Nitric oxide-dependent activation of pig oocytes

Transcription

Molecular and Cellular Endocrinology 242 (2005) 16–22
Nitric oxide-dependent activation of pig oocytes: Role of calcium
Jaroslav Petr a , Radko Rajmon b , Vilma Lánská b,∗ , Markéta Sedmı́ková b , František Jı́lek b
b
a Research Institute of Animal Production, Přátelstvı́ 815, Prague 10, Uhřı́něves, Czech Republic
Czech University of Agriculture in Prague, Faculty of Agrobiology, Food and Natural Resources, Department of Veterinary Sciences,
Kamýcká 129, 16521 Prague 6, Suchdol, Czech Republic
Received 9 February 2005; received in revised form 22 April 2005; accepted 11 May 2005
Abstract
Pig oocytes matured in vitro are parthenogenetically activated after treatment with nitric oxide (NO)-donor SNAP. The chelation of
intracellular calcium ions with BAPTA-AM suppressed the SNAP-induced activation in a dose-dependent manner. Activation by a NO-donor
is dependent on the influx of calcium from extracellular spaces, because the blockage of calcium channels by verapamil had significantly
reduced the activation rate in SNAP-treated oocytes. The blockage of inositol triphosphate receptors had no effect on the activation of oocytes
by a NO-donor. On the other hand, the blockers of ryanodine receptors, procaine and ruthenium red, inhibited the activation of oocytes induced
by a NO-donor.
These data indicate that the activation of pig oocytes by a NO-donor is calcium-dependent. The calcium for the activation is mobilized from
extracellular and intracellular spaces. For the mobilization of intracellular calcium stores, it is the ryanodine receptors and not the inositol
triphosphate receptors that play a key role.
© 2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Oocyte; Nitric oxide; Calcium; Pig; Parthenogenetic activation
1. Introduction
Matured mammalian oocytes spontaneously blocked their
meiotic maturation at the stage of metaphase II. Further
progress in meiosis beyond this spontaneous block and oocyte
activation is dependent on the activating stimulus. Under natural conditions, this stimulus is brought into the oocyte by
the sperm (Yanagimachi, 1988). The precise nature of this
stimulus is not known. Nevertheless, the activation is important for many topics, including the cloning of mammals using
the nuclear transfer, because the successful embryonic development of the clone depends on an appropriate activating
stimulus.
Sperm induces oscillation of the intracellular levels of
free calcium ions in the fertilized oocyte and that is why
the activation of mammalian oocytes is considered to be a
calcium-dependent process (Swann and Ozil, 1994). On the
∗
Corresponding author. Tel.: +420 224382952; fax: +420 234381841.
E-mail address: [email protected] (V. Lánská).
0303-7207/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.mce.2005.05.004
other hand, nitric oxide has recently been suggested as an
intracellular signal triggering activation of the oocyte (Kuo
et al., 2000).
Nitric oxide (NO) represents an important signalling
molecule and is synthesized in cells by nitric oxide synthase
(NOS) from molecular oxygen and l-arginine in a process
which also generates l-citruline (Kwon et al., 1990; Lamas
et al., 1992; Herrero and Gagnon, 2001).
NO plays a key role in many physiological processes
including reproduction (Biswas et al., 1998). It is involved in
the regulation of follicle growth, ovulation, spermatogenesis
(Sengoku et al., 2001; Zini et al., 1996) and the implantation
of embryos in the uterus (Moncada et al., 1991). Its important role in regulation of oocyte maturation was confirmed by
observation of mice with a genetic knockout of NOS. Oocytes
retrieved from these mice blocked their maturation mainly at
the stage of metaphase I or exhibited abnormal oocyte maturation (Jablonka-Shariff and Olson, 1998). In mouse, NO
is able to both block and stimulate oocyte maturation (Bu
et al., 2003, 2004). The significance of NOS for meiotic
J. Petr et al. / Molecular and Cellular Endocrinology 242 (2005) 16–22
maturation of oocytes is also indicated by the expression of
NOS isotypes in mouse (Abe et al., 1999) and pig oocytes
(Hattori et al., 2000; Tao et al., 2004).
However, a NO-dependent signalling cascade does not
represent the primary stimulus for oocyte activation. This was
shown in studies which demonstrated the sharp increase in
intracellular levels of free calcium ions preceding an increase
in intracellular levels of NO in activated sea urchin eggs
(Leckie et al., 2003) or activated tunicate and mouse oocytes
(Hyslop et al., 2001). Nevertheless, we can conclude from
the above mentioned studies that a NO-dependent signalling
cascade is functional in oocytes and is activated during oocyte
activation after fertilization or parthenogenesis.
