Nitric oxide-dependent activation of pig oocytes
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Nitric oxide-dependent activation of pig oocytes
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 J. Petr et al. / Molecular and Cellular Endocrinology 242 (2005) 16–22 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 J. Petr et al. / Molecular and Cellular Endocrinology 242 (2005) 16–22 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) Percent of activated oocytes Total number of oocytes 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) Percent of activated oocytes Total number of oocytes Concentration of xestospongin C (M) Percent of activated oocytes Total number of oocytes 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) Percent of activated oocytes Total number of oocytes 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) Percent of activated oocytes Total number of oocytes 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 J. Petr et al. / Molecular and Cellular Endocrinology 242 (2005) 16–22 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). 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