The involvement of Src family kinases (SFKs) in the events leading to resumption of meiosis

Molecular and Cellular Endocrinology 282 (2008) 56–62

The involvement of Src family kinases (SFKs) in the events leading to resumption of meiosis R. Tomashov-Matar, M. Levi, R. Shalgi ∗ Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv 69978, Tel-Aviv, Israel

Abstract Ovulated mammalian eggs remain arrested at the second meiotic metaphase (MII) until fertilization. The fertilizing spermatozoon initiates a sequence of biochemical events, collectively referred to as ‘egg activation’, which overcome this arrest. The initial observable change within the activated egg is a transient rise in intracellular Ca2+ concentration ([Ca2+ ]i ) followed by cortical granule exocytosis (CGE) and resumption of the second meiotic division (RMII). To date, the mechanism by which the fertilizing spermatozoon activates the signaling pathways upstream to the Ca2+ release and the manner by which the signals downstream to Ca2+ release evoke RMII are not well documented. Protein tyrosine kinases (PTKs) were suggested as possible inducers of some aspects of egg activation. Src family kinases (SFKs) constitute a large family of evolutionarily conserved PTKs that mediate crucial biological functions. At present, the theory that one or more SFKs are necessary and sufficient for Ca2+ regulation at fertilization is documented in eggs of marine invertebrates. The mechanism leading to Ca2+ release during fertilization is less established in mammalian eggs. A controversy still exists as to whether SFKs within the mammalian egg are sufficient and/or necessary for Ca2+ release, or whether they play a role during egg activation via other signaling pathways. This article summarizes the possible signaling pathways involved upstream to Ca2+ release but focuses mainly on the involvement of SFKs downstream to Ca2+ release toward RMII, in invertebrate and vertebrate eggs. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Calcium; MPF; c-Src; Fyn; c-Yes; CaMKII; Tubulin

1. Introduction Ovulated mammalian eggs are arrested at the metaphase of the second meiotic division (MII). At fertilization, the sperm activates the egg to re-enter the cell cycle and begin the embryonic development. The initial series of biochemical events taking place within the fertilized egg is collectively referred to as “egg activation”. It includes cortical granule exocytosis (CGE), resumption of the second meiotic division (RMII) to its completion by extrusion of the second polar body (PBII), DNA replication and the first mitotic cleavage. While an increase in the intracellular concentration of free calcium ([Ca2+ ]i ) is a universal egg-activating signal, the patterns of [Ca2+ ]i release vary among species. In mammalian eggs, fertilization triggers an initial [Ca2+ ]i rise, accompanied by repetitive Ca2+ transients, commonly referred to as Ca2+ oscillations (Kline and Kline, 1992). In most cases the calcium ∗

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is released from intracellular stores, the most prominent of which appears to be the endoplasmic reticulum (ER). Despite the universal phenomenon of [Ca2+ ]i release at fertilization, the signaling mechanism(s) by which the spermatozoa initiate [Ca2+ ]i responses in eggs has not yet been elucidated. There are two cycles of chromosome segregation in mammalian eggs. During the first meiotic division (MI), a reductional division segregates homologous chromosomes, while an equational division segregates sister chromatids during MII. The last event is critical for generating early embryos of the appropriate ploidy. Progression through the meiotic divisions, like the progression through divisions of the mitotic cell cycle, is controlled by maturation promoting factor (MPF), a heterodimer consisting of cyclin-dependent kinase I and a regulatory B-type cyclin (Draette et al., 1989; Gautier et al., 1990; Labbe et al., 1989; Marangos and Caroll, 2004). MPF activity increases just before germinal vesicle breakdown (GVBD) and remains elevated during the first meiotic M-phase. When the spindle of the first meiotic division is assembled, the metaphase-anaphase transition and the extrusion of the first polar body (PBI) are

