Signal transduction pathways leading to Ca2+ release in a vertebrate model system: Lessons from Xenopus eggs

Seminars in Cell & Developmental Biology 17 (2006) 285–292

Review

Signal transduction pathways leading to Ca2+ release in a vertebrate model system: Lessons from Xenopus eggs Ken-ichi Sato a,∗ , Yasuo Fukami a , Bradley J. Stith b,∗∗ a

Laboratory of Molecular Biology, The Research Center for Environmental Genomics, Kobe University, Kobe 657-8501, Japan b The Department of Biology 171, University of Colorado-Denver, P.O. Box 173364, Denver, CO 80217, USA Available online 2 March 2006

Abstract At fertilization, eggs unite with sperm to initiate developmental programs that give rise to development of the embryo. Defining the molecular mechanism of this fundamental process at the beginning of life has been a key question in cell and developmental biology. In this review, we examine sperm-induced signal transduction events that lead to release of intracellular Ca2+ , a pivotal trigger of developmental activation, during fertilization in Xenopus laevis. Recent data demonstrate that metabolism of inositol 1,4,5-trisphosphate (IP3 ), a second messenger for Ca2+ release, is carefully regulated and involves phospholipase C (PLC) and the tyrosine kinase Src. Roles of other potential regulators in this pathway, such as phosphatidylinositol 3-kinase, heterotrimeric GTP-binding protein, phospholipase D (PLD) and phosphatidic acid (PA) are also discussed. Finally, we address roles of egg lipid/membrane microdomains or ‘rafts’ as a platform for the sperm–egg membrane interaction and subsequent signaling events of egg activation. © 2006 Elsevier Ltd. All rights reserved. Keywords: Egg activation; Fertilization; Lipid/membrane rafts; Tyrosine phosphorylation; Signal transduction

Contents 1. 2. 3. 4. 5. 6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IP3 metabolism and Ca2+ release at fertilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of phospholipase C: how is IP3 production regulated? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Do PLD and PA act as upstream regulators of PLC␥ and Src? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Roles for egg rafts in sperm–egg interaction and Src-dependent Ca2+ signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lessons from Xenopus system: species-specific or universal? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Abbreviations: [Ca2+ ]i , intracellular-free Ca2+ concentration; IP3 , inositol 1,4,5-trisphosphate; IP4 , inositol 1,3,4,5-tetrakisphosphate; PLC, phospholipase; PI, phosphatidylinositol; PIP2 , phosphatidylinositol 4,5-bisphosphate; DG, diacylglycerol; PKC, protein kinase C; SH2, Src homology 2; PLD, phospholipase D; PA, phosphatidic acid; PH, pleckstrin homology; UPIII, uroplakin III; UPIb, uroplakin Ib; IICR, IP3 -induced Ca2+ release; CICR, Ca2+ -induced Ca2+ release; PIP3 , phosphatidylinositol 3,4,5-trisphosphate; G␣␤␥, trimeric GTP-binding protein ∗ Corresponding author. Tel.: +81 78 803 5953; fax: +81 78 803 5951. ∗∗ Corresponding author. Tel.: +1 303 556 3371; fax: +1 303 556 4352. E-mail addresses: [email protected] (K.-i. Sato), [email protected] (B.J. Stith). 1084-9521/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2006.02.008

Fertilization involves a species-specific recognition and fusion of egg and sperm, to combine parental genomes into that of a new individual. Major goals in the study of fertilization are to understand the molecular mechanisms of the egg–sperm recognition, binding and fusion, and the associated signal transduction events that culminate in the induction of embryonic development [1,2]. More recently, much attention has been focused on transmembrane signaling cascades involving sperm receptor(s) on egg plasma membranes, molecular switches believed

