Events Of Oocyte Activation Research Articles

Calcium Oscillatory Patterns and Oocyte Activation During Fertilization: a Possible Mechanism for Total Fertilization Failure (TFF) in Human In Vitro Fertilization?

This paper reviews the effects of calcium oscillatory patterns in oocytes and early embryo development. Total fertilization failure (TFF) is the failure of fertilization in all oocytes in a human IVF cycle, even after treatment with intracytoplasmic sperm injection (ICSI). It is not well understood and currently attributed to oocyte activation deficiency. Calcium signaling is important in oocyte activation events. Calcium oscillations, in particular, have been reported in animal and human oocytes after fertilization. Abnormal calcium oscillations after fertilization may be the principal mechanism for TFF. While studies also establish strong associations between abnormal calcium oscillatory patterns and suboptimal developmental outcomes, critical basic parameters and their mechanism of action have yet to be identified. Empirical use of artificial oocyte activation (AOA) methods has shown initial success in helping patients overcome TFF. The AOA methods attempt to raise calcium levels after fertilization, but the efficacy and safety of these AOA methods are still in early stages of addressing TFF. Additional information about calcium oscillatory patterns and the effects of AOA in human ART may allow the prevention of TFF or allow treatment of TFF patients effectively and safely.

Synaptotagmin 1 regulates cortical granule exocytosis during mouse oocyte activation.

Synaptotagmin 1 (Syt1) is an abundant and important presynaptic vesicle protein that binds Ca2+ for the regulation of synaptic vesicle exocytosis. Our previous study reported its localization and function on spindle assembly in mouse oocyte meiotic maturation. The present study was designed to investigate the function of Syt1 during mouse oocyte activation and subsequent cortical granule exocytosis (CGE) using confocal microscopy, morpholinol-based knockdown and time-lapse live cell imaging. By employing live cell imaging, we first studied the dynamic process of CGE and calculated the time interval between [Ca2+]i rise and CGE after oocyte activation. We further showed that Syt1 was co-localized to cortical granules (CGs) at the oocyte cortex. After oocyte activation with SrCl2, the Syt1 distribution pattern was altered significantly, similar to the changes seen for the CGs. Knockdown of Syt1 inhibited [Ca2+]i oscillations, disrupted the F-actin distribution pattern and delayed the time of cortical reaction. In summary, as a synaptic vesicle protein and calcium sensor for exocytosis, Syt1 acts as an essential regulator in mouse oocyte activation events including the generation of Ca2+ signals and CGE.

Oocyte activation deficiency: a role for an oocyte contribution?

Infertility affects between 10 and 16% of couples worldwide. Twenty to 30% of cases of infertility are due to a male factor, 20-35% to a female factor, and 25-40% are due to both male and female factors. In ∼10-25% of cases, the precise underlying cause remains unclear. IVF or ICSI followed by embryo transfer can be very appropriate treatment options in cases of female tubal damage, ovulatory failure or male-factor infertility. While the use of IVF has been reported to be suitable for many infertile couples, normal IVF cycles can fail in some cases. While ICSI can represent a powerful alternative in cases of IVF failure, complete fertilization failure can still occur in 1-5% of ICSI cycles. This can be due to a variety of factors and while commonly attributed to deficiency of sperm factors, it is very likely that abnormalities in crucial oocyte factors could also play a key role. A critical literature review using PubMed was performed between April 2014 and July 2015 targeting studies concerning sperm and oocyte factors that could account for oocyte activation deficiency, and including studies of in vitro oocyte maturation in human oocytes, and animal models. Accumulating evidence indicates that phospholipase C zeta (PLCζ) is the sperm oocyte activation factor, although recent studies claim that another sperm protein known as post-acrosomal WWP-binding domain protein could also play a significant role in the activation of oocytes. The present review discusses our current understanding of these two proteins but emphasizes that defects in the molecular machinery within the oocyte that interacts with such sperm proteins may also represent an underlying cause of fertilization failure and infertility, especially in cases where there is no obvious indication for sperm deficiency. Abnormalities in such mechanisms are highly likely to exert influence over the pulsatile release of calcium within the ooplasm, the critical signal that controls oocyte activation events. These molecular targets within the oocyte are rarely, if ever, considered clinically. We therefore recommend that future diagnostic assays should be developed to consider the inositol triphosphate receptor, protein kinase C, proteins associated with stored operated calcium entry calcium/calmodulin-dependent protein kinase II and mitogen-activated protein kinase. Development of such assays would represent a significant step forward in the diagnosis of oocyte activation deficiency and may identify a series of potential therapeutic targets. The present review provides a general overview of how a combination of sperm and oocyte factors can underlie oocyte activation deficiency, but pays particular attention to the less appreciated role of the oocyte. Enhanced research within this realm is much warranted and may establish new approaches for the diagnosis and treatment of infertility.