In our previous study we showed that the stimulation of
a NO-dependent signalling cascade is able to activate pig
oocytes (Petr et al., 2005). Pig oocytes treated with NOdonors are activated but they do not exhibit any exocytosis
of cortical granules and their parthenogenetic development
is restricted to several cleavages. This is in contrast to oocyte
activation with calcium ions which is accompanied by the
exocytosis of cortical granules and development beyond the
blastocyst stage (Jolliff and Prather, 1997).
Free calcium ions, which appeared in the cytoplasm of the
activated egg, can enter the oocyte from extracellular stores.
This influx from extracellular space plays an important role
in oocyte activation (Igusa and Miyazaki, 1983; Swann and
Ozil, 1994). However, intracellular calcium stores can also
contribute significantly to this increase of intracellular calcium levels (Igusa and Miyazaki, 1983). These intracellular
stores of calcium can be mobilized through ryanodine or inositol triphosphate receptors (Clapham, 1995).
Inositol triphosphate receptors (IP3 R) play an important
role in the regulation of meiosis in the oocytes of many mammals, e.g. mouse (Carroll et al., 1994), rabbit (Fissore and
Robl, 1993), hamster (Miyazaki, 1991), cattle (Yue et al.,
1995) and pig (Macháty et al., 1997). The existence of ryanodine receptors (RyR) was proved in cattle (Yue et al., 1995),
human (Sousa et al., 1996) and pig (Macháty et al., 1997)
oocytes. Macháty et al. (1997) demonstrated an increase in
intracellular calcium levels in pig oocytes after the activation of RyR or IP3 R and Petr et al. (2002) demonstrated
activation of pig oocytes and their parthenogenetic development after the activation of RyR or IP3 R. However, the
interaction of NO- and calcium-dependent signalling cascades in oocytes, which were activated using NO, remained
unclear.
To test the hypothesis that NO-induced activation of pig
oocytes is a calcium-dependent process, we manipulated
a calcium-dependent signalling cascade in oocytes treated
with a nitric oxide donor S-nitroso-N-acetylpenicillamine
(SNAP) on various levels. We used membrane – permeable,
selective chelator of intracellular calcium stores 1,2-bis (2aminophenoxy) ethane - N, N, N , N -tetraacetic acid tetrakis
(acetoxymethyl ester) (BAPTA-AM) for the chelation of
intracellular calcium ions. The influx of calcium through the
calcium channel from extracellular spaces was blocked by
17
verapamil. IP3 R were blocked using heparin or xestospongin C. RyR were blocked using procaine or ruthenium
red.
2. Materials and methods
2.1. Isolation and culture of oocytes
Pig ovaries were obtained from a local slaughterhouse
from gilts at an unknown stage of the estrous cycle and transported to the laboratory within 1 h in a saline solution (0.9%
sodium chloride) at 39 ◦ C. Fully grown oocytes were collected from follicles by aspirating those that were 2–5 mm
in diameter with a 20 gauge needle. Only oocytes with compact cumuli were chosen for further study. Before culture
the oocytes were washed three times in a maturation culture
medium.
The oocytes were cultured in a modified M199 medium
(GibcoBRL, Life Technologies, Paisley, Scotland) containing sodium bicarbonate (0.039 mL of a 7.0% solution per
milliliter of the medium), calcium lactate (0.6 mg/mL), gentamicin (0.025 mg/mL) HEPES (1.5 mg/mL), 13.5 IU eCG:
6.6 IU hCG/mL (P.G.600 Intervet, Boxmeer, Holland) and
10% of fetal calf serum (GibcoBRL, Life Technologies, Germany, Lot No. 40F2190F).
For in vitro maturation, the oocytes were cultured for 48 h
in 3.5 cm diameter Petri dishes (Nunc, Roskilde, Denmark)
containing 3.0 mL of the culture medium at 39 ◦ C in a mixture
of 5.0% CO2 in air.
2.2. Evaluation of oocyte activation
At the end of the culture, the oocytes were mounted on
slides, fixed with acetic alcohol (1:3, v/v) for at least 24 h, and
stained with 1.0% orcein. The oocytes were examined under
a phase contrast microscope. Activation was considered to
have occurred if the oocytes were in the pronuclear stage.
