (AA-NAT) responsible for
acetylation of serotonin.
The role of MEL in embryo development is not known. Every mammalian embryo from the
very beginning of its development undergoes the influence of MEL circulating in maternal
organism. The situation is different in birds where the embryo is formed as an individual unit
separated from the maternal organism and should be self-sufficient in respect of nutritive and
biologically active substances, i.e. all the substances necessary for its development should be
either stored in the yolk or synthesised from the very beginning of its development. Our
studies concern the possibility of the early MEL synthesis in the early avian embryo, before
the pineal gland differentiation.
Using RT-PCR and sequencing methods, and Japanese quail oocytes and embryos as
biological material, we have shown in the oocytes the presence of two AA-NAT transcripts,
differing by the presence or absence of an intron (85 bp) (Accession no AY 197 460). The
ratio of both transcripts changed during oogenesis. In the adult pineal gland only the mature
product (without intron) was present while in the embryonic pineal gland till ~ 8th day of
incubation there were always 2 products found (with and without intron). In the embryo
pineal gland we observed the transition period between the 8th - 10th day of development when
the transcription of AA-NAT switched off from the embryonic (2 transcripts) to adult (1
transcript) pattern. The presence of all three MEL receptor transcripts was found both in the
adult and embryonic pineals, opening the possibility of feed-back regulation of MEL
synthesis. Mel-1c transcript was found in maternal RNA (in the oocytes) and the transcription
of Mel-1a and Mel-1b started during cleavage stages, in the uterine embryos. MEL and the
AA-NAT-like activity were present in the egg yolk. The presence of serotonin, the direct
substrate for AA-NAT enzyme, in the egg yolk has already been known for some years.
Our results point to the possibility of MEL action and synthesis in the avian embryo much
earlier than it was supposed, well before the pineal gland differentiation and beginning of its
activity. The role and significance of the autocrine MEL could be different from that of MEL
secreted by the pineal gland (regulation and synchronisation of the circadian rhythms), e. g.
protection against the free radicals formed abundantly during intensive metabolism and rapid
cleavage divisions, and/or transmission of morphogenetic signals between cells before
formation of neural network. Such role has already been attributed to serotonin and serotoninlike substances in the sea urchin embryos. The reported works are the first on the MEL
presence and synthesis during oogenesis and early embryonic stages.
EFFECT OF THYROID HORMONES ON AVIAN OVARIAN FUNCTION
Andrzej Sechman, Helena Paczoska-Eliasiewicz, Janusz Rząsa
Thyroid hormones (TH) are essential for the control of several physiological processes such
as growth, metabolism and thermoregulation. In recent years it has become clear that in
mammals adequate levels of circulating TH are of primary importance for normal female
reproductive functions. It has been shown that changes in level of triiodothyronine (T3), the
active form of TH, result in altered pituitary gonadotropin secretion, and directly modulate
FSH and LH action on steroid biosynthesis in follicular and luteal cells. In birds with seasonal
reproduction TH are responsible for the onset of the reproductive season, development of the
ovary and post-seasonal ovarian regression. In the domesticated birds, such as a chicken, the
seasonality of reproduction was eliminated and the role of TH in the ovarian function is not
fully elucidated. Our earlier investigations demonstrated: (1) presence of TH in wall of
chicken ovarian follicles; (2) changes in iodothyronines concentration in the ovarian follicles;
(3) presence of TH receptors in all compartments of the chicken ovary. To better characterize
the role of T3 in avian ovarian function, the effects of T3 was examined in in vivo and in vitro
experiments. The in vivo experiments carried out on Hy-Line Brown laying hens showed that
injections of T3 (50 µg/kg b.wt/day) for 6 consecutive days decreases ovarian weight on the
average by 42%. Moreover, both single (bolus) and multiple (for few days) treatment of
laying hens with the similar dose of T3 significantly decreases levels of estradiol (E2) in blood.
This is consistent with results of in vitro experiments which for the first time showed that T3
decreases basal and LH-induced E2 secretion by the theca layer of white prehierarchical and
yellow preovulatory follicles (F3-F1). On the other hand, T3 enhances progesterone (P4)
production from the granulosa layer of F3-F1 follicles, and augments LH-induced P4 secretion
by these follicles. In summary, our results clearly demonstrate that TH affects avian ovarian
function by regulation of follicular steroidogenesis. The effect of TH can be regarded as a part
of the complex multihormonal regulation of follicle development in ovary and may contribute
to explain the relationship between thyroid and ovarian function in the female of the
domesticated birds.