Elevated Expression of the Testis-specific Gene WBP2NL in Breast Cancer.

Breast cancer is one of the most common causes of cancer death in women; therefore, the study of molecular aspects of breast cancer for finding new biomarkers is important. Recent studies have shown that WW domain-binding protein 2 (WBP2) is important for the oncogenic property of breast cancer. WWP2 N-terminal-like (WBP2NL) is a testis-specific signaling protein that induces meiotic resumption and oocyte activation events. Our previous study revealed that WBP2NL gene expression is elevated in actively dividing cells and it might be associated with cellular proliferation and tumorigenic process. However, the clinical relevance and importance of WBP2NL gene in cancer has not been understood yet. Therefore, we were interested in analyzing the expression of WBP2NL gene in human breast cancer tissues and breast cancer cell lines, for the first time. We used reverse transcription-polymerase chain reaction (RT-PCR) and semi-nested RT-PCR to evaluate the expression of WBP2NL in malignant breast cancer and adjacent noncancerous tissue (ANCT) samples, as well as MCF-7 and MDA-MB-231 cell lines. The WBP2NL gene was expressed in 45 out of 50 (90%) breast cancer tissues and overexpressed in the MDA-MB-231 cell line. We suggest that WBP2NL may play roles in breast cancer activation maybe through binding to a group I WW domain protein. The elevated expression of WBP2NL gene in breast cancer and MDA-MB-231 cell line leads us to suggest that WBP2NL might be considered as a novel prognostic factor for early diagnosis of breast cancer.

Chemical activation in Rhinella arenarum oocytes: effect of dehydroleucodine (DhL) and its hydrogenated derivative (2H-DhL).

Mature oocytes are arrested in metaphase II due to the presence of high levels of active maturation promoting factor (MPF). After fertilization, active MPF levels decline abruptly, enabling oocytes to complete meiosis II. One of the first and universal events of oocyte activation is an increase in cytosolic Ca2+ that would be responsible for MPF inactivation. Mature oocytes can also be activated by parthenogenetic activation. The aims of this work are to test the ability of dehydroleucodine (DhL) and its hydrogenated derivative 11,13-dihydro-dehydroleucodine (2H-DhL) to induce chemical activation in amphibian oocytes and to study the participation of calcium in the process. Results indicated that DhL and 2H-DhL induced oocyte activation in a dose-dependent manner. After 90 min of treatment, DhL 36 μM was able to induce 95% activation, while 2H-DhL 36 μM was less active, with only 40% activation. Our results suggest that DhL induced the inhibition of MPF activity, probably by an increase in intracellular Ca2+ concentration. Extracellular Ca2+ would not be significant, although Ca2+ release from intracellular stores is critical. In this sense, IP3Rs and RyRs were involved in the Ca2+ transient induced by lactones. In this species, RyRs appears to be the largest contributor to Ca2+ release in DhL-induced activation. Although more studies are needed on the mechanism of action through which these lactones induce oocyte activation in Rhinella arenarum, the results of this research provide interesting perspectives for the use of these lactones as chemical activators in in vitro fertilization and cloning.

How do spermatozoa activate oocytes?

Although the spermatozoon is 500,000 times smaller in volume than the oocyte, it induces rapid and dramatic changes in oocyte physiology that lead to meiosis re-initiation. These oocyte activation events are described here, as is the evidence for a soluble activating factor in the spermatozoon. Since changes in plasma membrane conductance, calcium ion release and maturation-promoting factor inactivation are common to all animal oocytes at activation, it is expected that the sperm-borne trigger is also ubiquitous. One likely candidate, phospholipase C (PLC) zeta 1, induces calcium release in mammalian oocytes; however, work on other deuterostomes suggests that the sperm factor is non-specific and multifactorial, regulating several activation events. Human, sea urchin and ascidian gametes are remarkably similar and comparative studies across the deuterostomes may help in elucidating basic principles in fertilization. Questions to be answered include the identification of PLC zeta 1 in invertebrate spermatozoa and the characterization of other targets in mammalian oocytes, such as the adenosine diphosphate ribose/nitric oxide pathway.

