Coetaneous Serum Supplementation of in vitro Culture Media

Journal of Animal and Veterinary Sciences
2015; 2(5): 47-52
Published online September 28, 2015 (http://www.openscienceonline.com/journal/javs)
Coetaneous Serum Supplementation of in vitro
Culture Media Improves Morphogenesis and
Development Gastrulation Embryos Rabbit
J. S. Vicente1, A. Parrilla-Ocon1, A. M. Saeed2, F. Marco-Jimenez1
1
2
Institute for Animal Science and Technology, Polytechnic University of Valencia, Valencia, Spain
Animal Production Research Institute, Animal Biotechnology Department, Dokki, Giza, Egypt
Email address
[email protected] (F. Marco-Jimenez)
To cite this article
J. S. Vicente, A. Parrilla-Ocon, A. M. Saeed, F. Marco-Jimenez. Coetaneous Serum Supplementation of in vitro Culture Media Improves
Morphogenesis and Development Gastrulation Embryos Rabbit. Journal of Animal and Veterinary Sciences.
Vol. 2, No. 5, 2015, pp. 47-52.
Abstract
The current study was conducted to investigate the role of blood serum from coetaneous pregnancy rabbit does on in vitro
development of rabbit late blastocyst. Embryos at 6 days post-coitum were recovered from nulliparous rabbit does. Normal
recovered late blastocysts were cultured in TCM 199 (Control group), TCM199 supplemented with 6 days post-coitum serum
blood (10% v/v, Serum group) or TCM199 supplemented with 9 ηg of progesterone water soluble (Steroid group). After 48h
culture, a significant differences on the rate of embryos reaching the advanced gastrulation stages 4 to 7 were observed. Medium
supplemented with coetaneous serum showed the highest rate with a 73.7% vs media with supplemented with progesterone o
without supplementation (47.4% and 48.4%, P<0.05, respectively). Furthermore, blastocysts dimensions were significantly
increased after 48h of culture in coetaneous serum or progesterone supplemented medium, and dead rates of blastocyst was
higher in no supplemented medium than in supplemented one. In conclusion, coetaneous serum supplementation improves the
gastrulation progress of rabbit blastocyst of 6 days old, reaching at 48h of culture, gastrulation states analogous to in vivo. While,
progesterone supplementation facilitate the blastocyst growth but not the gastrulation process. Supplementation of medium with
coetaneous blood serum may facilitate the study of autonomous part of embryonic differentiation and to analyze by steps the
interaction complex mechanism between conceptus and endometrium leading to success or failure of implantation process.
Keywords
Blastocyst, Progesterone, Serum, Conceptus, Implantation
1. Introduction
The embryonic mortality in the first stage of pregnancy can
reach to 50% in rabbits [1], [2], [3] or 33% in cattle of total
prenatal dead [4]. Establishment of pregnancy begins at the
conceptus stage and includes pregnancy recognition signaling,
implantation, and placentation [5], [6], [7], processes in which
embryonic and endometrial cells under the action of endocrine
and paracrine factors are sequentially triggered [8]. Study the
role of each cell type, peptide, growth factor are important to
characterize the causes from a biological point of view to
understand the way that prenatal mortality occurs.
Estrogen and progesterone play an important role in the
development and implantation of preimplantation embryos [9].
Elimination of these hormones from pregnant animals causes
deleterious effects on the development and implantation of the
embryos. Therefore, it appears to be important to maintain the
concentrations of estrogen and progesterone at appropriate
levels relative to each other. Elevated ratios
progesterone-estradiol in early luteal phase inhibit blastocyst
metabolism and implantation in mice [10] or retarded
embryonic development in sheep and cattle [11]. While
increasing concentrations of progesterone in vivo and in vitro
enhanced metabolism and conceptus elongation in bovine
embryos [11], [12], [13], [14] and in vivo in sheep [15].
To study the different factors affecting early embryo
development, standard culture conditions can be used to
blastocyst stage, nevertheless blastocyst growth into an
elongated conceptus does not occur in vitro, endocrine
stimulus such as ovarian steroids and endometrial secretions
seem be necessaries [16]. Therefore, studies of the
48
J. S. Vicente et al.:
Coetaneous Serum Supplementation of in vitro Culture Media Improves Morphogenesis and
Development Gastrulation Embryos Rabbit
gastrulation process have been carried out mainly in
laboratory species after euthanized the females at different
moments gestation. Gastrulation in rabbit has been well
studied and, at least, 7 gastrulation stages have been identified
and characterized, using morphological and molecular
methods [17], [18], [19], [20]. Rabbit model offer the
possibility to follow an accurate embryo development
chronology.