Poster presentation
AVIAN BIOTECHNOLOGY: MATURATION, OVULATION AND IN VITRO
FERTILIZATION OF THE BABCOCK300
Zine Laabidine Arhzaf
ECWSR, B. P: 9056 Ocean Rabat, 10 000 Morocco, Email: [email protected]
Fax: 0021237202689
The purpose of our work is to have in vitro maturation, and study various procedures to
induce in vitro ovulation and fertilisation in avian species (case of the Babcock300). And also,
to assess embryo development up to 24 hours after fertilisation. A total of 20 hens were
placed in individual cage. The hen is scarified 12 minutes after laying time; then the periovulatory follicle was collected before ovulation. The peri-ovulatory follicles were excised
from the ovary, washed in warm PBS (41degc), and re-suspended by their stalk to a receptacle
”glass” half filled with Dulbecco’s modified Eagle medium. The receptacles were kept in an
incubator (41degc), up to the time of ovulation. A total of 13 oocytes were recovered, placed
in an incubator (41degc), and then subjected to in vitro fertilisation by addition of
spermatozoa on to the germinal disk from which 11 embryos were obtained (84,6%), with
various degrees of development (from HH 4 to HH 7). Our research opens at international
level the door for further studies on rare birds transgenic manipulations.
Key words: Maturation, ovulation, fertilisation in vitro, transgenic, avian preservation.
ACID GLYCOSIDASES FROM HEN OVIDUCT
Dorota Błędniak, Maria Droba, Bogusław Droba
Department of Basic Natural Sciences of Agriculture, University of Rzeszów,
35-601 Rzeszów, Poland, e-mail: [email protected]
In all parts of the hen (Gallus domesticus) oviduct (infundibulum, magnum, isthmus, shell
gland, vagina), specific activity of seven acid glycosidases: β-hexosaminidase (β-HEX), αand β-galactosidase (α- and β-GAL), α- and β-mannosidase (α- and β-MAN), α-glucosidase
(α-GLU) and α-fucosidase (α-FUC) was investigated. The activity of all enzymes was the
highest in the isthmus and amounted to 336.1 for β-HEX, 6.1 for β-GAL, 2.8 for α-MAN, 2.8
for β-MAN, 2.5 for α-GAL, 1.7 for α-FUC and 1.3 mU/mg protein for α-GLU. Multiple
forms of acid glycosidases were separated by strong anion exchange chromatography (High
Pack Q, Bio-Rad) at pH 6.0. β-HEX activity in all parts of the oviduct was separated into
forms that were unbound and bound to the anion exchanger, the latter being eluted with NaCl
gradient at 4.8 mS. β-GAL activity was bound to the column and separated into two to three
forms with NaCl gradient. Only one form of β-GAL occurred in the isthmus. Up to 90% of
total α- and β-MAN activity in all parts of the oviduct except vagina was not bound to the
column, while all α-GLU and α-FUC activity was eluted with NaCl gradient at 8.3 mS. αGAL activity was separated into forms that were unbound and bound to the anion exchanger,
the latter being eluted in different parts of the oviduct at different concentrations of NaCl.
Chromatofocusing of the isthmus sample resulted in the appearance of more or less separate
multiple forms of acid glycosidases with pI values of 6.30, 6.05, 5.85, 5.65 and 5.34 for βHEX; 5.33, 5.24, 5.08 and 4.82 for β-GAL; 6.67, 6.51 and 6.10 for α-MAN; and 6.56 for
β-MAN. Secretory activity of the isthmus can be responsible for the high β-HEX activity in
the shell membrane [Win and Ball, Poultry Science 54: 799-805, 1975]. Our study was the
first to show high β-GAL activity in the shell membrane.
ROLE OF DOPAMINE RECEPTORS IN REALIZATION OF MELATONIN
EFFECTS ON HYPOTHALAMIC-HYPOPHYSEAL-GONADAL SYSTEM OF
BIRDS
Mykola E. Dzerzhynsky, Igor M. Varenyuk, Nataliya V. Nuzhyna
Department of cytology, histology and developmental biology, Kiev Taras Shevchenko
National University, Kiev, Ukraine, E-mail: [email protected]
It is known that melatonin is a regulator of diurnal and seasonal rhythms of an organism. This
hormone affects the reproductive system, too. Until now, all the mechanisms of melatonin
influence on the reproductive system are not known. Recent papers indicate that melatonin
can partially act through other neuromediators, e.g. through dopamine. Dopamine is known to
trigger D1 or D2 receptors. However, this process is not investigated thoroughly in birds,
especially regarding its diurnal variations.