Sperm‐borne protein, PAWP, initiates zygotic development in Xenopus laevis by eliciting intracellular calcium release

We previously reported postacrosomal sheath WW domain binding protein (PAWP) as a candidate sperm borne, oocyte-activating factor. PAWP enters the oocyte during fertilization and induces oocyte activation events including meiotic resumption, pronuclear formation, and egg cleavage. However, in order to provide proof that PAWP is a primary initiator of zygotic development it is imperative to show that PAWP initiates intracellular calcium signaling, which is considered essential for oocyte activation. Utilizing Xenopus laevis as our model, we injected recombinant PAWP or Xenopus sperm into metaphase II-arrested oocytes and observed a significant rise in intracellular calcium levels over controls. Concurring intensities and durations of PAWP and sperm-induced calcium waves, detected by infrared two-photon laser-scanning fluorescence microscopy, were prevented by coinjection of a competitive PPGY-containing peptide derived from PAWP but not by the point-mutated form of this peptide. This study also correlates PAWP and sperm-induced calcium release with meiotic resumption in Xenopus. The similar mode of oocyte activation, and the ability of the competitive peptide in blocking both sperm- and PAWP-induced calcium release, provide evidence for the first time that sperm-anchored PAWP is a primary initiator of zygotic development.

Intracytoplasmic sperm injection in the bovine induces abnormal [Ca2+]i responses and oocyte activation

Fertilisation by intracytoplasmic sperm injection (ICSI), a technique that bypasses the membrane fusion of the gametes, has been widely used to produce offspring in humans and mice. Success with this technique has lent support to the hypothesis that in mammalian fertilisation, a factor from the sperm, the so-called sperm factor, is responsible for oocyte activation and that the fusion process is not involved in the generation of the hallmark [Ca2+]i signalling seen following fertilisation. However, the success of ICSI has largely eluded large domestic species, such as the bovine, porcine and equine, casting doubt on the current model of oocyte activation at fertilisation in these species. Using Ca2+ imagery and a series of treatments to manipulate the chemical structure of the sperm, we have investigated the early events of oocyte activation in response to ICSI in the bovine. Our results demonstrate, for the first time, that following ICSI, the majority of bovine oocytes are unable to mount [Ca2+]i oscillations, although, in few cases, the initiation of [Ca2+]i oscillations can occur in a manner indistinguishable from in vitro fertilisation. We also show that bull sperm possess a full complement of sperm factor. However, either the release and/or activation of the sperm factor are compromised after ICSI, leading to the delivery of a defective Ca2+ stimulus, which results in premature termination of embryo development.

Interactions of sperm perinuclear theca with the oocyte: implications for oocyte activation, anti-polyspermy defense, and assisted reproduction.

Perinuclear theca (PT) is the cytoskeletal coat of mammalian sperm nucleus that is removed from the sperm head at fertilization. PT harbors the sperm borne, oocyte-activating factor (SOAF), a yet-to-be-characterized substance responsible for triggering the signaling cascade of oocyte activation, thought to be dependent on intra-oocyte calcium release. The present article reviews the current knowledge on the biogenesis and molecular composition of sperm PT. Possible functions of sperm PT during natural and assisted fertilization, and in the initiation of embryonic development are discussed. Furthermore, evidence is provided that SOAF is transferred from the sperm PT to oocyte cytoplasm through the internalization and rapid solubilization of the post-acrosomal PT. It is shown that during natural fertilization the sperm PT dissolves in the oocyte cytoplasm concomitantly with sperm nuclear decondensation and the initiation of pronuclear development. SOAF activity is preserved in the differentially extracted sperm heads only if the integrity of PT is maintained. After intracytoplasmic sperm injection (ICSI), activation occurs only in those oocytes in which the injected spermatozoon displays complete or partial dissolution of PT. In the latter case, the residual PT of the sub-acrosomal and/or post-acrosomal sperm region may persist on the apical surface of the sperm nucleus/male pronucleus and may cause a delay or arrest of zygotic development. We propose that the sperm PT harbors SOAF in the post-acrosomal sheath, as this is the first part of the sperm cytosol to enter the oocyte cytoplasm and its disassembly appears sufficient to initiate the early events of oocyte activation. Dissolution of the sub-acrosomal part of the PT, on the other hand, appears necessary to insure complete DNA decondensation in the internalized sperm nucleus and initiate DNA synthesis of both pronuclei. The release of the SOAF from the sperm head into oocyte cytoplasm at fertilization ultimately leads to the activation of oocyte mechanism including the completion of the meiotic cell cycle, pronuclear development and anti-polyspermy defense.