This study was carried out to investigate the effect of
coetaneous blood serum or progesterone supplementation in
culture medium on in vitro development of day 6
preimplantation rabbit blastocysts in order to use as a model in
further studies.
2. Materials and Methods
2.1. Animals and Study Sample
All experimental procedures involving animals were
approved by the Research Ethics Committee of the UPV and
licensed by European Community Directive 86/609/EC.
Rabbits from a maternal selected line were used at the
experiment. Maternal line is based on New Zealand White
rabbits selected since 1980 by family index selection for litter
size at weaning [21].
Animals were kept individually in flat deck cages under 16
h light/8 h dark conditions and were fed with a commercial
diet ad libitum and had free access to water. Multiparous
females between third and sixth parity were inseminated with
a semen pool belong to the same selected line.
2.2. Embryo Collection and in vitro Cultured
Donor does from maternal line were slaughtered at 6 days of
pregnancy. Embryos (n=107) were recovered by perfusion of
each oviduct and uterine horn with 20 mL pre-warmed
Dulbecco Phosphate Buffered Saline supplemented with 0.2%
of Bovine Serum Albumin. The blastocysts were randomly
distributed in round-bottomed microwell plates (Nunc,
Roskilde, Denmark) with 750 µL of three media. The control
medium was TCM-199 with 25mM hepes buffer and antibiotics
(100 IU of G sodium penicillin and 25 µg/mL of
dihydrostreptomycin) (Control group). The second medium
contained the same components as above described plus 9
ηg/mL of progesterone water soluble (Steroid group), the same
levels found in rabbit blood serum at 6 day of pregnancy. And
the third medium contained the control medium, but
supplemented with 10% (v/v) of heat-inactivated coetaneous
rabbit serum (Serum group), pre-filtered in a 0.2 µm pore
diameter. The culture microwell plates with 2 blastocyst by hole
were kept under a humified atmosphere of 5% CO2 at 38°C.
2.3. Serum Preparation
Whole blood was collected with the aid of a
Vacutainer-heparin tube (LH/Li Heparin Tube TAPVAL®, Mon
Lab, SL. Barcelona, Spain) from 8 donor does of 6 day-old
blastocysts. Blood was centrifuged (1500 g, 10 min at 4ºC) and
sera were detoxified at 56°C for 30 minutes before frozen and
stored at -8°C. Serum levels of progesterone (steroid C21,
preg-4-ene-3, 20-dione) was determined by direct enzyme
immunoassay technique following the manufacturer’s
instructions (Rabbit Progesterone Elisa Test, Endocrine
Technologies, Inc. Newark, USA). Sensitivities of the tests used
were 0.1 ng/mL for progesterone). Progesterone levels of serum
were measured and the blood serum concentration of
progesterone were 9.0±1.22 ηg/mL respectively. A pooled
coetaneous serum was used to supplement the third medium.
2.4. Morphometry and Gastrulation Analysis
Embryos were morphological and physically evaluated for
their developmental progression during the in vitro culture by
microscopy. Every 24 hours, diameter and area of the
trophoblast blastocyst and perimeter and area of the
embryoblast were registered using Image J software.
Gastrulation stage and viability (hatched or collapsed) were
noted down as well.
Gastrulation stages were assigned according to the
cataloguing made by Blum et al. [19]. Gastrulation stages
were grouped to analysis according to Hensen´node formation,
stages 0 to 3 were grouped as early stage (without
Hensen´node) and the stages 4 to 7 were grouped as advanced
gastrulation development (Figure 1).
Hatched or collapsed blastocysts were stained with a
differential vital dye (propidium iodide and SYBR-14, -Life
technologies inc-). We observed that all collapsed embryos
were died and all hatched were alive (Figure 2).
2.5. Statistical Analysis
All analyses were performed with SPSS 16.0 software
package (SPSS Inc.). Values were considered statistically
different at P < 0.05.
Perimeter and area of blastocyst and gastrula were analyzed
by an analysis of variance using as fixed effect culture time by
embryo group. Percentage of collapsed (dead embryos) and
gastrulation stage (early and advanced) were analyzed using a
chi-square test with Yate’s correction.