The study was carried out on males of Coturnix coturnix japonica, which were maintained in
the photoperiod of 14L:10D. Single injection of physiological solution (into a muscle, and
into the III’d ventriculum of the brain), or melatonin (orally), or R(+)SCH-23390hydrochloride (blocker of dopamine D1-receptors, injection into the III’d ventriculum of the
brain), or R(+)SCH-23390-hydrochloride and then melatonin, or galoperidol (blocker of
dopamine D2-receptors, injection into a muscle), or galoperidol and then melatonin, or
R(+)SCH-23390-hydrochloride and galoperidol and then melatonin were administered to the
birds in the morning (at 7.00), in the afternoon (at 13.00), in the evening (at 19.00) or at night
(at 1.00). Afterwards, the hypothalamus, the adenohypophysis, and gonads were examined
morphometrically.
The study showed that melatonin treatment altered structure and functional state of the
hypothalamic-hypophyseal-gonadal system of birds differently when administered at the
different periods of the day. Introduction of melatonin in the afternoon stimulates
gonadotropocytes of the hypophysis and gonads. Thus, melatonin activity is in part
accomplished through dopamine system of the brain, namely through both D1 and D2
dopamine receptors. In a turn, the administration of melatonin in the morning, in the evening,
and at night oppresses gonads by different means. In the morning, melatonin oppresses the
reproductive system by stimulating the allocation of dopamine that function through D1 and
D2 receptors; in the evening – through D1 receptors; while at night the dopamine system is
disconnected from the melatonin chain.
Thus, melatonin affects the reproductive system by cooperation with dopaminergic system of
the brain. The character of this interaction changes depending on the time of the day.
Probably, this is one of the reasons for different results yielded by melatonin administration at
the different hours of the day.
DISTRIBUTION OF ACID GLYCOSIDASES IN THE MALE GENITAL
TRACT OF THE PHEASANT
Małgorzata DŜugan
Department of Basic Natural Sciences of Agriculture, University of Rzeszów, 35-959
Rzeszów, Poland, E-mail: [email protected]
The aim of the experiment was to compare acid glycosidase activities in the reproductive
organs of the pheasant (Phasianus colchicus). The study was carried out on 7 mature birds in
the reproductive season (May). Birds were obtained from a pheasantry at the age of 11
months and were of similar body weight (1270±150 g). Enzyme activities of β-Nacetylhexosaminidase (β-HEX), α- and β-galactosidase (α- and β-GAL), α- and βmannosidase (α- and β-MAN), α-fucosidase (α-FUC) and α-glucosidase (α-GLU) were
determined according to Barrett and Heath (Lysosomes: a laboratory handbook. Ed.
J.T.Dingle, Elsevier North-Holland, 1977). Significant (p<0.001) differences in acid
glycosidases activity dependent on the enzyme origin were observed. The highest activity of
the studied enzymes was found in epididymis (36.7, 26.8, 6.0, 3.3, 1.1 and 0.8 mU/mg protein
for β-HEX, β-MAN, β-GAL, α-GAL, α-FUC and α-GLU, respectively), lower activity in
deferent ducts, and the lowest activity in testis. Exceptionally for α-MAN, the highest activity
(39.8 mU/mg of protein) was found in the deferent ducts. The enzymes were separated into
multiple forms using anion-exchange chromatography (High Pack Q, Bio-Rad, pH 6.0, linear
gradient 0 – 0.5M NaCl). Elution profiles of β-HEX, α-MAN, α-GAL and α-GLU from the
tested reproductive organs were similar, usually one form unbound (minor) and one form
bound (major) to anion exchanger were observed. For β-GAL and α-FUC additional forms
binding to column were obtained from testis and deferent duct. The increase in the relative
contribution of unbound form was observed for β-MAN from epididymis and testis. The high
activity of acid glycosidases (especially α- and β-MAN and α-FUC) in excurrent ducts of the
testis of pheasant may be evidence for the role of these enzymes in sperm maturation during
their passage through the excurrent ducts of birds.
LEPTIN AS A REGULATOR OF CHICKEN EMBRYOGENESIS
Agnieszka Grzegorzewska1, Tomasz Jacek1, Marcin Lis2 and Helena PaczoskaEliasiewicz1
1
Department of Animal Physiology, 2Department of Animal Hygiene and Breeding
Environment, University of Agriculture, Al. Mickiewicza 24/28, 30-059 Krakow,
The role of leptin in avian embryogenesis is poorly known. The genes coding leptin and leptin
receptor have been cloned in chicken and turkey. In both cases high sequence identity has
been found between chicken and turkey genes. So far, only the long isoform of leptin receptor
has been identified though the possibility of existence of other isoforms has been suggested.
