Calcium (Ca2+) signals are involved in the regulation of oocyte maturation and play a critical role during fertilization. In the egg, Ca2+ is stored in the lumen of the endoplasmic reticulum and a signal is generated when the stored Ca2+ is released through specialized channels in the membrane of the endoplasmic reticulum to elevate the free Ca2+ concentration in the cytoplasm. Extracellular Ca2+ is also important, indicated by the fact that the mobilization of luminal Ca2+ is typically followed by Ca2+ entry across the plasma membrane. The transmembrane Ca2+ flux replenishes the endoplasmic reticulum, and thus, it is essential to sustain prolonged Ca2+ signals. It also seems to be responsible for the stimulation of important signaling cascades required for complete egg activation. Characterization of the pathway that mediates Ca2+ entry implies that its major components include STIM1, a protein that senses the filling status of the stores, and ORAI1, a channel protein located in the plasma membrane. Defining the mechanism and functions of Ca2+ entry will not only lead to a better understanding of egg physiology but may also help improving the efficiency of a number of assisted reproductive technologies.
The calcium ion (Ca2+) is the most universal signaling messenger that regulates a wide array of cellular processes (Berridge et al. 1998). It carries messages by reversibly attaching to Ca2+-binding sites of proteins; Ca2+ binding changes the shape and charge of proteins and confers new functions to them. A series of proteins and signaling molecules then make up a signaling cascade that transduces signals between or within cells. Because prolonged exposure to high Ca2+ concentration is detrimental, cells maintain cytosolic Ca2+ levels low, ∼20 000-fold lower than that in the extracellular space. For this purpose, Ca2+ can be chelated by specialized proteins, stored in intracellular compartments or pumped out the cell. For signaling, the Ca2+ concentration in the cytosol is elevated briefly and this can be achieved by releasing Ca2+ from the intracellular stores or letting external Ca2+ in through ion channels located in the plasma membrane. Cells employ a set of signaling, homeostatic, and sensory mechanisms to shape Ca2+ signals in space and time (Bootman et al. 2001). In the female gamete, Ca2+ signals drive important biological processes and Ca2+ released from the endoplasmic reticulum contribute primarily to the generation of the signals. Although extracellular Ca2+ is also essential for proper signaling, relatively little is known about the mechanism that mediates the entry of Ca2+ across the plasma membrane. The aim of this review is to summarize the information that is available about Ca2+ entry in mammalian eggs with special emphasis on the most recent findings.
The source of Ca2+ during signaling
In some cell types, Ca2+ signaling depends primarily on the entry of extracellular Ca2+ (Clapham 2007). In excitable cells, certain external stimuli are able to depolarize the plasma membrane. Membrane depolarization then activates voltage-gated Ca2+ channels, thus creating an influx of Ca2+ across the plasma membrane. The Ca2+ that enters the cell will initiate various cellular events either directly by increasing intracellular Ca2+ levels or indirectly by inducing Ca2+ release from the sarcoplasmic reticulum.
Cells that cannot use membrane depolarization to trigger a Ca2+ influx rely on the release of Ca2+ from intracellular stores to elevate their intracellular free Ca2+ concentrations (Van Den Brink et al. 1999). Stimulation of cell surface receptors in such cells activates the phosphoinositide signaling system leading to Ca2+ release from the endoplasmic reticulum and the generation of a Ca2+ signal. These cells, however, can also use the external source of Ca2+ during signaling. The release of Ca2+ from the internal store is generally followed by a Ca2+ influx across the plasma membrane. The Ca2+ that enters the cells is believed to be necessary to replace Ca2+ pumped out of the cell during the period of high Ca2+ levels (Rink & Hallam 1989); it refills the intracellular stores, provides Ca2+ for long-lasting plateau signals, and also sustains repetitive Ca2+ oscillations (reviewed by Prakriya (2009)).