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accompanied by a transient inactivation of MPF (Brunet et al., 2003; Hashimoto and Kishimoto, 1986; Jones, 2004). The spindle of the second meiotic division forms under the influence of the increasing MPF activity, and it is at the MII stage that the egg arrests (Verlhac et al., 1996). Relief from this arrest is provided by the fertilizing spermatozoon. Inactivation of MPF during exit from both MI and MII stages is controlled by the anaphase promoting complex (APC), a multi-subunit E3 ligase that ubiquitinates cyclin B1, thereby targeting it to the 26S proteasome for degradation (Fang et al., 1999; Peters, 2002). Eggs are arrested at the MII stage until fertilization, owing to the “cytostatic factor” (CSF; Kubiak et al., 1993; Tunquist and Maller, 2003). The molecular nature of CSF has not been fully elucidated. The importance of the Mos/MAP kinase pathway in establishing CSF arrest was demonstrated in the Mos knockout mouse model (Colledge et al., 1994; Hashimoto et al., 1994), as well as by the ability of Mos and MAP kinase to induce metaphase arrest in blastomers and by the failure of the egg to arrest at MII caused by ablating this pathway (Tunquist and Maller, 2003). CSF inhibits the onset of anaphase by attenuating cyclin B1 and securing its degradation. In mammalian eggs, cyclin B1 degradation occurs during CSF-induced cell cycle arrest. Since cyclin B1 has a half-life of 1–2 h (Nixon et al., 2002), spontaneous activation is prevented due to the continuous synthesis of cyclin B1. The relief from CSF-mediated arrest is provided by a sperm-induced increase of [Ca2+ ]i . It is suggested that the effects of calcium are mediated by calmodulin-dependent protein kinase II (CaMKII; Lorca et al., 1991, 1993; Markoulaki et al., 2003), but it is not known how the activation of APC is affected by CaMKII. 2. Src family kinases The human Src family protein tyrosine kinases (Src family PTKs or SFKs) contains eight members: Src, Yes, Fyn, Lck, Fgr, Lyn, Hck and Blk (Brown and Cooper, 1996; Thomas and Brugge, 1997). Src, Yes, Fyn and Yrk (a non-human SFK) are found in a broad range of tissue types while Lck, Fgr, Lyn, Hck and Blk are restricted to cells of specific haematopoietic lineages. There are two splice-variants of Fyn: T, which is restricted to T-cells and B, found primarily in brain, though it is also expressed in lymphocytes, monocytes, platelets, fibroblasts, endothelial cells and neurons (Sudol et al., 1993). The Src family kinases (SFKs) belong to the non-receptor kinases that have no extracellular portion and no integral plasma membrane-spanning domain. Activated SFKs catalyze the transfer of ␥-phosphates from ATP molecules to tyrosine residues of target proteins, thereby transmitting extracellular signals to downstream cellular components (Brown and Cooper, 1996; Thomas and Brugge, 1997; Abram and Courtneidge, 2000). Src was originally identified as an oncogene product of Rous Sarcoma virus, named viral-Src (v-Src), that caused transformation of chicken cells (Jove and Hanafusa, 1987). However, the on-off switch of the kinase activity of c-Src is strictly regulated, thus abolishing c-Src ability to transform cells, but rather implicating it in normal cellular functions such as cell growth, differentiation and survival (Jove and Hanafusa, 1987).