3. Results
All 6-old day blastocyst recovered showed a stage 0 before
to begin the culture period. Supplementation of culture
medium with coetaneous blood serum (Serum) enhanced the
development rate of gastrula to reach an advanced stage
compared with the control and steroid groups. After 48h of
culture the percentage of blastocyst reaching advanced
gastrula stage were 73.7% (28/38), 47.4% (18/38) and 48.4%
(15/31) in coetaneous serum, control and progesterone
supplemented group, respectively (P<0.05, data not shown in
tables, Figure 1).
On the other hand, the percentage of collapsed embryo (died
blastocyst during culture) were higher in control than in
coetaneous serum group (39.5% - 15/38- vs 13.3% - 5/38,
P<0.05), while 8 blastocyst died in progesterone supplemented
group (25.8%, data not showed in tables, Figure 2).
Journal of Animal and Veterinary Sciences 2015; 2(5): 47-52
49
Fig. 1. Gastrulation stages of the rabbit embryo observed in vitro in control, progesterone and coetaneous serum groups.
groups Stages 0 to 3 were grouped as early
stage (without Hensen´node). Stages
tages 4 to 7 were grouped as advanced stage (with Hensen´node).
Fig. 2. Hatched (alive, A) and collapsed (died B) blastocyst stained with SYBR-14
SYBR 14 and propidum iodide after 48 of in vitro culture.
Embryo blastocysts and gastrula
strula of all experimental
groups grew significantly during the first 24 hours of in vitro
culture, increasing a 133% and 130% their perimeters and
195% and 164% their areas respectively, and after 48h the
increments reached the 171-177%
177% to perimeters and
300-318%
318% to areas in the supplemented groups (coetaneous
serum and progesterone groups, Table 1). Nevertheless,
because blastocyst hatched or collapsed at 24 and 48 hours of
culture, so only those remained in spherical shape,
morphometric measurements were
wer taken. In these blastocysts
were observed, the favourable effect of coetaneous serum
supplementation on blastocyst size at 24h and 48h of culture
and on gastrula dimensions after 48h of culture (Table 1).
While progesterone group support the growth of blastocyst
bl
but not the gastrula at 48h of and no supplemented medium
allowed only the growth in the first 24h of culture Noted that,
few blastocysts remaining at 48h in control and progesterone
groups.
50
J. S. Vicente et al.:
Coetaneous Serum Supplementation of in vitro Culture Media Improves Morphogenesis and
Development Gastrulation Embryos Rabbit
4. Discussion
spherical shape, morphometric measurements were taken and
differences were observed at gastrula level. So, progesterone
could be affecting the proliferation of trophoblast cells.
Progesterone supplemented medium enhanced mainly the
growth of blastocyst increasing substantially the size but not
enhanced an improvement in gastrula size or in the
gastrulation process.
The effects of progesterone can be different related to
embryonic stage and environment. Inhibitory effects
following supplementation of the culture medium with
progesterone has been reported in mice, rabbit and rat early
embryos, wen progesterone was added in pharmacological
does [23], [24], [25]. But a combination of progesterone and
uterine proteins has even been shown to stimulate the
development of mouse and rabbit early embryos [26]. By
contrast, it well known that rabbit blastocyst accumulates
progesterone into blastocoel fluid, probably originate from
uterine fluid and it can use as a metabolic precursor of
estradiol and estrone, those may have importance as pig
embryos like an embryonic signal for maternal recognition of
pregnancy [27], [28], [29].
After 48 hours of culture, more than 47% of alive spherical
and hatched blastocyst from all media reached the advanced
gastrulation stages described in the literature to in vivo seven
day (stages 4 and 5, beginning neurulation, Figure 1) showing
an autonomous gastrulation progress but, only 8 blastocyst
from coetaneous serum group reached the stage 6 or 7,
equivalent to 8 days (21%, Figure 1). Media without or with
progesterone do not support a normal gastrulation progress for
48 hours, other constituents of coetaneous blood serum allow
that the last stages can be reached, may be the presence of
growth factors similar to secrete by endometrial cells.