Mechanisms underlying oocyte activation and postovulatory ageing.

Mammalian oocytes undergo significant growth during oogenesis and experience extensive cytoplasmic and nuclear modifications immediately before ovulation in a process commonly referred to as oocyte maturation. These changes are intended to maximize the developmental success after fertilization. Entry of a spermatozoon into the oocyte, which occurs a few hours after ovulation, initiates long-lasting oscillations in the free intracellular calcium ([Ca(2+)](i)) that are responsible for all events of oocyte activation and the initiation of the developmental programme that often culminates in the birth of young. Nevertheless, the cellular and molecular changes that occur during maturation to optimize development are transient, and exhibit rapid deterioration. Moreover, fertilization of oocytes after an extended residence in the oviduct (or in culture) initiates a different developmental programme, one that is characterized by fragmentation, programmed cell death, and abnormal development. Inasmuch as [Ca(2+)](i) oscillations can trigger both developmental programmes in mammalian oocytes, this review addresses one of the mechanism(s) possibly used by spermatozoa to initiate these persistent [Ca(2+)](i) responses, and the cellular and molecular changes that may underlie the postovulatory cellular fragmentation of ageing mammalian oocytes.

Signalling mechanisms of mammalian oocyte activation.

Intensive study over the past 10 years has resulted in major advances in our understanding of the signalling mechanisms required for the transformation of the oocyte into an embryo capable of developing into a new individual. This review focuses on the signal transduction events that occur subsequent to the initial sperm-oocyte plasma membrane interaction, with particular emphasis on the mechanism(s) by which sperm initiate these signalling pathways. Downstream signalling/effector molecules that respond to sperm-induced calcium oscillations and appear to have important roles in oocyte activation are then discussed. Finally, the mechanisms of physiological oocyte activation are compared with events of oocyte activation that occur after ICSI.

Calcium Release at Fertilization: Artificially Mimicking the Oocyte's Response to Sperm.

The mechanism of sperm-induced calcium release has been the subject of many studies since the development in the late 1950s of in vitro culture systems that support mammalian fertilization. Despite efforts to elucidate the nature of the signal from the sperm that triggers both the early and late events of oocyte activation, the precise mechanism remains unresolved. Now, with the advent of somatic nuclear transfer technologies, the need to better understand this unique process has been recognised. Nuclear transfer embryos must be induced to commence development artificially because the activating signal from the sperm is absent. The primary activating stimulus is a large increase in the concentration of intracellular-free calcium and numerous physical and chemical treatments have been found to induce calcium changes that initiate the events of oocyte activation. Although live cloned offspring have been produced in a number of species, the overall efficiencies of the nuclear transfer procedures described thus far are unacceptably low and phenotypic anomalies are common. With the aim of improving these efficiencies, researchers are developing artificial activation treatments which induce oocyte responses that mimic those induced by fertilizing sperm. One strategy is to replicate the pattern of calcium change more closely. Another strategy is to couple an activating stimulus with treatments that inhibit maturation (or M-phase) promoting factor (MPF) activity, which regulates meiotic progression in oocytes. This paper reviews what is understood of calcium release at fertilization and describes the treatments that have been used to induce oocyte activation artificially in parthenogenetic and nuclear transfer studies. The relative effectiveness of the strategies employed to mimic the oocyte's response to sperm are discussed.