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2.1. SFKs in invertebrate eggs It was more than 20 years ago when Kinsey’s group first described a Src-type kinase activity in sea urchin eggs (Kamel et al., 1985). Before that, fertilization-dependent activation of PTK in sea urchin eggs was reported by Dasgupta and Garbers (1983). Since then, sea urchin and some deuterostome invertebrate eggs served as the most popular models for studying SFKs activity. Several studies have demonstrated that sperm-induced tyrosine phosphorylation of egg’s Fyn proteins occur as early as 5 min after insemination (Kinsey, 1995). A more recent study revealed that SFKs (p57 kinase) are activated within 15 s of fertilization (Abassi et al., 2000). Moreover, inhibition of SFK activity in eggs resulted in a delayed, or a complete block of [Ca2+ ]i elevation in response to insemination. It has been suggested that sperm-induced sea urchin egg activation involves a rapid and transient activation of egg SFKs, followed by an SFK-dependent activation of egg PLC␥ that is responsible for the subsequent inositol 1,4,5-trisphosphate (IP3 )-dependent [Ca2+ ]i transient and egg activation events (Giusti et al., 2003). The molecular identity of sea urchin egg SFKs has also become evident. Kinsey et al. (1995) showed the presence of a 57-kDa protein and of Fyn (Kinsey, 1996; Kinsey and Shen, 2000) in sea urchin eggs. Giusti et al. (2003) found that an SFK, that is expressed in sea urchin eggs membrane is highly related to the mammalian c-Src, named Strongylocentrous purpuratus SFK, or to its Lytechinus variegates homologue, both of which show fertilization-dependent activation and/or phosphorylation, although they have not been molecularly cloned. SFKs have also been implicated in fertilization of other sea invertebrate eggs, such as starfish and ascidians. Injection of an active Src protein into starfish eggs resulted in complete egg activation accompanied by a [Ca2+ ]i transient. Moreover, injection of the Src SH2 domain blocked the active Src-dependent [Ca2+ ]i transient (Giusti et al., 1999), as well as the sperminduced [Ca2+ ]i transient in ascidian eggs, suggesting that egg SFKs promote Ca2+ signaling at fertilization (Runft and Jaffe, 2000). The molecular identity of starfish and ascidians egg SFKs, prone to be activated upon fertilization, has not yet been demonstrated. It is noteworthy that injection of PLC␥ SH2 domain into eggs of all sea invertebrates described above, has been shown to block a future [Ca2+ ]i transient intended to occur in response to either a later injection of the active Src protein, or insemination. 2.2. SFKs in vertebrate eggs An SFK, immunochemically related to the mammalian Fyn, is activated within 30 s of insemination of zebrafish eggs (Wu and Kinesy, 2000, 2002). The involvement of a receptor-like protein tyrosine phosphatase-␣ (rPTP␣) in the sperm-induced activation of the zebrafish egg SFK has been suggested; namely, rPTP␣-constitutively associated with the eggs SFK may be activated upon fertilization. The activated rPTP␣ can dephosphorylate the C-terminal negative-regulatory phosphotyrosine of the egg SFK, and thereby activate the SFK. The interaction between rPTP␣ and egg SFK seems to be mediated by the SFK SH2 domain, explaining the SFK