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to transduce the extracellular egg–sperm binding/fusion signal into the intracellular space, and cytoplasmic components involved in the reception as well as propagation of the fertilization signals. This paper reviews recent evidence concerning the sperm-induced egg activation during fertilization in Xenopus laevis. We emphasize the roles of the second messengers produced, the enzymes involved in their production such as phospholipases and tyrosine kinase Src, and the egg membrane rafts during fertilization. A series of events for sperm-induced egg activation has been revealed by both in vivo egg experiments and in vitro cell-free approaches. We refer the reader to other references [3–7] for discussion of more general events of Xenopus fertilization that are not emphasized here, and for discussion of fertilization in other animals, as well as plant systems, see other papers in this issue. 2. IP3 metabolism and Ca2+ release at fertilization A sperm-induced elevation of intracellular-free Ca2+ concentration ([Ca2+ ]i )1 is central to the induction of fertilization events in Xenopus laevis and all other species examined [1,8–18]. The mechanism of the initial rise in [Ca2+ ]i at the sperm-binding site is controversial and may vary from species to species. A number of studies, however, have confirmed the general importance of the second messenger inositol 1,4,5-trisphosphate (IP3 ) as a trigger of the initial rise in [Ca2+ ]i [8–16]. Phospholipase C (PLC) hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2 ) to IP3 and sn-1,2-diacylglycerol (DG). IP3 binds to its receptors [19,20] located in the endoplasmic reticulum to gate this Ca2+ channel and allow flooding of Ca2+ into the cytoplasm. On the other hand, DG is believed to activate protein kinase C (PKC) [21], which might be responsible for cortical granule exocytosis, cortical contraction, sperm chromatin decondensation, and reformation of the nuclear envelope [3,22,23]. The immature Xenopus oocyte undergoes maturation by the action of the steroid hormone to become the fertilizable egg [24]. During maturation, the oocyte moves from the prophase of the first meiosis to metaphase of second meiosis and acquires a higher sensitivity to IP3 to promote a robust and propagative Ca2+ release at fertilization [25]. At fertilization, the increase in IP3 mass is estimated to be ∼200 fmol/egg [26,27] and it is associated with an increase in [Ca2+ ]i from ∼200–400 nM to ∼1.2–1.4 ␮M [5,28]. IP3 increases about four-fold over basal levels and peaks at ∼5 min after insemination [26,27,29,30]. A [Ca2+ ]i wave travels from the sperm-binding site to the opposite side of the egg [28,30–33]. Using a unique rapid sampling and biosensor system, peak IP3 concentration has been estimated to be 175–430 nM (average of 190 nM) near the sperm entry point and 120–700 nM (average of 370 nM) at half way around the egg from the sperm entry point (value taken just after the Ca2+ wave passed that point) [18]. Models for the Ca2+ wave involve Ca2+ stimulation of Ca2+ release [34–36], whereas others suggest a wave of PLC activation [18,37]. In support of the PLC model, a wave of PKC activation, a downstream 1

See abbreviations.