Mercury chloride induces changes in the Mg 2+-stimulated membrane currents in oocytes of the prawn Palaemon serratus

In the prawn Palaemon serratus, seawater Mg 2+ ions trigger oocyte activation events in the absence of spermatozoon. The effect of mercury chloride on the conductance of unactivated prawn oocytes and the functioning of K + Ca and Na + Ca channels sequentially involved in the electrical response of prawn oocytes stimulated with external Mg 2+ was examined. In unactivated oocytes, 20 μM Hg 2+ induced a significant increase in membrane conductance after 15 min of incubation. Moreover, micromolar amounts of Hg 2+ ions (1–20 μM) affected, in both a dose- and voltage-dependent manner, the functioning of Ca 2+-dependent K + channels implicated in the first phase of the Mg 2+-induced electrical response of prawn oocytes. Treatment of oocytes with 5 and 10 μM Hg 2+ ions significantly diminished the maximal value of this initial Mg 2+-triggered outward K + current, while 20 μM Hg 2+ completely inhibited this response after 20 min of incubation. Maximal treatments of 10 and 20 μM Hg 2+ greatly increased the inward current involved in the second phase of the Mg 2+-induced electrical response of prawn oocytes, consequently inducing an increase in membrane conductance. This final increase in conductance elicited by Hg 2+ ions reflects a change in the selectivity of Ca 2+-dependent Na + channels.

Sheep oocyte activation after intracytoplasmic sperm injection (ICSI).

The effect of calcium concentration on fertilization and activation was examined in oocytes injected in vitro with sperm. Oocytes were subjected to sperm injection, to sham injection or remained uninjected, and were then cultured for 19 h in bicarbonate-buffered synthetic oviduct fluid (BSOF) without calcium, or containing either calcium chloride or calcium ionophore. There was no difference in fertilization rates after ICSI when oocytes were cultured in vitro in media containing calcium chloride or calcium ionophore but the rate was lower in calcium-free media. There was also no difference in the fertilization rate after ICSI when oocytes were cultured in vivo compared with that observed in vitro in media containing calcium chloride or calcium ionophore. In calcium chloride-treated oocytes, activation was induced by mechanical injection, and in calcium ionophore-treated oocytes, by the ionophore. In uninjected oocytes, calcium itself did not cause oocyte activation. It is concluded that it is possible to induce activation by the injection process, but that manipulation alone is inadequate to cause proper oocyte activation unless calcium is also present. No difference in oocyte activation between ICSI and sham injection was found, indicating that the sperm may play no role in the early events of oocyte activation.

Development of calcium signalling mechanisms during maturation of human oocytes.

Sperm-induced Ca2+ signals mediate the events of oocyte activation at fertilization. In this study, the development of mechanisms involved in the generation of Ca2+ signals in human oocytes was investigated. The thiol reagent, thimerosal, which induces oscillations of intracellular Ca2+ ([Ca2+]i) similar to those seen during fertilization, was used to mobilize Ca2+ in in-vivo matured, immature and in-vitro matured human oocytes. There was an increase in the sensitivity to thimerosal during maturation of human oocytes, with oocytes from small antral follicles being relatively insensitive, compared with those from luteinized follicles, which displayed a large spike followed by sustained oscillations in [Ca2+]i. These oscillations were inhibited by caffeine which suggests that they were mediated by the inositol trisphosphate receptor Ca2+ release system. When immature oocytes were cultured in vitro they acquired the capacity to undergo a single large spike in [Ca2+]i, however, subsequent sustained oscillations were not observed, indicating that these oocytes failed to develop fully competent Ca2+ signalling mechanisms during culture in vitro. This finding may be a key factor in the poor developmental competence of in-vitro matured human oocytes.

Activation of porcine oocytes via an exogenously introduced rat muscarinic M1 receptor.

Thirty hours after the beginning of in vitro maturation, porcine oocytes were microinjected with mRNA coding for the rat muscarinic M1 receptor. They were then incubated for 15 h to allow sufficient time for completing maturation, translation of the mRNA, and insertion of the receptor into the plasma membrane. They were then treated with acetylcholine, the receptor's agonist, and its effect on inducing various activation-related changes was examined. Acetylcholine treatment triggered the release of Ca2+ from internal stores that could be blocked by atropine, the receptor's antagonist. The Ca2+ release was probably mediated via a G protein, since prior injection of guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S) totally inhibited the effect of the agonist. Pertussis toxin (PT) had no effect on the Ca2+ transients induced by acetylcholine, suggesting that the signal transduction pathway involved a PT-insensitive G protein. Electron microscopy revealed that in the injected oocytes, acetylcholine induced cortical granule exocytosis. The oocytes were released from meiotic arrest as evidenced by the decrease in H1 kinase activity measured in the oocytes during the histone H1 kinase assay. After resuming meiosis they entered interphase: 58.8% of the injected oocytes formed pronuclei after incubation with the agonist. Injection without subsequent acetylcholine treatment, or acetylcholine incubation without prior injection with the receptor mRNA, did not cause these changes. The results provide further evidence that the components of a G protein-mediated signal transduction pathway exist in porcine oocytes and that the activation of this pathway via an exogenously supplied G protein-coupled receptor results in a full complement of oocyte activation events. Whether this pathway transduces the activating signal at sperm-induced oocyte activation requires further examination.