Expression of leptin mRNA has been detected in developing chicken embryo as early as day 3
of incubation. Expression pattern of leptin receptor mRNA in brain, liver and yolk sac has
been shown during turkey embryogenesis. It has been also reported that leptin stimulates
neovascularization of chicken chorioallantoic membrane, while in the Japanese quail
recombinant leptin injected in ovo advances embryonic and postembryonic development. In
the present study we examined in chickens the effect of in ovo administration of leptin on: 1)
the course and duration of embryogenesis and 2) expression of leptin receptor gene in
chorioallantoic membrane (CAM). We performed two experiments using fertilized Ross
Broiler Breeder eggs and recombinant ovine leptin prepared by genetic engineering (Gertler
et al., 1998). Experiment 1 was performed on 360 eggs divided into 5 equal groups. On day 5
of incubation three experimental groups were injected with leptin (0,5 µg, 5 µg and 50 µg
/egg), one control group with the solubilizing solution (aqua pro injectione) and one was not
injected. The significant acceleration of pipping and hatching time was observed in group
injected with 5 µg of leptin while the synchronization of hatching was seen in group injected
with 50 µg of leptin. Experiment 2 was performed on 120 eggs divided into two equal groups:
experimental group injected with 5 µg of leptin/egg and control group with solubilizing
solution. On day 12, 15 and 18 of incubation total RNAs of CAM fragments were isolated
using TRI-Reagent and RT-PCR reactions were carried out with primers specific for the
cytoplasmatic domain of the long form of leptin receptor. Data were standardized to 18S
rRNA and resultant images were quantified by densitometry using Scion Image analysis
software. Expression of leptin receptor mRNA in CAM was present in males and females at
all examined stages of embryogenesis. In group injected with leptin expression of leptin
receptor gene on day 15 and 18 of embryogenesis was lower than in control group but only in
females it was at statistical level. The observed decrease may suggest that female embryos are
more sensitive to leptin action. In summary, our results indicate that in chicken leptin may act
as a regulator of embryogenesis by affecting development of chorioallantoic vascular system.
SPERM PENETRATION OF THE PERIVITELLINE LAYER OF THE OVUM
AFTER ARTIFICIAL INSEMINATION OF BROILER BREEDER HENS
DEPENDING ON AGE
Małgorzata Gumułka
Department of Small Animal Breeding, University of Agriculture, Al. Mickiewicza
24/28, 30-059 Krakow, Poland, E-mail: [email protected]
Recently, the sperm penetration assay (SPA) has been used in birds to determine the number
of sperm that penetrate the outer layer of the ovum – inner perivitelline layer (IPVL), prior to
fertilization (Wishart and Staines, 1999; Brillard, 2003).
In all species of poultry fertility progressively decreased with the age but factors that
influence the age-related decline in reproduction are still poorly understood. So, it seemed
interesting to find out how the intensity of sperm penetration of the IPVL of eggs and the
related probability of fertilization changes during consecutive days after AI as hens age.
Broiler breeder hens (n=30-52) were AI at 31, 36, 43, 50, 56 and 62 wks of age. Additional
AI, with ejaculates of roosters aged 36 wks (R2), were performed at 62 wks of age. Hens were
inseminated for two consecutive days (D0 and D1) with an insemination dose of 125 · 106
spermatozoa /0.06 ml containing pooled ejaculates. Eggs for SPA were collected on the first
day after the second insemination (D2). SPA was determined in laid eggs by fixing and
staining the intact IPVL section with Schiff’s reagent (Bramwell et al.,1995). Holes after SP
were counted in four areas of 0.299 mm2 each using a Nikon-Eclipse E-800 microscope at
200x magnification. The values obtained were expressed as a number of holes per mm-2 of the
germinal disc area (SP/mm2 GDIPVL) and determined until no SP were observed in three
consecutively laid eggs.
Together with age of the flock decrease of sperm penetration holes in the perivitelline layer of
the eggs was observed. The greatest mean number of holes was found in eggs laid by hens on
day three (D3) post AI. After insemination at 50, 56 and 62 wks of age, it was respectively
12.2, 33 and 43.8% lower than in eggs laid at peak egg production (31-36 wks of age). Even
greater differences between peak egg production and the second half of reproductive life were
observed for SP/mm2 GDIPVL in eggs laid on the D2. At four days (D4) after AI, differences
in the intensity of SP in eggs laid after hen insemination at 43 and 50 vs. 56 and 62 weeks of
age were higher than those observed on D3.