event of PLC activation, has also been found to be associated with the Ca2+ wave [38]. PIP2 antibodies also decrease, but do not eliminate, the [Ca2+ ]i wave [32]. It is also shown that an antibody against the type I receptor for IP3 is capable of inhibiting Ca2+ release in fertilized Xenopus eggs [39]. The selfpropagating Ca2+ wave is believed to be responsible for several biochemical and cellular events of egg activation [22,40,41]. Kline [40] noted that the fertilization membrane depolarization potential (especially sensitive to [Ca2+ ]i localized at the plasma membrane), exocytosis of cortical granules, elevation of the fertilization envelope, sperm decondensation and pronuclear formation were completely inhibited by prior microinjection of the Ca2+ chelator O,O -bis(2-aminophenyl)ethyleneglycolN,N,N ,N -tetraacetic acid (BAPTA). Buffering [Ca2+ ]i with BAPTA resulted in an elevation of the peak IP3 value achieved at fertilization, and the time required to attain the peak increased from ∼5 to 7 min [27]. From these data, one important function of the increase in [Ca2+ ]i at fertilization may be to initiate a negative feedback loop to help turn off the release of Ca2+ through stimulation of IP3 metabolism. In support of this assumption, 3 H-labeled IP3 microinjected into Xenopus eggs showed a relatively short half life of ∼1 min (Ciapa and Stith, unpublished results) whereas, in the presence of BAPTA, the half life increased to 10 min. By viewing peak sizes of various inositol phosphate(s) in a series of samples collected at various times after 3 H-IP3 injection, the pathway of degradation in the IP3 -activated egg was: IP3 → inositol 1,3,4,5tetrakisphosphate (IP4 ) → inositol 1,4-bisphosphate → inositol 1-phosphate → myo-inositol (Ciapa and Stith, unpublished results). Thus, it is suggested that an inositol 3 -kinase acts at fertilization to produce the IP4 . Half maximal release of Ca2+ is achieved at 88 nM IP3 whereas that for IP4 is 3.44 ␮M [42]. Thus, although IP4 may have other functions, IP3 metabolism to IP4 may turn off the Ca2+ release signal. It is interesting to note that this type of metabolic pathway is also known to be present in mammalian cells [43]. 3. Regulation of phospholipase C: how is IP3 production regulated? Since IP3 is believed to be the trigger for sperm-induced initial [Ca2+ ]i release, the mechanism of the activation of the enzyme that produces IP3 , PLC, has been of particular interest. As noted, even while blocking [Ca2+ ]i -dependent fertilization events (gravitational rotation, contraction wave, cleavage) with either BAPTA or heparin, an IP3 receptor blocker, sperm still stimulate PLC and elevate IP3 and DG [27,44]. Thus, there is a support for the model that sperm do not have to elevate [Ca2+ ]i to stimulate PLC. In fact, Ca2+ ionophore increased egg IP3 and DG to values only ∼20–30% as large as that induced by sperm [26,44]. We suggest that the initial sperm-dependent activation of PLC does not require elevated [Ca2+ ]i , however, subsequent stimulation of PLC for Ca2+ wave may be due at least in part to the elevated [Ca2+ ]i . Interestingly, various levels of polyspermy induce equivalent increases in IP3 [26]. Whether 1, 3, 10 or 75 sperm enter the egg, the IP3 peak value is similar. That is, the binding of one sperm is

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as potent as the binding of many sperm in the stimulation of PLC. As each sperm may activate only one or a few receptors [45,46], this result is a bit surprising unless one takes into account the belief that much of the IP3 increase occurs during the subsequent [Ca2+ ]i wave [18,37]. Thus, small initial IP3 production at one sperm entry site, or larger initial IP3 production at multiple sperm entry sites would not affect the total production of IP3 , which is largely dependent upon the regenerative [Ca2+ ]i wave. Although multiple waves after polyspermy would be expected and thus one might estimate a higher IP3 peak, annulations of colliding Ca2+ waves or refractive periods after wave passing may minimize the IP3 contribution of multiple waves [36]. One might compare the IP3 data obtained with polyspermy data and a possible Xenopus sperm factor such as PLC␨ [47]. If more some 75 sperm enter the egg and contribute 75-fold higher levels of active PLC␨-like enzyme than that found in the monospermic egg, polyspermy would be expected to be associated with faster increase in and higher maximum levels of higher levels of IP3 production. Since the entry of ∼75 sperm did not result in faster or higher levels of IP3 than that of the monospermic case [26], these data do not support the idea that a PLC-like Xenopus sperm factor is a trigger of IP3 production. It should be remembered that the production of IP3 at the sperm-binding site is less than that produced during subsequent calcium waves, but the exact ratio is still not known. Furthermore, it may be possible to argue that there is not sufficient substrate to produce higher levels of IP3 in the polyspermic zygote. However, PIP2 levels are at least ∼800–1000 fmol per egg (although all may not be accessible to PLC) [30], and the IP3 increase after the entry of one sperm is ∼200 fmol [26], there should be sufficient substrate to produce higher levels of IP3 after polyspermy. Another aspect of sperm-derived factor(s) in Xenopus fertilization, as we discuss below (Sections 4 and 5), is that sperm may contain some biologically active components unrelated to PLC, which seem to be essential for sperm–egg interaction and subsequent egg activation events. Moving from data on the [Ca2+ ]i regulation of PLC, what other biochemical signaling path might be involved in the activation of PLC at fertilization? One mechanism would involve tyrosine kinase-dependent activation of PLC␥, as found in some invertebrate species [48]. However, microinjection of the PLC␥ Src homology 2 (SH2) domain recombinant protein did not inhibit fertilization in Xenopus [39] or mouse [49]. Runft et al. [39] were careful to state that this result does not preclude tyrosine kinase activation of PLC␥ but that it may preclude a mechanism of PLC activation that requires SH2 domains of PLC␥. In Xenopus, however, PLC␥ is rapidly tyrosine-phosphorylated, associated with the tyrosine kinase Src, and activated within minutes of insemination ([29,50,51,64]). Preincubation of unfertilized eggs with specific tyrosine kinase inhibitor (PP2) results in a decrease of tyrosine phosphorylation as well as enzymatic activation of PLC␥ in response to insemination [9,29]. Concomitant decrease of IP3 production and lack of Ca2+ release have also been demonstrated with tyrosine kinase inhibition [29]. On the other hand, artificial activation of Src or introduction of active Src can mimic sperm and promote tyrosine phosphorylation as well as enzymatic activation of PLC␥ [9,51].