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SH2 domain-dependent SFK function. Another SFK member – c-Yes – was also identified in zebrafish eggs; it is maternally expressed and localized to the cortical region of the unfertilized egg. The kinase activity of c-Yes decreases at fertilization, increases progressively during epiboly and is then maintained at high level throughout gastrulation (Tsai et al., 2005). Xenopus eggs microinjected with an ATP-competitive inhibitor, pyrazolopyrimidine (PP1/PP2), a Src-specific inhibitor, but not with PP3 (the inactive form of the inhibitor), show a delay or defect in the sperm-induced [Ca2+ ]i transient (Sato et al., 2000a, 2001). PP1/PP2 effectively block the [Ca2+ ]i transient in unfertilized Xenopus egg extracts induced by purified Src (Tokmakov et al., 2002). PP2 also blocks mouse egg activation events induced by microinjection of a truncated form of c-Kit PTK (Tr-kit; Sette et al., 2002). Since Tr-kit has no ATP-binding site, the mechanism by which Tr-kit promotes egg activation involves activation of SFKs that can be inhibited by PP2. A constitutively active (CA) form of Fyn is capable of activating mouse eggs as judged by the phosphorylation of PLC␥ downstream to Tr-kit (Sette et al., 2002). Although tyrosine phosphorylation-dependent activation of PLC␥ was established in somatic cells, the question of how PLC is activated in the absence of SH2 domain-mediated protein-protein interaction remains unanswered. Phospholipase D (PLD) could be an upstream regulator of the egg PLC␥. Phosphatidic acid (PA) and choline are produced through the PLD-induced hydrolysis of phosphatidylcholine. Over 20 years ago some researchers have suggested that PA activates PLC and increases Ca2+ (Moolenaar et al., 1986). However, the mechanism by which PA activates PLC has not been determined. In vitro studies produced conflicting evidence as to the identity of the PLC isoform that can be activated by PA and as to the altered kinetic constant. PA may also directly activate c-Src which, in turn, would activate PLC␥ (Sato et al., 1997). In a mammalian cell system, a catalytically active PLD is required for the activation of c-Src by mitogens (Ahn et al., 2003). Addition of PA to neutrophils caused an activation of Fyn and Syk, tyrosine phosphorylation dependent activation of PLC␥, an increase in IP3 mass and Ca2+ release. These events were inhibited by the tyrosine kinase inhibitor, herbimycin A (Siddiqui and English, 1997, 2000). The effect of PP2 on fertilization has not yet been reported, neither have the egg SFKs, nor other PTKs responsible for sperm-induced egg activation, been characterized. Although, DuPont et al. (1996) reported that genistein blocked mouse egg activation at fertilization, the fact that genistein was added to the IVF culture medium raises the possibility that sperm function was also abrogated. Moreover, a CA c-Src protein or mRNA encoding for c-Src were unable to activate mouse eggs (Kurokawa et al., 2004a,b). Reports indicated that Fyn and c-Yes proteins are expressed in mouse eggs and that injection of Fyn SH2 domain or a mixture of Fyn and c-Yes SH2 domains into eggs did not inhibit Ca2+ release at fertilization (Mehlmann and Jaffe, 2005; Meng et al., 2006). The expression and localization of Fyn, c-Src and c-Yes were also demonstrated in rat eggs (Talmor et al., 1998; Talmor-Cohen et al., 2004a), but only of Fyn and c-Yes in mouse eggs (Sette et

al., 2002; Mehlmann and Jaffe, 2005). The three SFKs are localized at the eggs cytoplasm while c-Yes and Fyn are expressed at the cortex as well. Fyn was also found at the spindle, implicating an association with the eggs microtubules (Talmor-Cohen et al., 2004a,b). Activating the eggs with ionomycin in the presence of PP2 or SU6656 blocked RMII, but not CGE. Injecting the eggs with a CA Fyn protein caused RMII in a dose depended manner, but did not cause CGE (Talmor-Cohen et al., 2004a,b). The findings of Talmor-Cohen et al. (2004a,b) and of Sette et al. (2002) should be regarded with reservation since the constructed Fyn protein was not myristoylated, which left it a cytosolic protein, incapable of binding to membranes. This diminished its efficiency in finding a suitable substrate, although it did not hinder its kinase activity. To overcome this obstacle, the protein kinase was microinjected into the egg at a relatively high concentration. The presence of various SFKs within mammalian eggs (Kurokawa et al., 2004a,b; Mehlmann and Jaffe, 2005; Talmor et al., 1998; Talmor-Cohen et al., 2004a) can cause compensatory or combinatorial effects that may account for the difficulty in demonstrating a conclusive role for specific SFKs in egg activation. Supporting information for the compensatory phenomena is achieved from knock-out mice. Knock-out mice such as cYes (−/−) or Fyn (−/−) can survive with severe developmental abnormalities but they are fertile (Stein et al., 1994; Umemori et al., 1994). The c-Src (−/−) knock-out mice develop normally but die soon after birth (Soriano et al., 1991). Mice with double knock-outs, such as c-Src (−/−)/c-Yes (−/−), do not survive (Stein et al., 1994). We have to bear in mind that the best way to knock-down/out a specific protein during a certain developmental phase such as fertilization, is by using the siRNA technique while the techniques of introducing a dominant negative (DN) product or an SH2 interrupting domain do not abolish the protein activity completely. Unfortunately, the siRNA technique is difficult to implement because SFKs are present within the egg as proteins from the germinal vesicle stage and their turnover is very slow. Moreover, it has been reported that SFKs activities do not change during fertilization (Livingston et al., 1998), however, c-Yes activity decreases upon fertilization (Tsai et al., 2005). At present, the hypothesis that SFKs are necessary and/or sufficient for [Ca2+ ]i release at fertilization is well supported in invertebrate eggs, but not in mammalian eggs. There is a possibility that mammalian eggs have adapted different pathways for generating IP3 . It is not clear whether SFKs have a role in generating the [Ca2+ ]i transient in mammalian eggs, and/or whether they affect other signaling pathways during egg activation. The following parts of this article summarized the involvement of SFKs upstream and downstream to [Ca2+ ]i release during egg activation, focusing on mammalian eggs. 3. The role of SFKs in the signal transduction during egg activation 3.1. SFKs upstream to Ca2+ release SFKs and PLC␥ are activated by sperm at fertilization of echinoderm, ascidian, fish and frog eggs. The necessity of