The origin of Ca2+ during signaling may vary depending on a number of factors. During fertilization for example, eggs of many species including most marine invertebrates, fish, and frogs show an action potential and Ca2+ influx as a result of this depolarization is part of the signaling mechanism responsible for their activation. Some of these (e.g. the echiuran worm Urechis) rely almost entirely on the Ca2+ entry for successful fertilization; in others such as sea urchins, the initial influx is followed by a release of Ca2+ from internal stores and the mobilized intraluminal Ca2+ provides the primary trigger for activation (Stricker 1999). The situation in mammalian eggs is slightly different. Hamster and mouse eggs do not show depolarization at fertilization; also, their resting membrane potential of −30 to −40 mV inactivates voltage-gated Ca2+ channels (Miyazaki & Igusa 1981a, Igusa et al. 1983). Due to these reasons, in such eggs, the fertilization Ca2+ signal is primarily provided by Ca2+ release from the intracellular stores and not by Ca2+ entry across the plasma membrane. Nevertheless, an increasing line of evidences now indicates that Ca2+ influx in mammalian eggs does have an important role to play.
Ca2+ influx during oocyte maturation
Before ovulation, mammalian oocytes are arrested at the first meiotic prophase in the ovarian follicles (Edwards 1965). During the control of meiosis, a high level of cAMP in the oocyte cytoplasm that originates from the surrounding granulosa cells is probably critical for maintaining the first meiotic arrest (Dekel & Beers 1978). Under physiological conditions, oocyte maturation is triggered by an LH surge (or it can occur spontaneously, after the removal from the follicle environment) that leads to the disruption of gap junctions between the oocyte and the granulosa cells (Gilula et al. 1978) and a drop in cAMP levels (Eppig & Downs 1988). cAMP in known to suppress the activity of the M-phase promoting factor (MPF) through cAMP-dependent protein kinase A (PKA); thus, a decrease in cAMP concentrations leads to the activation of MPF. Active MPF then stimulates the progression of the cell cycle from the G2 to the M-phase and germinal vesicle breakdown (GVBD) occurs (reviewed by Downs (2010)). Additional factors such as downregulation of cGMP levels (Norris et al. 2009, Vaccari et al. 2009) and activation of the MAP kinase cascade (Fan et al. 2009) are also believed to be crucial in the induction and progression of meiosis. A great deal of experimental data suggests, however, that Ca2+ signals also play a regulatory role in meiotic maturation. Early observations in the mouse indicated that extracellular Ca2+ was essential for meiotic resumption (De Felici & Siracusa 1982, Racowsky 1986). It was then reported that immature mouse and hamster oocytes showed a series of spontaneous Ca2+ oscillations after being released from antral follicles and the oscillations lasted until GVBD (Carroll & Swann 1992, Fujiwara et al. 1993). Although the Ca2+ oscillations in mouse oocytes were caused by an increase in inositol 1,4,5-trisphosphate (InsP3), Ca2+ influx inhibitors rapidly blocked the repetitive Ca2+ transients indicating the involvement of Ca2+ entry in the signaling process. Interestingly, the spontaneous oscillations did not serve as a trigger for meiotic resumption as inhibition of the signals with a Ca2+ chelator had no effect on the onset of GVBD (Carroll & Swann 1992). Other studies led to similar conclusions: GVBD was Ca2+ independent and proceeded without interruption in the absence of extracellular Ca2+ but completion of the first meiotic division did not occur without Ca2+ in the holding medium (Paleos & Powers 1981, Tombes et al. 1992). This again indicated the importance of Ca2+ entry across the plasma membrane in meiotic maturation. Species-specific differences clearly exist, as in bovine (Homa 1991) and pig oocytes (Kaufman & Homa 1993), chelating Ca2+ inhibited GVBD; in the pig, an influx of Ca2+ was also a prerequisite for development beyond the metaphase I stage (Kaufman & Homa 1993).