Oocytes remaining at metaphase II or arrested at anaphase II
or at telophase II were not considered as activated.
2.3. Experimental design
In experiment 1 we investigated the dependency of NOdonor induced oocyte activation on the presence of calcium
ions. Matured oocytes were mechanically devoided of cumulus cells by repeated pipetting through a narrow glass pipette.
Cumulus free oocytes were treated with BAPTA-AM (0, 5,
10, 25 and 50 ␮M) for 1 h and subsequently cultured for 10 h
in a medium supplemented with 2 mM SNAP and the respective concentration of BAPTA-AM. Control oocytes were
cultured for 11 h with BAPTA-AM without SNAP. Control
activation was performed on matured oocytes, which were
cultured for 1 h without BAPTA-AM and then cultured for
10 h in a medium with 2 mM SNAP without BAPTA-AM.
18
In experiment 2 we investigated the dependence of the
NO-donor induced oocyte activation on the influx of calcium
ions from extracellular space. Matured oocytes, denuded like
in experiment 1, were treated for 1 h with a blocker of calcium channels verapamil (0, 0.01, 0.02, 0.04 and 0.08 mM).
Subsequently, the oocytes were exposed for 10 h to a medium
supplemented with 2 mM SNAP and the respective concentration of verapamil. Control oocytes were cultured for 11 h
in verapamil without SNAP. Control of oocyte activation was
performed in matured oocytes, which were cultured for 1 h
without verapamil and then cultured for 10 h in a medium
with 2 mM SNAP without verapamil.
Experiment 3 was performed to investigate the dependency of NO-donor induced activation on the function of
ryanodine receptors. Matured oocytes, denuded like in experiment 1, were treated for 1 h with a blocker of ryanodine
receptors procaine (0.1, 0.5, 1, 5 and 10 nM). Subsequently,
the oocytes were exposed for 10 h to a medium supplemented
with 2 mM SNAP and a respective concentration of procaine.
Control oocytes were cultured for 11 h in procaine without
SNAP. Control of oocyte activation was performed in matured
oocytes, which were cultured for 1 h without procaine and
then they were cultured for 10 h in medium with 2 mM SNAP
without procaine.
Similar experiments were carried out with another blocker
of the ryanodine channel – ruthenium red. Matured oocytes,
denuded like in experiment 1, were treated for 1 h with ruthenium red (100, 200, 400 and 600 mM). Subsequently these
oocytes were exposed for 10 h to a medium supplemented
with 2 mM SNAP and the respective concentration of ruthenium red. Control oocytes were cultured for 11 h in ruthenium red without SNAP. Control of oocyte activation was
performed in matured oocytes, which were cultured for 1 h
without ruthenium red and were then cultured for 10 h in a
medium with 2 mM SNAP without ruthenium red.
In experiment 4 we investigated the dependence of NOdonor induced activation of pig oocytes on inositol trisphosphate receptors. Matured oocytes, denuded like in experiment
1, were treated for 1 h with an inhibitor of IP3 R xestospongine
C (5, 10, 20 and 40 ␮M). Subsequently the oocytes were
exposed for 10 h to the medium supplemented with 2 mM
SNAP and the respective concentration of xestospongine C.
Control oocytes were cultured for 11 h in xestospongine C
without SNAP. Control of oocyte activation was performed
in matured oocytes, which were cultured for 1 h without
xestospongine C and were then cultured for 10 h in a medium
with 2 mM SNAP without xestospongine C.
The second IP3 R inhibitor – heparin – is cell impermeable and it was microinjected into the oocytes. Microinjections were performed on an inverted microscope with phase
contrast (IMT-2, Olympus, Japan) using mechanical micromanipulators (MN-151, Narishige, Japan) and microinjectors
(IM-5, Narishige, Japan). NOS and calmodulin were diluted
in water, and this solution was injected into the oocytes
in a volume of 20 pL. In control experiments, the oocytes
were injected with 20 pL of water. Immediately before the
microinjection, the oocytes were mechanically devoided of
cumulus cells. Cumulus-free oocytes were microinjected into
a droplet of the culture medium covered with paraffin oil.