The number of sperm penetration holes in eggs laid on first three days after AI illustrates the
possibility of accepting spermatozoa by sperm storage tubules (SST) in hens’ oviduct, and the
possible increase of their release from SST with hens’ age does not affect the results obtained
during this period. In light of the present study it is suggested that in the final period of egg
production a lower number of spermatozoa is stored in sperm storage tubules, which reduces
fertility of females.
LEPTIN-INDUCED THYROID APOPTOSIS IN IMMATURE CHICKENS
Tomasz Jacek and Helena Paczoska-Eliasiewicz
30-059 Krakow, Poland, E-mail:[email protected]
In mammals, leptin acts both as hormone and growth factor exerting wide pleiotropic effects.
By inhibitig food intake, enhancing energy expenditure and regulating metabolism leptin
maintains whole-body energy balance and controls reproductive processes. In birds, the
biological role of leptin is poorly known. Recombinant leptin markedly inhibits food intake as
reported in mammals and its blood concentration is under nutritional control. Our recent
studies have shown that injections of recombinant leptin during sexual maturation of the
domestic hen: (i) advanced the onset of puberty, (ii) decreased plasma concentration of T4,
(iii) enhanced the negative relationship existing during sexual maturation between ovarian and
thyroid activity and (iv) had no effect on plasma concentration of T3, the metabolically active
form of T4 produced mainly by peripheral deiodination of T4. The aim of the present study
was to examine whether changes observed in thyroid activity during sexual maturation in
leptin-treated pullets might be the result of leptin action on the epithelial cells of thyroid
follicles. Experiment was performed on 48 Isa Brown pullets fed ad libitum and kept in
individual cages under a photoperiod of 14L:10D. Experimental group was injected daily
(s.c.) with recombinant chicken leptin at a dose of 256 µg/kg body weight from 79 day of age
until the onset of puberty evidenced by first oviposition. Control group (n=24) received PBS
with BSA. The left and right lobe of thyroid glands isolated from six randomly chosen 106day-old pullets of both groups were weighed, embedded in paraffin and 6 µm tissue sections
were examined for histological (H&E staining) and apoptotic (TUNEL staining) changes in
the epithelium of thyroid follicles. The results (mean ± SE) are presented in the table given
below:
Leptin
dose
(µg/kg
b.wt./day)
Weight of
thyroid gland
0 (n=24)
168 ± 7.0
(n=6)
(mg)
197 ± 10.9*
(n=6)
**
P<0.01,*P<0.05 vs control
256 (n=24)
106 day-old pullets
Thickness of
Number of
follicular
epithelial
epithelium
apoptotic cells
(µm)
(50 µm x 50 µm)
Onset of puberty
(day of age)
4,6 ± 0.26
(n=12)
14.9 ± 0.41
(n=12)
124.4 ± 1.7
(n=18)
3.4 ± 0.12**
(n=12)
16.7 ± 0.79*
(n=12)
118.4 ± 1.4*
(n=18)
Our results suggest that during sexual maturation of the domestic hen leptin might inhibit
thyroid activity by proapoptotic action on the epithelial cells.
ARYLSULPHATASE DURING POST-NATAL DEVELOPMENT AND
INVOLUTION OF TESTES AND EPIDIDYMIDES FROM JAPANESE QUAIL
Radosław Józefczyk1, Maria Droba1, Bogusław Droba1, Andrzej Witkowski2
1
Department of Basic Natural Sciences of Agriculture, University of Rzeszów,
35-601 Rzeszów, 2Department of Basic Natural Sciences of Animal Production,
University of Agriculture, 20-950 Lublin, Poland,
During post-natal development of the Japanese quail (Coturnix coturnix japonica), testicular
activity of arylsulphatase decreased 5-fold between 8 days of age and the onset of sexual
maturity at 2 months of age. Involution of the testes and epididymides was induced in
sexually mature quail by introduction of a shortened photoperiod (from 16 L : 8 D to 6 L : 18
D). Enzyme activity was determined at 6, 10, 15, 20, 24 and 31 days of the experiment.
Arylsulphatase activity in the testes increased until day 20 (3-fold in relation to the control)
and then persisted at the same level. Multiple forms of arylsulphatase from the testes and
epididymides were separated by strong anion exchange chromatography (High Pack Q, BioRad) at pH 6.0. Arylsulphatase from the testes and epididymides was separated into forms
that were unbound (I) and bound (II) to the anion exchanger. In pubertal quail, form I
accounted for 95% of enzyme activity, while in immature testes weighing 2-10 mg, 90% of
arylsulphatase activity accounted for form II. The preparation of epididymides weighing
approx. 5 mg was started in 27-day-old quail. Until puberty at 2 months of age, arylsulphatase
activity in the epididymides was found to increase 5-fold. During epididymidal involution,
arylsulphatase activity was found to increase 2-fold on day 10 of the experiment and to
decrease 3-fold on day 31 compared to the activity of this enzyme in the epididymides of the
control quail. Elution profiles of arylsulphatase activity during post-natal development and
involution of epididymides were highly similar to the profile of the activity of this enzyme
from the testes. It is worth adding that the analysed epididymides weighing approx. 5 mg were
the earliest to show clear predominance of form II of arylsulphatase (up to 60% of total
activity).