The mechanism that mediates Ca2+ entry during maturation is not known in detail. The result of early studies indicated the involvement of unspecific voltage-gated calcium channels, and an increase in the number of such channels together with a gradual depolarization of the plasma membrane was also shown to occur during oocyte growth when the female gamete gains meiotic competence (Murnane & De Felice 1993). This implies that such channels may carry a Ca2+ current to control the onset of maturation. Another study described the presence of functional L-type voltage-gated Ca2+ channels in mouse oocytes that were missing from GVBD-arrested oocytes, suggesting that such channels were necessary for maturation (Lee et al. 2004). In addition, inward currents through L-type voltage-gated channels were detected in immature bovine (Tosti et al. 2000) and sheep (Boni et al. 2005) oocytes, although in cattle their activity decreased by the end of maturation. Finally, in pig oocytes, P-type voltage-gated Ca2+ channels were identified and their activation by membrane depolarization led to a prolonged increase in cytoplasmic Ca2+ levels and eventually maturation of the oocyte (Gioia et al. 2005). These findings may indicate the involvement of Ca2+ influx via voltage-gated channels in the G2/M transition.
Similarly, there is no complete agreement on the exact function of Ca2+ as a transducer of the maturation signal. As mentioned earlier, cAMP is a crucial molecule in the cascade that controls the maintenance of the arrest at prophase of the first meiotic cell division. It activates PKA that in turn inhibits MPF through the phosphorylation of mediating proteins (Downs 2010, Luciano et al. 2011). The question is whether Ca2+ signals exert any control over the downregulation of cAMP levels at the beginning of maturation. In spontaneously maturing mouse oocytes, the phosphoinositide signaling pathway may play a regulatory role (Lefevre et al. 2007), whereas gonadotropin-induced oocyte maturation was suggested to be Ca2+ independent (Mehlmann et al. 2006). What seems to support the regulatory function of Ca2+ is the observation that the preovulatory LH surge generates a Ca2+ signal in the cumulus cells that is transmitted to the oocyte (Mattioli et al. 1998, Webb et al. 2002). This Ca2+ signal may be involved in controlling intracytoplasmic cAMP levels by modulating the activity of Ca2+-sensitive adenylate cyclases during both spontaneous and gonadotropin-induced oocyte maturation (Silvestre et al. 2011).
Ca2+ influx during fertilization
At the time of ovulation, mammalian eggs are arrested at the metaphase stage of their second meiotic cell division. They are released from this second meiotic arrest and embryo development is stimulated during fertilization, when the sperm triggers a prolonged series of Ca2+ oscillations in the egg cytoplasm. Research has shown that the oscillations are the result of the release of Ca2+ from the egg's intracellular stores; the release is induced by the protein phospholipase Czeta (PLCζ) that diffuses from the sperm into the ooplasm after gamete fusion (Saunders et al. 2002). The released Ca2+ binds calmodulin and Ca2+-bound calmodulin activates Ca2+/calmodulin-dependent protein kinase II (CaMKII) that stimulates a signaling cascade leading to the downregulation of the MPF and eventually exit from meiosis (Whitaker 2006).
The series of sperm-induced Ca2+ transients were first demonstrated indirectly, in hamster eggs, as periodic hyperpolarizations of the egg's plasma membrane (Miyazaki & Igusa 1981a). The hyperpolarizations were caused by a potassium conductance, which in turn was stimulated by the transient elevations in the intracellular free Ca2+ levels. The transients were then observed directly in mouse eggs using the Ca2+-sensitive photoprotein aequorin (Cuthbertson et al. 1981, Cuthbertson & Cobbold 1985). The series of Ca2+ transients stopped upon the removal of extracellular Ca2+ and its frequency also depended on the Ca2+ concentration outside of the cell (Igusa & Miyazaki 1983). Based on these observations, it was hypothesized that Ca2+ entry was a key element of the spiking mechanism in mammalian eggs. Subsequent findings that identified InsP3 as a Ca2+-mobilizing second messenger (Streb et al. 1983), the demonstration that its level increased during fertilization (Turner et al. 1984), and the discovery that its injection into the egg stimulated parthenogenetic activation (Whitaker & Irvine 1984) established that InsP3 was the molecule that had a key role in generating the fertilization Ca2+ signal by triggering the release of Ca2+ from intracellular stores. The sperm-induced Ca2+ transients seemed to be associated with a continuous Ca2+ influx, and a linkage between the Ca2+ influx and Ca2+ release from the intracellular stores was also suggested. However, the identity of the transport system through which Ca2+ passed through the plasma membrane was not known. Voltage-gated Ca2+ channels present in the plasma membrane of hamster eggs (Miyazaki & Igusa 1981b) were ruled out mostly because the frequency of the hyperpolarization responses increased on hyperpolarization. A model of Ca2+ oscillations was later proposed where the fertilizing sperm increased the permeability of the plasma membrane and the Ca2+ influx was essential not only for providing favorable conditions for the discharge of Ca2+ but also for the refilling of the Ca2+ pools to release Ca2+ again (Miyazaki 1991).