All the procedures for one experimental group (30 oocytes)
were performed within approximately 10 min. In preliminary
studies, we tested the activation of in vitro matured oocytes
after further culture (24 h), or after piercing with an injection
capillary and a subsequent 24 h. None of these treatments
activated any oocyte. We also tested the activation of in vitro
matured oocytes after injection with water or with three commonly used injection buffers. Solutions of 10 mM HEPES
or 100 ␮M EGTA, or a solution containing 140 mM KCl,
1.0 mM MgCl2 with 5.0 mM HEPES were used. Injected
oocytes were cultured for 24 h. All these treatments induced
activation in only a very limited portion of the oocytes cultured for 24 h after the procedure. Injection of water resulted
in activation in 1% of the oocytes. Activation after injection
with buffers did not differ significantly. To avoid any interaction of the injected drug with components of the buffers, we
chose water as the vehicle for microinjection.
Matured and denuded oocytes were microinjected with
heparin (0.5, 1 or 2 mg of heparin per mL). Control oocytes
were microinjected with heparin and then cultured for 11 h
in the SNAP-free medium. To control their activation, the
matured oocytes were then microinjected with water and cultured for 11 h in a medium with 2 mM SNAP.
2.4. Statistical analysis
Data from all experiments were subjected to statistical
analysis. Each experiment was performed four times. The
results were pooled for presentation and evaluated by chisquare analysis (Snedecor and Cochran, 1980). The mean
percentage of oocytes or embryos reaching the given stage
of maturation or development in all trials did not vary from
the pooled percentage by more than 2.5%. A P value of less
than 0.05 was considered significant.
3. Results
In our experiments we confirmed observations made in
our previous study (Petr et al., 2005) which demonstrated
activation of in vitro matured pig oocytes using NO released
from the NO-donor SNAP.
The 48-h culture of oocyte resulted in maturation to the
stage of metaphase II in 96% of the oocytes. The remaining oocytes reached the stage of metaphase I, anaphase I or
telophase I. These oocytes matured in vitro were treated with
2 mM SNAP for 10 h and approx. 70% of them were parthenogenetically activated (n = 120). None of the oocytes cultured
for the same time in a SNAP-free medium were activated.
One-hour treatment of matured oocytes with cellpermeable chelator of intracellular calcium stores BAPTAAM followed by a 10 h-treatment with the NO-donor SNAP
and BAPTA-AM decreased the activation rate of the oocytes
19
Table 1
Effect of the calcium chelator BAPTA-AM on the activation of pig oocytes
with the NO-donor SNAP
Table 4
Effect of the ryanodine receptor inhibitor ruthenium red on the activation of
pig oocytes using NO-donor SNAP
Concentration of
BAPTA-AM (␮M)
Percent of activated oocytes
Total number of oocytes
Concentration of ruthenium
red (mM)
0
5
10
25
50
71A
120
56B
120
30C
120
10D
120
0D
120
0
100
200
400
600
73A
120
61B
120
55B
120
35C
120
23D
120
The oocytes matured in vitro for 48 h and were then treated with BAPTAAM for 1 h and with BAPTA-AM and 2 mM NO-donor SNAP for another
10 h.
A–D Statistically significant differences in the percentage of activated oocytes.
The oocytes were matured in vitro for 48 h and then treated with ruthenium
red for 1 h and with ruthenium red and 2 mM NO-donor SNAP for another
10 h.
A–D Statistically significant differences in the percentage of activated oocytes.
Table 2
Effect of the calcium channel blocker verapamil on oocyte activation by the
NO-donor SNAP
Table 5
Effect of the inositol triphosphate receptor inhibitor xestospongin C on activation of the pig oocytes using NO-donor SNAP
Concentration of verapamil
(mM)
Concentration of
xestospongin C (␮M)
0
0.01
73A 69A
120 120
0.02
0.04
70A
120
53B
120
0.08
17C
120
0
5
10
20
40
68A
120
75A
120
69A
120
73A
120
76A
120
The oocytes were matured in vitro for 48 h and were then treated with verapamil for 1 h and with verapamil and 2 mM NO-donor SNAP for another
10 h.
A–C Statistically significant differences in the percentage of activated oocytes.
The oocytes were matured in vitro for 48 h and were then treated with
xestospongin C for 1 h and with xestospongin C and 2 mM NO-donor SNAP
for another 10 h.
A No statistically significant differences in the percentage of activated
oocytes.
in a BAPTA-AM-dose dependent manner (Table 1). Treatment of matured oocytes with BAPTA-AM for 11 h had
no effect on the oocytes which remained at the stage of
metaphase II.