Intracellular receptors and agents that induce activation in bovine oocytes

At fertilization, a dramatic change in the intracellular physiology of the oocyte occurs. Intracellular calcium transients have been observed in various species of oocytes including the bovine. These calcium transients have been associated with the early events of oocyte activation. Several reports have evaluated the effects of inositol 1,4,5-trisphosphate (IP3) on oocyte activation and its potential role in the events of fertilization. Recent results indicate the existance of both IP3 and ryanodine receptors in bovine oocytes. The events associated with activation and the existance of specific receptors within bovine oocytes will be discussed.

Activation of the g protein in porcine oocytes

Fertilization in most animal species involves a transient increase in the intracellular free Ca” concentration of the oocyte in the form of a CaPt wave starting from the site of sperm attachment. It is thought that the origin of this Ca” is inositol triphosphate (IP,) induced Ca” release from the endoplasmic reticulum. IP, is derived from the hydrolyses of phosphatidylinositol4,5biphosphate (PIP,) which is catalyzed by the enzyme phospholipase C under regulation of a G protein. Thus the G protein mediated PIP, hydrolysis seems to play an important role in oocyte activation. G proteins or guanine nucleotide binding proteins are a class of membrane proteins that couple membrane receptors to membrane effector enzymes. They are present in a variety of cells, and in oocytes their activation by GTP-7-S (a hydrolysisresistant analog of GTP) causes resumption of meiosis in many species. The present study was undertaken to determine the possibility of porcine oocyte activation through a G protein. Oocytes from slaughterhouse-obtained porcine ovaries were matured in Waymouth medium supplemented with 2.5 mM pyruvate, 10 IU/ml PMSG, 10 IU/ml hCG, 10% (v/v) heat-treated fetal calf serum (Sigma Chemical Co., St. Louis, MO) and 10% (v/v) porcine follicular fluid for 20 h, then in the same medium without hormonal supplement for an additional 16 h. Mature oocytes were stripped of their cumulus cells by vortexing in 0.3 M mannitol solution containing 0.3 mg/ml hyaluronidase. Then 40 pl of 1 mM GTP-7-S (Sigma Chemical Co., St. Louis, MO) was injected near the center of the oocytes using a microinjector (Narishige Co. Ltd, Tokyo, Japan). The carrier medium in which GTP-7-S was dissolved consisted of 100 PM EGTA and 10 mM Hepes buffered at pH 7.0. Control oocytes were injected with either 40 pl of 1 mM GDP-p-S, a metabolically stable analog of GDP (which inactivates G proteins) or carrier medium. Injected oocytes were incubated in Hepes-buffered Tyrodes medium (HbT) for six hours then they were evaluated for some of the early and late events of oocyte activation. Transmission electron microscopy revealed that in contrast to GDP-p-S, GTP-rS caused the release of the vast majority of the cortical granules. It also triggered changes in the protein profiles (i.e., dephosphorylation of a 25 kDa protein) as determined by one-dimensional polyacrylamide gel electrophoresis (1D SDS PAGE). Following GTP-7-S injection 71.4% (190/266) of the mature oocytes formed pronuclei, which was significantly higher (P<O.OOl) than the rates of pronuclear formation in oocytes injected with GDP-p-S (23.7%; 67/282) or carrier medium (25.9%; 52/201). Furthermore, following 7 d of incubation in ligated porcine oviducts 13.1 ok ( 10/76) of the transferred injected oocytes developed to the compact morula/blastocyst stage. We conclude that a signal transducing pathway involving a G protein does exist in the porcine oocyte, and by stimulating the G protein with GTPq-S porcine oocytes can readily be activated.