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Taken together, these data provide strong support for the idea that a Src-dependent PLC␥ activation is responsible for sperminduced Ca2+ release in fertilized Xenopus eggs. However, several unanswered questions remain: (1) how is PLC␥ activated by an SH2 domain-independent mechanism (see Section 4)? (2) How is Src activated in fertilized eggs (see Section 5)? (3) Is there any role for other PLC isoforms? As to the third question, Runft et al. [39] has demonstrated that a specific antibody against a subunit of Gq protein, which would block Gqdependent activation of PLC␤ activation, does not inhibit normal fertilization in Xenopus. However, as previously reported, introduction of GTP␥S can promote parthenogenetic egg activation in Xenopus [52], suggesting an involvement of GTP-binding protein(s) such as heterotrimeric GTP-binding protein(s) and downstream PLC␤ subspecies. Further investigation of GTPbinding protein(s) that can participate in the GTP␥S-dependent egg activation would be of interest. 4. Do PLD and PA act as upstream regulators of PLC␥ and Src? Although tyrosine phosphorylation-dependent activation of PLC␥ is well established in somatic cells, the question of how PLC is activated in the absence of SH2 domain-mediated protein-protein interaction remains unanswered. Due to our previous finding that choline mass increased at fertilization [44], we became interested in phospholipase D (PLD) as an upstream regulator of egg PLC␥. Phosphatidic acid (PA) and choline are produced through the PLD-induced hydrolysis of phoshatidylcholine. Over a period of 20 years, many references have suggested that PA activates PLC and increases [Ca2+ ]i (e.g., [53]). However, the mechanism by which PA activates PLC has not been determined. The literature suggests that PA may bind directly to various PLC isoforms. PA, but not phosphatidylserine, phosphatidylcholine, or phosphatidylethanol, stimulated the in vitro activity of partially purified PLC from Xenopus oocytes [54]. However, the in vitro work involved preparations of PLC that may contain Src kinase, which would also be activated by PA (see below). Furthermore, other in vitro studies produced conflicting results as to the PLC isoform that can be activated by PA and which kinetic constant is altered. PA may also directly activate Src [55], which, in turn, would activate PLC␥. In support of this concept, it has been demonstrated in mammalian cell systems that catalytically active PLD is required for Src activation by mitogens, and that PLD1 can activate Src in vitro [56]. PA caused a rapid tyrosine phosphorylation of neutrophil proteins (other anionic lipids such as phosphatidylserine, PIP2 or phosphatidylinositol were ineffective [57]). Dicapryl PA addition to neutrophils caused an activation within 10 s of two tyrosine kinases: Fyn and Syk, tyrosine phosphorylation of PLC␥, and an increase in IP3 mass and [Ca2+ ]i . These events were inhibited by the tyrosine kinase inhibitor herbimycin A [58,59]. With these data, we can suggest a model for [Ca2+ ]i release in Xenopus fertilization involving PLC, PLD, PA, and tyrosine kinase Src. Our unpublished data indicate that (1) by employing new methodology to measure the mass of PA [60], a 2.5-fold