ISOLATION, CHARACTERIZATION, AND cDNA SEQUENCING OF
KAZAL-TYPE PROTEINASE INHIBITOR FROM SEMINAL PLASMA
OF TURKEY (Meleagris gallopavo)
Mariola Kotłowska1, Mariusz Olczak2, Mariola Wojtczak1, Jan Glogowski3, Jan
Jankowski4, Wiesław Wątorek2, Andrzej Ciereszko1
1
Department of Semen Biology, 3Department of Molecular Andrology, Institute of
Animal Reproduction and Food Research, Polish Academy of Sciences, 10-747
2
Institute of Biochemistry and Molecular Biology, Wroclaw University, 50-137
Wroclaw, Poland; 4Deparment of Poultry Science, University of Warmia and Mazury,
10-719 Olsztyn, Poland.
Our previous study examined serine proteinase inhibitors in turkey seminal plasma with
relation to their distribution within the reproductive tract. We found three serine proteinase
inhibitors in turkey seminal plasma: inhibitor of low migration rate similar to α1-antiproteinase-like protein and two other inhibitors of moderate (Ia) and fast migration rate (Ib)
that may be similar to low molecular weigh proteinase inhibitors described for chicken
seminal plasma. Additional knowledge concerning the characterization of inhibitors in turkey
semen is required for better understanding of turkey semen physiology. The objective of this
study was to isolate and characterize serine proteinase inhibitors of low molecular weigh
present in turkey seminal plasma.
Two serine proteinase inhibitors (Ia, Ib) have been isolated by affinity chromatography
(column X/K 16/10 of chymotrypsin-Sepharose 4B), anion exchange chromatography
(column X/K 16/10 of HiLoad Q-Sepharose) and RP-HPLC (BioBasic-8 column). Using this
three-step procedure, we obtained 450-fold purified inhibitor Ia and 180-fold purified
inhibitor Ib. The molecular weight of native inhibitors were estimated using gel filtration in an
X/K HiLoad 16/60 Superdex 75 column and were determined to be 6.3 kDa for Ia inhibitor
and 7.2 kDa for inhibitor Ib. Both inhibitors effectively inhibited trypsin but not
chymotrypsin. They formed a complex with trypsin that was not dissociated in SDS except
boiling, indicating that a covalent bond was not formed. N-terminal Edman sequencing of Ia
inhibitor allowed identification of the first 22 N-terminal amino acids
SIPPACDKYSHLPGCPRDYNNV and the same 15 amino acid we obtained for Ib inhibitor.
This sequence was used to construct primers and obtain a cDNA sequence from the testis. The
mRNA coding the protein is 401 nt in the length including a single open reading frame of 246
nt that encodes 81 amino acid residues. Analysis of this sequence confirmed the similarity to
bird caltrin (Galus galus) and mammalian acrosin-trypsin inhibitor (HUSI -II).
To our knowledge this is the first report confirming the presence of Kazal-type proteinase
inhibitors in turkey seminal plasma. These inhibitors appear to be specific for turkey
reproductive tract and their physiological function may be related to the inactivation of
acrosin from dead or damaged spermatozoa.
SEMEN QUALITY EVALUATION IN ONE-YEAR-OLD BIŁGORAJ
GANDERS
Justyna Kugla–Owczarska1, Helena Puchajda–Skowrońska1 , Marek Opałka2, Luiza
Dusza2
1
Department of Poultry Breeding, 2Department of Animal Physiology, University of
Warmia and Mazury, 10-719 Olsztyn, Poland, E-mail: [email protected]
Low fertility of geese is a factor limiting not only the breeding progress and improvement but
also production intensification. The relatively low reproduction indices in geese, as compared
with other species of domestic fowls, are due to a short reproduction cycle and a decrease in
the sexual activity of ganders. The latter of the two, observed especially in the second half of
the reproduction season, has a significant effect on egg fertilization rate.
The aim of the present study was to evaluate semen quality. The following parameters were
analyzed: ejaculate volume, and concentration and quality of spermatozoa obtained from
individual ganders in successive weeks of the reproduction period.