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SFKs and PLC␥ for egg activation has been demonstrated by researchers using a variety of pharmacological inhibitors (Abassi et al., 2000; Runft and Jaffe, 2000; Wu and Kinesy, 2002; Sato et al., 2000a) as well as dominant-interfering strategies (Giusti et al., 2003; Runft et al., 2004; Mehlmann and Jaffe, 2005; Meng et al., 2006). An active Src protein triggers Ca2+ release, when injected into starfish eggs, but cannot rescue eggs that had already been injected with a DN PLC␥ protein, thus placing the SFKs upstream to PLC␥ (Giusti et al., 2000b). Likewise, pharmacological studies have implicated PLC and tyrosine kinase activity in the Ca2+ release pathway at fertilization of vertebrates’ eggs (DuPont et al., 1996). However, SH2-mediated dominant interference of PLC␥ or of SFKs has no effect on sperm-induced Ca2+ release at fertilization of amphibian and mammalian eggs (Runft et al., 1999; Mehlmann et al., 1998; Mehlmann and Jaffe, 2005; Meng et al., 2006), indicating that the possible involvement of PLC␥ and SFKs in mammalian eggs is regulated differently than in invertebrates eggs. Moreover, another report (Kurokawa et al., 2004a,b) indicated that specific pharmacological (PP2, lavendustin A) and peptide (peptide A) inhibitors of SFKs had no effect on Ca2+ release in mouse eggs and that injection of a recombinant Src protein or of Src mRNA encoding a CA form of Src did not stimulate Ca2+ release. Injection of a CA form of Fyn protein combined with Tr-kit caused phosphorylation of PLC␥ in mouse eggs (Sette et al., 2002), although no Ca2+ release was demonstrated. It is worth investigating the ability of the CA forms of the entire Fyn or c-Yes proteins to evoke Ca2+ oscillations. Moreover, the ability or inability of the DN forms of the full length Fyn or c-Yes (not of specific domains of these proteins) to trigger Ca2+ increase in sperm activated eggs, will supply valuable information regarding the involvement of SFKs in the process of fertilization. Although, less efforts were directed towards resolving the role of SFKs in fertilization of mammalian eggs than in non-mammalian eggs, it appears that SFKs play a different role during activation of mammalian eggs.