The calcium channel blocker verapamil was also shown
to block parthenogenetic activation induced in pig oocytes
by the NO-donor SNAP (Table 2). With respect to the fact
that verapamil blocks the influx of calcium ions into the cells
through the L-type calcium channel, the results of this experiment indicate that NO-dependent activation of pig oocytes
is dependent on the influx of calcium ions from extracellular
spaces. An 11-h exposure of the matured oocytes to verapamil had no effect. All the oocytes remained at the stage of
metaphase II.
Both inhibitors of ryanodine receptors (RyRs) used in
our study – procaine and ruthenium red – blocked the
effects of NO-donor SNAP on parthenogenetic activation
(Tables 3 and 4). This indicates that the mobilization of
calcium ions from RyRs is necessary for parthenogenetic
activation induced in matured pig oocyte by stimulation of the
NO-dependent signalling cascade. Treatment of oocytes with
procaine or ruthenium red for 11 h did not have any effect and
the treated oocytes remained at the stage of metaphase II.
Contrary to the inhibition of RyRs, the blockage of inositol
triphosphate receptors using inhibitors heparin or xestospongin C had no statistically significant effect on the parthenogenetic activation induced in oocytes treated with NO-donor
SNAP (Tables 5 and 6). For this observation, we can conclude that calcium release from InsP3 R is not necessary
for NO-dependent parthenogenetic activation of pig oocytes.
Treatment of the oocytes with xestospongin C lasting 11 h
had no effect on the oocytes which remained at the stage of
metapahase II. A microinjection of heparin and subsequent
11 h culture in a SNAP-free medium also had no effect on the
oocytes which remained at the stage of metaphase II.
Table 3
Effect of the ryanodine receptor inhibitor procaine on the activation of pig
oocytes using NO-donor SNAP
Concentration of procaine
(nM)
0
0.1
0.5
68A 72A
120 120
56B
120
1
5
10
50B
120
21C
120
12C
120
The oocytes were matured in vitro for 48 h and then treated with procaine
for 1 h and with procaine and 2 mM NO-donor SNAP for another 10 h.
A–C Statistically significant differences in the percentage of activated oocytes.
4. Discussion
In our study we demonstrated the dependency of NOinduced parthenogenetic activation of pig oocytes on calcium.
This was clearly shown when the parthenogenetic activation induced by the NO-donor was successfully blocked
after chelation of intracellular calcium ions from the oocytes
using the calcium chelator BAPTA-AM. This implies that
Table 6
Effect of the inhibitor of inositol triphosphate receptors heparin on the activation of pig oocytes using the NO-donor SNAP
Concetration of injected
heparin (mg/mL)
0
72A
120
0.5
69A
120
1
2
70A
120
73A
120
The oocytes were matured in vitro for 48 h and were then microinjected
with heparin. They were then cultured in a SNAP-free medium for 1 h and
subsequently cultured with 2 mM NO-donor SNAP for another 10 h.
A No statistically significant differences in the percentage of activated
oocytes.
20
parthenogenetic activation of pig oocytes using the NO-donor
SNAP is successful only in the presence of a sufficient amount
of intracellular calcium.
The oocyte activation is a calcium-dependent process
(Homa et al., 1993). Calmodulin (Lorca et al., 1991; Xu
et al., 1996) and calmodulin-dependent kinase II (Lorca et
al., 1993) are clearly involved in this process, because they
induce inactivation and disintegration of the molecules which
are responsible for the matured oocyte remaining at the stage
of metaphase II. On the other hand, it was shown that NO
is able to activate oocytes in invertebrates (Kuo et al., 2000)
and mammals (Petr et al., 2005). Further studies concluded
that NO did not represent the primary signal for activation
of oocytes in chordates (Hyslop et al., 2001) or sea urchins
(Leckie et al., 2003). The increase of intracellular levels of
NO in a sea urchin fertilized oocyte is slightly delayed after
the increase of intracellular levels of calcium ions induced by
penetration of the sperm into the oocyte and it seems that NO
only modulates the levels of free calcium ions in the fertilized
egg (Leckie et al., 2003). This indicates that Ca- and NOdependent signalling cascades are interconnected in oocytes.
The same interconnection of these two signalling cascades
also occurs in various types of somatic cells (Berridge et al.,
2000).
The NO-dependent signalling cascade is regulated by calcium through several mechanisms. Production of NO in the
cell depends on activation of NOS by calmodulin which is
itself activated by calcium ions (Nathan, 1992; Lamas et al.,
1992). On the other hand, NO influences the levels of intracellular calcium through the regulation of calcium ion channels
and pumps which modulate the influx and efflux of calcium
between the cell and extracellular spaces (Berridge et al.,
2000).