The experiment was performed on 20 one-year-old Biłgoraj ganders. During the period
preceeding the reproduction season (December – January), the ganders were accustomed to
massage and semen collection procedure. In consecutive months (February - May), semen
samples were collected from each gander twice a week (Monday - Friday), by dorsalabdominal massage. Ejaculate volume, sperm concentration and morphology were analyzed.
At the beginning of the reproduction season (February) ejaculate volume was 0.16 ml. In
consecutive months it increased by 0.04 – 0.03 ml, to reach the highest level (0.2 ml) in
March and April. The highest variation of this parameter was recorded in February (v% =
60.18). In the other months under analysis the coefficient of variation of ejaculate volume
was 26.13 to 33.97%. The ejaculates collected in April were characterized by the highest
sperm concentration (174 400/mm3), and those collected in May – by the lowest (9 560/mm3).
As regards particular forms of spermatozoa, the proportion of normal sperm cells was 55.48%
in February, 62.09% in March, 62.72% in April, and 60.41% in May. The coefficient of
variation of this parameter ranged from 3.31% in February to 17.21% in May. The highest
proportion of damaged sperms was noted in February (abnormally shaped heads – 12.48%,
broken necks – 7.92%, spirally twisted spermatozoa – 4.96%). The most dead spermatozoa
were found in May (9.44%), and the least in March (6.55%).
The results showed that the quality of semen collected from the ganders widely differed over
the entire reproduction cycle. The best-quality semen was acquired at the peak of
reproduction (March - April).
HATCHING VALUE OF TURKEY EGGS STORED FOR LONGER PERIODS
OF TIME
Emilia Mróz, Aneta Orłowska, Katarzyna Michalak
Department of Poultry Breeding, University of Warmia and Mazury, 10 – 719
The problems related to egg hatchability remain topical despite wide experience and manyyear practice in artificial incubation. This concerns first of all a too low number of healthy
hatchlings. One of the factors affecting hatchability is egg storage time.
The objective of the present study was to determine the effects of prolonged from 7 to 12 days
egg storage time on embryo death, distribution of embryo mortality during incubation, and
hatching rate. The experimental materials comprised hatching eggs obtained from white
heavy broad-breasted turkeys in the 12th to 18th week of the laying season. The experiment
was performed on eggs stored for 7 to 12 days. 126 eggs were randomly selected for
incubation from each storage period. A total number of eggs selected from a given storage
period was 378 (126 eggs x 3 hatches). The eggs were given consecutive numbers, and their
weights were determined one or two days before incubation. The eggs were candled after 9 to
11 days of incubation, in order to eliminate unfertilized eggs and eggs containing dead
embryos. The eliminated eggs were broken and subjected to embryopathological
examinations. The numbers of dead and unhatched embryos, as well as the distribution of
embryonic mortality, were determined after incubation. The numerical data were analyzed
statistically by one-factor analysis of variance in an orthogonal and non-orthogonal design.
The significance of differences was determined by the Duncan test.
Due to the experimental period, egg weight was not differentiated and remained within
relevant norms for turkey hatching eggs (75 – 95 g). Egg fertilization rate was high (94.1 –
96.5%). Hatching rate was lower in the case of eggs laid and stored for more than 9 days (75.1
– 78.5%), in comparison with those stored for 7 to 9 days (82 – 84.6%). As regards fertilized
eggs, hatching rate was also lower in the batches stored for 10 to 12 days (79.7 – 82.9%) than
in those stored for 7 to 9 days (85.6 – 87.6%). Embryo mortality rate varied from 10.2 to 14%
over the entire incubation period, and was lower in the group of eggs stored for 7 to 9 days
(10.2 – 10.4%). The proportion of unhatched embryos was higher in the egg batches stored
for 10 to 12 days, as compared with the other ones (6.1 – 6.4 and 1.9 – 4.1, respectively).The
first peak of embryonic mortality, regardless of egg storage time, was recorded after 2 to 3
days of incubation. It reached the highest level (30%) in the group of eggs stored for 12. days.
Another peak of embryonic mortality was noted on the 28th day of incubation. It reached the
highest level (53%) in the group of eggs stored for 10 days. Additional peaks of embryonic
mortality (mortality rate > 5%) were also observed on the 10th, 21st, 22nd and 25th day of
incubation in the batches of eggs stored for 7, 8, 9 and 11 days. The inter-peak period lasted
for 14 to 23 days, and was longer in the groups of eggs stored for longer than 9 days. The
length of storage period affects the hatching value of eggs, increases embryo death and
decreases hatching rate. In the group of eggs stored longer than 10 days embryonic mortality
was higher on the first days of incubation.
Key words: turkeys, egg storage time, hatchability, embryo mortality.