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none reached the blastocyst stage. The higher the SH2 concentration was, the earlier the arrest was; the highest concentration used, arrested the eggs already at the pronuclear stage with both male and female pronuclei present. In contrast with the reports described above (Sette et al., 2002; Talmor-Cohen et al., 2004a), Meng et al. (2006) found no affect on RMII. The authors provided two possible reasons for this discrepancy: 1. The fertilizing spermatozoa might be better activators of multiple signaling pathways than parthenogenetic treatments and therefore suppression of SFKs is not enough for blocking the development of spermatozoa activated eggs; 2. RMII requires the catalytic-domain of SFKs, and not the SH2 domain. We can add one more explanation: the discrepancy could be attributed to the compensatory activity of SFK proteins, where members of the family can make up for the inactive protein as described in Section 2.2. We have investigated the effect of SFKs inhibition on RMII in eggs activated by SrCl2 under conditions that allow the occurrence of two Ca2+ peaks (Tomashov-Matar et al., 2005; Fig. 1), as well as in eggs activated by ionomycin allowing a single Ca2+ peak (Talmor-Cohen et al., 2004a). The pace and occurrence of RMII in SU6656 treated, SrCl2 -activated eggs, were decreased in a dose-dependent manner. The rate of RMII of SrCl2 -activated eggs (84%; positive control group), decreased to 50%, 7% or was totally inhibited after exposure to 2.5 ␮M, 5.0 ␮M or 10.0 ␮M SU6656, respectively. The majority of the activated eggs at the positive control group extruded PBII, but most of them reached only anaphase stage in the presence of 2.5 ␮M or 5.0 ␮M SU6656 (Fig. 1). We can conclude that, in the presence of SFKs inhibitor, RMII is blocked in either SrCl2 (Ca2+ oscillations) or ionomycin (a single Ca2+ rise) activated eggs (Talmor-Cohen et al., 2004a). In a previous study we have shown that Fyn, c-Yes and c-Src are distributed throughout the egg cytoplasm (Talmor-Cohen et al., 2004a) but that c-Yes, like Fyn, tends to concentrate at

3.2. SFKs downstream to Ca2+ release The Ca2+ increase causes vertebrate eggs to enter anaphase and resume meiosis culminating in extrusion of PBII, by decreasing the activity of CSF and MPF. The role of SFKs in later stages of mammalian fertilization has been addressed primarily via parthenogenetic activation. Studies of rat and mouse eggs demonstrate that agents like SU6656 and PP2 that simultaneously inhibit all three members—Fyn, c-Yes and c-Src, also inhibit the MII/anaphase transition induced by parthenogenetic activation in vitro (Mehlmann and Jaffe, 2005; Talmor-Cohen et al., 2004a). These observations indicate that RMII triggered by parthenogenetic activators depends, at least in part, on SFKs activity. Microinjection of CA Fyn protein stimulated RMII in rat and mouse eggs (Sette et al., 2002; Talmor-Cohen et al., 2004b), however, a CA c-Src protein or CA cRNA of c-Src did not trigger mouse egg activation (Kurokawa et al., 2004a,b; TomashovMatar et al., 2007). Meng et al. (2006) reported recently that injection of SH2 domain of Fyn arrested the development of most mouse zygotes during the first two cleavage divisions and

Fig. 1. MII eggs were incubated in the presence or absence of SU6656 at the indicated concentrations, prior to activation by SrCl2 (2 mM). Activation was scored 60 min after exposure to SrCl2 by the occurrence of meiotic resumption. Data is expressed as percent of eggs successfully resuming meiosis. Each column represents at least 35 eggs per treatment at any given experiment (three independent experiments).