The dependence of the oscillations on extracellular Ca2+ was also demonstrated in mouse eggs. By using fluorescent Ca2+ indicators to monitor intracellular free Ca2+ levels, these experiments revealed that the sperm-induced Ca2+ oscillations rapidly stopped or their frequency significantly decreased upon chelation of external Ca2+ (Kline & Kline 1992, Shiina et al. 1993). In addition, depleting the intracellular stores with thapsigargin, a plant-derived inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase, activated a Ca2+ influx across the plasma membrane in unfertilized eggs and suppressed the train of Ca2+ spikes in fertilized ones (Kline & Kline 1992). This indicated that a mechanism known in somatic cells as store-operated Ca2+ entry was functional in mouse eggs as well and implied that it was essential to refill the stores and maintain the Ca2+ oscillations. Further evidence regarding the existence of Ca2+ influx during fertilization came from experiments using the sulfhydryl reducing agent dithiothreitol (DTT). In unfertilized mouse eggs, DTT increased the rate of divalent cation influx while in fertilized eggs it was able to accelerate the frequency of the sperm-induced Ca2+ transients (Cheek et al. 1993). This again implied that the influx of external Ca2+ played an important part in signaling during fertilization.
The precise linkage between the Ca2+ influx and the train of Ca2+ spikes was determined using the manganese (Mn2+)-quench technique (McGuinness et al. 1996). When added to the extracellular medium, Mn2+ acts as a Ca2+ surrogate: it can cross the plasma membrane and bind the Ca2+ indicator dye fura-2. Binding quenches the fluorescence of fura-2 and this enables the measurement of divalent cation influx. This study revealed that in fertilized mouse eggs, the rising phase of each Ca2+ transient was followed by the stimulation of a Ca2+ influx across the plasma membrane. In addition, a smaller yet notable increase in Ca2+ influx persisted during the periods between the spikes. Again, although the presence of voltage-gated Ca2+ channels had previously been reported in mouse eggs (Murnane & De Felice 1993, Day et al. 1995), these cells show only negligible hyperpolarizations during Ca2+ transients (Igusa et al. 1983). Thus, the authors hypothesized that the spike-associated Ca2+ influx was probably controlled by the filling status of the internal stores. A similar conclusion was reached by Mohri et al. (2001) after injecting a sperm extract into mouse eggs to stimulate repetitive Ca2+ oscillations. The injections were followed by a persistent Ca2+ influx that was activated during the initial Ca2+ release from the intracellular stores, possibly by a store-operated mechanism. The continuous Ca2+ influx was apparently responsible for refilling the stores and sustaining the Ca2+ oscillations.