THE INFLUENCE OF PHYTOESTROGENS ON TESTOSTERONE
SECRETION BY LEYDIG CELLS IN BIŁGORAJ GANDERS
Marek Opałka1, Barbara Kamińska1, Helena Puchajda-Skowrońska2, Luiza Dusza1
1
Department of Animal Physiology, 2Department of Poultry Breeding , University of
Warmia and Mazury in Olsztyn, 10-719 Olsztyn, Poland,
The phytoestrogens, possessing estrogen-like activity, have been found to influence human
and animal reproduction by changing the functions of reproductive organs. These plant
hormones present in legumes, which are the compounds of goose feed. Since there are few
data concerning phytoestrogens and testicular steroidogenesis in domestic fowl, the aim of the
study was to examine the effect of phytoestrogens on testosterone (T) secretion by isolated
Leydig cells in ganders. In addition, the study served to investigate the possibility of
phytoestrogen action by estrogen or androgen receptors and by protein tyrosine kinases
pathway.
The testis were obtained from six sexually matured Biłgoraj ganders. Isolated Leydig cells
(1×105/ml) were incubated for 20 h (37°C) with or without: LH (20 ng/ml), phytoestrogens:
genistein, dadzein, equol and coumestrol (0.5, 5, 50 µM), estradiol (5, 50, 500 pg/ml),
cyproterone acetate - androgen receptor antagonist (10 µM), hydroxytamoxifen - estrogen
receptor inhibitor (10 µM), lavendustin A - protein tyrosine kinases inhibitor (0.5, 5, 50 µM).
Medium concentrations of T were measured by radioimmunoassay.
Genistein (5 and 50 µM), dadzein (50 µM) and equol (50 µM) inhibited basal and LHstimulated T secretion by Leydig cells. Coumestrol (5 and 50 µM) only decreased basal T
release. Estradiol and lavendustin A did not change T production. The effects of cyproterone
acetate and hydroxytamoxifen on phytoestrogen inhibition of T secretion by Leydig cells
were not observed.
In conclusion, phytoestrogens inhibited basal and LH-stimulated T secretion by Leydig cells.
The influence of genistein seems to be the strongest and the effect of coumestrol appears the
weakest. Moreover, the results suggested that phytoestrogen action in gander Leydig cells
depends on neither androgen and estrogen receptors nor inhibition protein tyrosine kinases
system.
COMPARISON OF CARDIAC WORK IN EMBRYOS OF BROILER
CHICKEN AND DUAL PURPOSE BREED
Krzysztof Pawlak, Barbara Tombarkiewicz, Jerzy Niedziółka
Department of Animal Hygiene and Breeding Environment, University of Agriculture,
al. Mickiewicza 24/28, 30-059 Krakow, Poland, E-mail: [email protected]
Embryonic development is an important stage of bird life since it exerts a major effect on the
biological value and performance of adult bird. For this reason, the possibility of monitoring
embryonic development is highly desirable. Cardiac work is one the first signals of activity of
the developing organism in the egg. The purpose of the present study was to compare the
cardiac work of chick embryo of broiler chickens and dual purpose breed.
20 broiler eggs (Coob; group I) and 20 eggs of dual purpose breed (Rosa; group II) were used
in the experiment. Incubation was carried out in a laboratory incubator under standard
conditions. The cardiac work of the developing chicken embryo was measured with the
balistocardiograph. The signal coming from the embryo was recorded at the same intervals of
time throughout the incubation period. The signal from each egg was registered for 60
seconds and analyzed by a computer. Digital signal processing was used to determine the
number of heart rate. The results were statistically analyzed by t-test.
A repeatable signal of the heart work of all embryos was recorded for the first time on 9 day
of incubation. The number of heart rate is one of the most frequent parameters used to
characterize the cardiac work of the avian embryo. In the present experiment it was found that
in both groups the highest frequency of cardiac work occurs on 9 day of incubation (in group I
– on the average 260 contractions per minute and in group II - 243 contractions per minute).
The lowest heart rate was recorded on the final day of incubation (185 and 158 contractions
per minute in group I and II, respectively). Analysing the trends for the average number of
heart rate on subsequent days of incubation it was affirmed that in both groups heart
contractions decreased from 9 to 12 day and then they became stable between 12 and 16 day
of incubation. After 16 day of incubation the decrease in the frequency of heart work was
observed. In the present experiment it was found that the heart rate of broiler chicken embryo
was always higher in comparison with the embryo of the dual purpose chicken breed. The
average differences between group I and II were 18 contractions per minute and were
statistically significant.
Studies supported by grant: DS-3210/ZHZiŚH
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