The embryo develops in tubal and uterine
microenvironments that are mainly controlled by progesterone,
that it may act directly or indirectly promote the production
and secretion of endometrial cytokines which contribute to
embryonic survival and development [22]. Nevertheless, the
role of ovarian steroids in the development of mammalian
blastocyst is very incomplete, it still remain without knowing
whether the presence of progesterone receptors in some
embryonic stages is indicative of whether it favours the
transcription processes related to development (metabolism
and/or differentiation). It is therefore, that obtain optimal
extrauterine environments could permit elucidate the
autonomous part of embryonic differentiation and to analyse
by steps the interaction complex mechanism between
conceptus and endometrium leading to success or failure of
implantation process. In this preliminary study, we obtained
good survival and gastrulation rates supplementing a
conventional embryo culture media with coetaneous serum,
rabbit blood serum adds growth factors as epidermal and
insulin growth factors, as well as favorable relationship
progesterone: 17-β estradiol. In this medium, gastrulation
process progresses in some embryos to similar stages
observed in vivo by other authors [19], [20]. While it is true
that a high percentage of blastocysts do not escape and stay
spheroid shape and surrounded by gloiolemma (52.6%). We
highlight the fact that hatching is an ordinary physiological
event in rabbit uterus and consequently a sign of well
development. At day 7 in vivo, it was quite difficult to observe
the gastrula in these embryos since they have begun the
endometrium attach process and lost their spherical shape. In
blastocyst at 24 and 48 hours of culture that remained in
Table 1. Embryos and gastrula size in the different culture media.
Culture time (hours)
Group
Control
0
24
Steroid
38
31
Embryo (LSM ± SE)
2
Area (mm )
a
7.6 ± 0.87
a
a
7.5 ± 0.97
9.6 ± 0.45
a
8.4 ± 0.87
Control
33
13.6 ± 0.94b
Control
22
31
8
Perimeter (mm)
9.7 ± 0.40
38
Steroid
Gastrula (LSM ± SE)
a
Serum
Serum
48
n
10.2 ± 0.36
15.9 ± 1.15
bc
16.4 ± 0.97
c
13.6 ± 1.90
bc
d
Steroid
8
23.9 ± 1.90
Serum
20
25.2 ± 1.20d
Area (mm2)
Perimeter (mm)
0.88 ± 0.101a
3.4 ± 0.18a
0.81± 0.114
a
12.8 ± 0.43b
13.8 ± 0.53
bc
14.2 ± 0.45
c
12.8 ± 0.88
b
17.0 ± 0.88
d
17.4 ± 0.55d
a
3.3 ± 0.21a
a
0.89 ± 0.101
3.3 ± 0.18a
1.40 ± 0.114bc
4.2 ± 0.21bc
bc
4.1 ± 0.27bc
b
4.4 ± 0.20c
ab
3.3± 0.46ab
1.33 ± 0.147
1.49 ± 0.112
0.89 ± 0.254
ab
1.12± 0.254
4.0 ± 0.46abc
1.61 ± 0.147c
4.8 ± 0.26c
n: number of farms. Control TCM199 culture, Steroid: TCM199 plus 9.0 ηg/mL progesterone. Serum: TCM19 plus 10% (v/v) serum does rabbits. N: number of
samples. abValues with different letters in the same column are significantly different (P<0.05). LSM ± SE: Data are expressed as Least square means ± standard
error.
Progesterone is associated with the greatest changes in
embryo growth: provoking differences in the distribution of
cells of the inner cell mass vs trophoblast [30], regulating via
the endometrium gastrulation and elongation of conceptus [11],
[13], preventing apoptosis and early embryo losses. Satterfield
et al. [15] showed treatment with progesterone increased sheep
blastocyst diameter by 220% on day 9 and advanced the time of
elongation of blastocysts to a filamentous conceptus on day 12,
also progesterone stimulation of blastocyst growth is
manifested after shedding of the zona pellucida. In accordance
with Satterfield et al. [15], the addition of serum from pregnant
rabbit does or progesterone at physiological level to the culture
media increased the size of embryos but only serum improve
the rate of embryos with Hensen´node.
Journal of Animal and Veterinary Sciences 2015; 2(5): 47-52
5. Conclusion
Current research is focused upon understanding the
importance of progesterone to initiate its normal
developmental and gastrulation program. The effect of
coetaneous blood rabbit serum or progesterone enhanced
growth of rabbit late blastocyst during in vitro culture but only
coetaneous serum supplementation allowed that the
gastrulation progress to neurulation. It should help to better
define the limits of the early mammalian embryo development
autonomy and it is necessary to design healthier in vitro
culture conditions.
Acknowledgements
English text version was revised by N. Macowan English
Language Service.
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