The Ca2+ entry that is stimulated by the filling status of the Ca2+ store seems to be under the control of protein kinase C (PKC). It was known for quite some time that phorbol esters such as 12-O-tetradecanoylphorbol acetate (TPA) or phorbol-12-myristate-13-acetate (PMA), potent stimulators of PKC, caused low-amplitude Ca2+ oscillations, ZP2 modification, zona hardening, and cortical granule exocytosis in mouse eggs (Cuthbertson & Cobbold 1985, Endo et al. 1986, Colonna et al. 1989, Ducibella et al. 1991). Similarly, 1-oleyl-2-acetyl-sn-glycerol (OAG), a synthetic analog of endogenous diacylglycerol (DAG, the physiological activator of PKC) promoted mouse egg activation (Colonna et al. 1989). Further evidence came from the observation that during Ca2+ oscillations in fertilized mouse eggs, fluorescently labeled PKCs translocated to the egg plasma membrane repeatedly and the pattern of translocation followed that of the Ca2+ transients and also the periodic increases in the rate of Ca2+ influx (Halet et al. 2004). In addition, inhibition of PKCs with bisindolylmaleimide I (BIM) not only blocked Ca2+ influx after store depletion induced by thapsigargin but also terminated the sperm-induced Ca2+ oscillations. On the other hand, the PKC agonist PMA stimulated store-operated Ca2+ entry and promoted the long-lasting Ca2+ signal at fertilization. In similar studies, PKC activation promoted Ca2+ influx and repetitive Ca2+ oscillations (Yu et al. 2008) and constitutively active PKC constructs triggered a persistent elevation in cytosolic Ca2+ levels after the mobilization of luminal Ca2+ (Madgwick et al. 2005). It was hypothesized that the Ca2+ entry channel or some accessory proteins at the plasma membrane became phosphorylated by PKC that in turn caused an increase in Ca2+ entry. Interestingly, the store depletion-activated Ca2+ influx in somatic cells was inhibited, rather than stimulated, by PKC activation (Parekh & Penner 1995, Haverstick et al. 1997), and later it was demonstrated that PKC suppressed Ca2+ entry by phosphorylating ORAI1, the protein that forms the store-operated Ca2+ entry channel (Kawasaki et al. 2010). The reason for this difference is not clear. It is possible that proteins of the store-operated Ca2+ entry cascade are not substrates for PKC in eggs (other isoforms of the ORAI protein such as ORAI2 and ORAI3 are not targeted by PKC inhibitory phosphorylation and mouse eggs may use such proteins as the pore-forming subunit as their store-operated Ca2+ channel) and PKC stimulates Ca2+ influx through a completely different mechanism. Alternatively, differences in the various PKC subtypes or variations in the molecular composition of store-operated Ca2+ entry channels in different cell types may explain the diverse effects of PKC on Ca2+ entry. In addition, the level of PKC activation may also be important: in Xenopus oocytes, low level of PKC activation promoted store-operated Ca2+ entry while high degree of PKC activity had an inhibitory effect (Petersen & Berridge 1994). Overall, these findings suggested that PKC controlled cytoplasmic Ca2+ levels in eggs, and it may do so through a store-operated entry mechanism.
The onset of a Ca2+ influx triggered by the depletion of the intracellular stores was later demonstrated in pig (Machaty et al. 2002) and human (Martín-Romero et al. 2008) eggs as well. This further extended the list of mammalian eggs in which the mechanism of store-operated Ca2+ entry was shown to be functional. The molecular components of the pathway were clarified soon after the identity of these proteins became known in somatic cell types. Limited RNAi screens in human HeLa (Liou et al. 2005) and Drosophila S2 (Roos et al. 2005) cells identified stromal interacting molecules (STIM) in the endoplasmic reticulum. STIM proteins (STIM1 and STIM2) are single transmembrane proteins that sense Ca2+ content with a canonical EF hand directed toward the lumen of the Ca2+ store. Upon store depletion, STIM1 translocates to the plasma membrane and activates Ca2+ influx; STIM2 is activated by smaller decreases in luminal Ca2+ and therefore functions to stabilize basal Ca2+ levels in the cytoplasm and the endoplasmic reticulum (Brandman et al. 2007). The proteins that form the channel pore were discovered through whole genome RNAi screens in Drosophila S2 cells (Feske et al. 2006, Vig et al. 2006, Zhang et al. 2006) and named ORAI after the gatekeepers of heaven in Greek mythology. Of the three ORAI proteins (ORAI1–3) identified, ORAI1 is the most potent; it is located in the plasma membrane and when stimulated by STIM1, it lets Ca2+ flow into the cytoplasm from the extracellular space.