The important role of NO interaction with calcium channels was also confirmed in our study which demonstrated the
lack of NO-induced parthenogenetic activation in oocytes
treated with verapamil the blocker of calcium channels. Verapamil is known to block L-type calcium channels (Atlas and
Adler, 1981). It is not able to completely block the influx of
calcium ions into the oocyte, because mammalian oocytes
express wide spectra of another type of calcium channels
(Lee et al., 2004). A certain amount of calcium ions can enter
the oocyte from the extracellular milieu even after treatment
with verapamil. However, L-type calcium channels evidently
play an important role in pig oocytes, because their blockage using verapamil induces a block of meiotic maturation
(Kaufman and Homa, 1993) and results in an abnormal distribution of intracellular calcium stores (Rozinek et al., 2003).
Based on data from our experiment, we can conclude that
the function of L-type calcium channel also plays the key
role in parthenogenetic activation of pig oocytes using the
NO-donor treatment.
NO could also regulate the mobilization of calcium ions
from their intracellular stores. Calcium from this source is
released through either ryanodine or inositol triphosphate
receptors (Clapham, 1995). In our experiments, we did not
prevent parthenogenetic activation in NO-donor treated pig
oocytes in which IP3 R had been blocked. This occurred
despite the fact that heparin was used in concentration which
were able to prevent the parthenogenetic activation of the
pig oocytes using inositol triphosphate (Petr et al., 2002).
The dose of xestospongin C also seems to be sufficient to
block IP3 R, because we used xestospongin C at concentrations which were effective for the blockage of calcium release
in mouse oocytes (Kang et al., 2003). On the other hand, the
blockage of RyRs prevented the parthenogenetic activation of
pig oocytes using the NO-donor. This indicates that the NOdependent activation of pig oocytes depends on mobilization
of intracellular calcium stores from RyRs but is independent
of the mobilization of calcium stores from IP3 R. The important role of RyRs could be due to activation of these receptors
after their S-nitrosylation by NO (Xu et al., 1998).
The importance of RyRs for oocyte activation is not clear.
RyR were not found in oocytes of some species (hamster
– Miyazaki et al., 1992) or are thought to be of negligible
importance in others (cattle – Yue et al., 1995). However,
RyRs can be very important in the activation of pig oocytes,
because stimulation of these receptors resulted in significantly increased intracellular calcium levels (Macháty et al.,
1997). Moreover, the stimulation of RyRs is clearly able to
induce parthenogenetic activation of matured pig oocytes,
although the development of eggs activated according this
protocol is very low (Petr et al., 2002).
Contrary to the situation in RyRs, IP3 Rs are thought to
be very important in the proper activation of mammalian
oocytes. In our previous study (Petr et al., 2002) we demonstrated that during parthenogenetic activation in pig oocytes,
the inositol triphosphate-sensitive calcium stores are mobilized first and their stimulation triggered the mobilization
of calcium from ryandodine-sensitive stores. This arrangement was described as the “two store model” by Tesařı́k and
Sousa (1996) in human oocytes. The parthenogenetic activation of pig oocytes using a NO-donor seems to be triggered
by mobilization of calcium from RyRs. This indicates that
the signalling cascade relevant to the “two store model” is
activated only partially at the level of RyRs and the higher
level of IP3 R is bypassed.
The developmental competence of pig parthenogenetic
embryos is much higher in embryos developing after stimulation of IP3 Rs than in embryos developing after the stimulation
of RyRs (Petr et al., 2002). With this fact in mind it is interesting to note that development of the parthenogenetic embryos
generated using NO-donor treatment is very poor and is comparable to the development of embryos generated using the
stimulation of RyRs bypassing the stimulation of IP3 Rs (Petr
et al., 2005).
Based on our data we can conclude that the parthenogenetic activation of pig oocytes using the NO-donor is
calcium-dependent. It needs an adequate intracellular calcium store, depends on the influx of calcium from extracellular stores and calcium is released mainly from RyRs but not
from IP3 Rs.
Acknowledgement
We thank Mrs. Lucy Westcott for editorial assistance with
this manuscript. This work was supported by a grant from
MZe CR MZE 0002701401, GACR 523/03/0029, GACR
523/03/H076 and MSM 6046070901.
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Nitric oxide-dependent activation of pig oocytes

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