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the egg cortex as well. The egg cortex is known to be rich in actin cytoskeleton. Of the three SFKs studied, only Fyn is also localized to the meiotic and mitotic spindles (Talmor et al., 1998). The subcellular distribution of the three kinases does not change upon activation. The localization of the three kinases to different compartments within the egg indicates that these proteins may have different functions within the egg. It is possible that the intracellular localization of c-Yes and Fyn to cortical structures and of Fyn to the microtubules (MTs) as well, imply their association with cytoskeletal elements. Inhibition of SFKs caused spindle disintegration, dispersion of tubulin fibers in the cytoplasm and, as a result, no alignment of the chromosomes at a metaphase plate structure (Talmor-Cohen et al., 2004b). For successful fertilization, the spindle of vertebrate eggs must remain stable and properly organized during the second meiotic metaphase arrest (Terret et al., 2003; Wassmann et al., 2003). Thus, we have suggested that SFKs mediate some functions needed for the organization of a proper MII spindle. Fyn is the only studied SFK that was found to be localized to the meiotic and mitotic spindles. The findings that the proper organization of the MII spindle is an SFK-dependent process, and that Fyn kinase is localized to the MT and can phosphorylate tubulin in vitro (Talmor-Cohen et al., 2004b), point at the proteins of this family as promising candidates for involvement in microtubule function. The question remains, where in the signal transduction pathway that leads to PBII extrusion, are SFKs involved? Is it upstream to MPF or does it affect MPF degradation? To address that, we have studied the effect of SFKs inhibition on the activity of CaMKII (Fig. 2). Using MII eggs, parthenogenetically activated by 2 mM SrCl2 (Tomashov-Matar et al., 2005) in the presence or absence of an SFKs inhibitor (SU6656; 5 ␮M; Fig. 2), CaMKII activity of SrCl2 -activated eggs was almost three-fold the activity of non-activated eggs. Exposing eggs to

Fig. 3. A model for the involvement of SFKs during mammalian egg activation (adapted from Talmor-Cohen (2003)). Sperm binding to a receptor on the eggs membrane, or fusion between the sperm and the egg membranes allowing the delivery of a sperm factor (SF), results in the “activation” of phospholipase C (PLC). PLC hydrolyzes phosphatidylinositol (4,5) biphosphate (PIP2) to form inositol 1,4,5-triphosphate (IP3 ) and diacylglycerol (DAG). IP3 triggers intracellular Ca2+ ([Ca2+ ]i ) release from the endoplasmic reticulum (ER) via the IP3 receptor (IP3 R). Both [Ca2+ ]i rise and DAG contribute to egg activation events: cortical granule (CG) exocytosis (CGE) and resumption of meiosis II (RMII). Based on the data presented in this work, we propose that Fyn and c-Yes activity modulate Ca2+ release and is also associated with resumption of the cell cycle in response to the fertilization signal. SFKs can modulate CGE via PKC but not via Ca2+ rise.

SrCl2 in the presence of SU6656 had no significant effect on CaMKII activity (Fig. 2), although [Ca2+ ]i rise, was observed (data not shown). We can conclude that inhibiting the elevation of CaMKII activity as well as reducing the rate and pace of RMII in SrCl2 -activated rat eggs by inhibiting SFKs activity, implies involvement of SFKs in the signal transduction pathway leading to RMII, probably involving MPF activity. 4. Conclusions

Fig. 2. MII eggs were parthenogenetically activated by 2 mM SrCl2 in the presence or absence of an SFKs inhibitor (SU6656; SU; 5 ␮M), while other MII, non-activated eggs were exposed either to CaMKII inhibitor (20 ␮M; W7; negative control) or to SU6656, or were not treated at all. The degree of CaMKII activity (Signa TECT assay system) found in non-activated eggs was set as a reference for the CaMKII activity of the SrCl2 -activated eggs or of eggs exposed to W7 or SU6656. Each column represents at least 10 eggs per treatment at any given experiment (three independent experiments).

This article focuses on the role of SFKs during mammalian egg activation. We may speculate that these kinases are involved in signaling events that implicate cytoskeletal reorganization via association and activation of cytoskeletal proteins. Although there is evidence for a role of SFKs in initiating the Ca2+ rise (through PLC␥) at fertilization of marine invertebrate and amphibians eggs, PLC␨, a sperm factor candidate involved in mammalian egg activation at fertilization, lacks Src-homology domains, implying that any involvement of SFKs in mammalian egg activation is downstream to Ca2+ rise (Fig. 3).