The presence of STIM1 has been demonstrated in pig (Koh et al. 2007) and mouse (Gómez-Fernández et al. 2009) eggs. In the pig, STIM1 expression was shown by PCR amplification using oligonucleotide primers based on a porcine EST sequence that showed high similarity to human and mouse STIM1. In resting cells, STIM1 co-localized with the endoplasmic reticulum and pharmacological store depletion triggered its redistribution in regions close to the plasma membrane. STIM1 overexpresssion led to an increase in Ca2+ influx after depletion of luminal Ca2+ whereas the influx was inhibited in the presence of store-operated Ca2+ channel blockers or after suppression of STIM1 expression with siRNAs (Koh et al. 2009). Additional experiments implicated the involvement of STIM1 in the signaling process during fertilization. The protein rapidly re-distributed at the plasma membrane following fertilization (Gómez-Fernández et al. 2009). In addition, downregulation of STIM1 expression using siRNAs in pig eggs completely abolished the repetitive Ca2+ oscillations at fertilization and had a negative effect on subsequent embryo development. Ionomycin added to fertilized eggs that stopped oscillating due to downregulated STIM1 levels was able to mobilize only a small amount of Ca2+ compared with that in control eggs, indicating that the stores were not properly refilled in the absence of STIM1. Restoring STIM1 levels in these eggs following fertilization did not rescue embryo development, suggesting that it was at earlier stages when the function of STIM1 was necessary. Finally, as expected, STIM1 downregulation was without effect on egg activation when development was stimulated parthenogenetically, by triggering a single rise in the egg's cytosolic free Ca2+ levels (Lee et al. 2012). These results seem to suggest that STIM1 is essential to maintain the repetitive Ca2+ signal by mediating store-operated Ca2+ entry during fertilization.
The channel component of the store-operated Ca2+ entry cascade, ORAI1, is also present and functional in pig and mouse eggs. Both indirect immunocytochemistry and overexpression of fluorescently tagged ORAI1 revealed that ORAI1 was present mostly in the cell cortex consistent with plasma membrane localization. Expression of ORAI1 decreased in pig eggs during maturation with STIM1 levels remaining fairly constant (Wang et al. 2012); this was different in mouse eggs where ORAI1 expression levels were unaltered during maturation while STIM1 was expressed only at very low levels in the GV-stage oocyte (Gómez-Fernández et al. 2012). Despite these species-specific differences in the expression levels of these signaling proteins, the effect of the changes seems to be the same in both species: to enhance the small store-operated Ca2+ influx measured in GV-stage oocytes to a much higher level by the time the oocytes complete maturation and are ready for fertilization. Downregulation of ORAI1 expression with siRNAs blocked Ca2+ entry induced by pharmacological depletion of the intracellular stores and also abolished the repetitive Ca2+ oscillations in fertilized eggs. Furthermore, embryo development stimulated by the abnormal fertilization Ca2+ signals in eggs with downregulated ORAI1 was severely impaired. On the other hand, overexpression of ORAI1 together with STIM1 resulted in a huge increase in Ca2+ influx induced by artificial depletion of the intracellular stores. These data suggest that the cascade that mediates store-operated Ca2+ entry involves STIM1 and ORAI1 and it is essential to sustain the long-lasting Ca2+ oscillations during fertilization.
Interestingly, inhibiting store-operated Ca2+ entry in mouse eggs using known inhibitors or by the expression of protein fragments that interfere with STIM1–ORAI1 interaction did not prevent the sperm-induced Ca2+ oscillations raising the possibility that Ca2+ entry other than that regulated by the stores is responsible for sustaining the train of sperm-induced Ca2+ spikes (Miao et al. 2012, Takahashi et al. 2013). This may indicate species-specific differences and suggest that, at least in mouse eggs, ORAI1 proteins are not essential to provide Ca2+ influx in order to maintain the long-lasting Ca2+ signal. Potential candidates for this role may include trp channels that in several cell types mediate Ca2+ entry. Such channels are present in eggs (Petersen et al. 1995, Machaty et al. 2002) and certain types can be sensitized via phosphorylation by PKC (Hardie 2007), which would explain the above-mentioned stimulatory effect of PKC on the fertilization Ca2+ signal.
The potential mechanism that may operate during fertilization to sustain the repetitive Ca2+ signal is described in Fig. 1. The sperm delivers PLCζ that hydrolyzes PIP2 into InsP3 and DAG. InsP3 binds to its receptor on the surface of the endoplasmic reticulum and triggers Ca2+ release from the intracellular store. Ca2+ is pumped out of the cell or bound to buffer proteins in the cytoplasm, thus replenishing of the store requires extracellular Ca2+. This may be mediated by STIM1: when it senses store depletion, STIM1 moves closer to the plasma membrane and opens store-operated Ca2+ channels that let Ca2+ flow into the cell. SERCA pumps load Ca2+ back into the endoplasmic reticulum, so it will be available when another Ca2+ transient is generated. DAG produced by PIP2 hydrolysis may activate PKC that promotes Ca2+ influx through the entry channel and thereby facilitates store refilling. However, additional experiments are clearly needed to clarify the exact mechanism involved in the process.
The importance of transmembrane Ca2+ fluxes has been further highlighted by the results of a study mentioned earlier (Miao et al. 2012). In those experiments using mouse eggs, Ca2+ influx was found necessary not only to maintain the sperm-induced Ca2+ oscillations but also for the activation of critical signaling pathways. Following IVF or ICSI, blocking Ca2+ influx inhibited late activation events such as second polar body emission even in the presence of multiple Ca2+ transients. In addition, after a single sperm-induced Ca2+ spike, Ca2+ influx itself was sufficient to induce polar body extrusion and pronuclear formation. A similar phenomenon was observed before in somatic cells where only Ca2+ transients with accompanying Ca2+ influxes through store-operated Ca2+ channels were able to stimulate gene expression (Di Capite et al. 2009). These results are unexpected and seem to contradict earlier studies where the number of Ca2+ oscillations was the primary determinant of the temporal order of egg activation events (Ducibella et al. 2002, Tóth et al. 2006). They also mean, if confirmed, that subcellular localization of the Ca2+ elevation is important for successful initiation of embryo development and further strengthens the notion that Ca2+ influx is an integral part of the Ca2+ signaling mechanism in the egg.
It is generally accepted that transient elevations in the intracellular free Ca2+ concentration in the cytoplasm of the female gamete play important regulatory roles during development. The origin of Ca2+ is mostly intracellular, but influx across the plasma membrane is also important to shape the signal and determine its spatial and temporal dynamics. Efforts to characterize the Ca2+ influx pathway involved received impetus in recent years; however, a number of important questions remain to be clarified. PKC is known to regulate Ca2+ fluxes across the plasma membrane but the pathway through which it operates is not known. It is not clear whether it controls store-operated Ca2+ entry or alternatively its action is mediated by an entirely different mechanism. Transient receptor potential (trp) channels are also present in eggs; in somatic cell types, such channels are capable of mediating transmembrane Ca2+ fluxes and they might have a potential role in eggs as well. Additional experiments are needed to determine the contribution of store-operated Ca2+ entry to the fertilization Ca2+ signal in other species and to resolve the apparent discrepancies in recent published results. Finally, the exact function of the Ca2+ influx during fertilization also needs to be verified: whether it is needed simply for replenishing the stores or has other roles such as activating additional signaling cascades during egg activation. The information obtained through such studies will deepen our general understanding about Ca2+ signaling in the egg and may improve the efficiency of assisted reproductive technologies.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.
The experiments conducted to investigate store-operated Ca2+ entry in pig eggs were supported by Agriculture and Food Research Initiative Competitive Grant no. 2011-67015-30006 from the USDA National Institute of Food and Agriculture.
The authors wish to thank Dr Karl Swann for providing helpful feedback during the preparation of the manuscript.
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