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Lately (Tomashov-Matar et al., 2007) we were able to demonstrate the occurrence of Ca2+ oscillations, though in a delayed manner, in eggs activated by either sperm, SrCl2 or PLC␨, when their SFKs were inhibited by SU6656. CA Fyn, c-Src and cYes were insufficient for inducing Ca2+ release in MII eggs. DN forms of both Fyn and c-Yes were unable to block Ca2+ release in SrCl2 and sperm activated eggs. We can conclude that SFKs are neither sufficient nor necessary for initiating Ca2+ oscillations. Any role they may have in Ca2+ release could be in modulating the process. To determine whether SFKs participate in the signal transduction pathway leading to CGE we employed SU6656. Activation of eggs by either SrCl2 , PLC␨ or sperm, triggers [Ca2+ ]i rise and CGE, whereas, activation by TPA leads to CGE via PKC activation. The presence of SU6656 in the culture medium decreased the relative CGE intensity of TPA activated eggs by 60% but had no effect on CGE intensity in eggs activated either by SrCl2 , PLC␨ or sperm. We can conclude that inhibition of SFKs does not abolish CGE, but rather modulates it, when CGE is induced by the PKC pathway. The most dramatic effect was on RMII. On one hand, SFKs inhibitor blocked RMII in a dose dependent manner in eggs activated by SrCl2 , ionomycin or puromycin. Additional supportive evidence was derived from experiments in which SrCl2 activation or fertilization was inhibited by microinjection of DN Fyn or DN c-Yes cRNAs. On the other hand, microinjection of CA Fyn or CA c-Yes cRNAs, but not of CA c-Src c-RNA, caused RMII (Tomashov-Matar et al., 2007; Fig. 3). In our future study we will focus on the signals and/or proteins involved in RMII. Acknowledgments This work was partially supported by a grant from the Israel Science Foundation (695/05) and the Ministry of Health to RS. References Abassi, Y.A., Carroll, D.J., Giusti, A.F., Belton, R.J., Foltz, K.R., 2000. Evidence that src-type tyrosine kinase activity is necessary for initiation of calcium release at fertilization in sea urchin eggs. Dev. Biol. 218, 206– 219. Abram, C.L., Courtneidge, S.A., 2000. Src family tyrosine kinases and growth factor signaling. Exp. Cell Res. 254, 1–13. Ahn, B.H., Kim, S.Y., Kim, E.H., Choi, K.S., Kwon, T.K., Lee, Y.H., et al., 2003. Transmodulation between phospholipase D and c-Src enhances cell proliferation. Mol. Cell Biol. 23, 3103–3115. Brown, M.T., Cooper, J.A., 1996. Regulation, substrates and functions of src. Biochim. Biophys. Acta 1287, 121–149. Brunet, S., Pahlavan, G., Taylor, S., Maro, B., 2003. Functionality of the spindle checkpoint during the first meiotic division of mammalian oocytes. Reproduction 126, 443–450. Colledge, W.H., Carlton, M.B., Udy, G.B., Evans, M.J., 1994. Disruption of cMos causes parthenogentic development of unfertilized mouse eggs. Nature 370, 65–68. Dasgupta, J.D., Garbers, D.L., 1983. Tyrosine protein kinase activity during embryogenesis. J. Biol. Chem. 258, 6174–6178. Draette, G., Luca, F., Westendorf, J., Brizuela, L., Rudermann, J., Beach, D., 1989. Cdc2 protein kinase is complexed with both cyclin A and B: evidence for proteolytic inactivation of MPF. Cell 56, 829–838. DuPont, G., McGuiness, O., Johnson, M.H., Berridge, M.J., Borgese, F., 1996. Phospholipase C in mouse oocytes: characterization of isoforms and their

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