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increase in PA is detected within 1 min of insemination; (2) PA peaks (1 min) well before the peak of IP3 is reached (5 min); (3) pretreatment of eggs with primary, but not secondary, alcohols inhibited the PA increase at fertilization and fertilization events. One well-known hallmark of the PLD work has been the unique ability of PLD to utilize primary alcohols (such as 1-butanol, with 2-butanol as a control) in a “transphosphatidylation” reaction that inhibits PA production: phospholipases such as PLC and phospholipase A2 do not utilize primary alcohols [61,62]. Furthermore, PA stimulation of PLC was not inhibited by 1-butanol. It is also worth noting that insemination after 1-butanol treatment of eggs did not prevent dark sperm entry spots or increased DAG production (molecular species data suggest DAG production from phosphatidylcholine not PIP2 ). Thus, sperm–egg fusion and stimulation of at least one biochemical path do not seem to be inhibited by 1-butanol, but subsequent [Ca2+ ]i release by insemination was. (4) PA addition to eggs increases the mass of IP3 independently of [Ca2+ ]i and primary alcohols or BAPTA do not prevent this activation; (5) the addition of PA also induces fertilization events and this induction can be blocked by an IP3 receptor blocker ((2-aminoethoxy)diphenylborane) or BAPTA; (6) PA addition to Xenopus eggs stimulates Src activation and PLC␥ tyrosine phosphorylation. These data corroborate the idea that PLD activity and its product PA play an important role in activating Src/PLC␥ pathway in Xenopus eggs [63,64]. In Xenopus eggs, at least one PLD isoform has been identified at the cDNA level: PLD1b (accession number: AY233395). Further analysis will evaluate the role of egg PLD1b in fertilization. PLD and its ability to raise PA levels may also play a role in membrane fusion such as that in sperm–egg fusion or during exocytosis [65]. 5. Roles for egg rafts in sperm–egg interaction and Src-dependent Ca2+ signaling A recent trend in cell and developmental biology is the hypothesis that cell regulation occurs through cell plasma membrane-located “rafts”. Rafts are lipid/membrane microdomains enriched in cholesterol and sphingolipids. A number of studies have suggested that such rafts would play a crucial role in extracellular signal reception, cell–cell or cell–substratum interaction, trafficking, and pathogen interaction [66]. In general, the physiological relevance as well as the existence of rafts in living cells is still in dispute [67,68], however, results obtained in Xenopus eggs support the idea that egg rafts serve as a platform for sperm–egg membrane interaction/fusion and subsequent ignition of sperm-induced egg activation signaling. Most importantly, the in vitro analysis of egg rafts in Xenopus has allowed us to understand the molecular basis of sperm-induced activation of PLC, IP3 production and Ca2+ release. As described in a previous section (Section 3), Xenopus egg fertilization involves a sequential activation of the tyrosine kinase Src and PLC␥. Src activation at Xenopus fertilization may be due to sperm–egg membrane interaction through integrin–disintegrin complexes on the gamete plasma membranes [69–72] or sperm factors such as phosphatidic acid (see Section 4) and proteases (see below [73]).

We initially found that Src is enriched in Triton X-100resistant, low-density membrane fractions (a criterion for rafts) of unfertilized Xenopus eggs. These fractions, like rafts, are enriched in cholesterol and the GM1 ganglioside [74]. Further analysis using fertilized eggs demonstrate that sperm-induced tyrosine phosphorylation on tyrosine-415 of the Xenopus Src protein, a phosphorylation associated with the activation of Src, and some other proteins occurs mainly in the rafts [74]. When rafts in eggs are disrupted with the cholesterol-depleting drug methyl-␤-cyclodextrin, the sperm-induced Src phosphorylation and Ca2+ release are inhibited [74]. These results are consistent with the scheme that egg rafts are required for proper sperm–egg interaction and Src-dependent Ca2+ release. In contrast to Src, PLC␥ is not present in rafts of unfertilized Xenopus eggs. It is demonstrated, however, that, after minutes of insemination, PLC␥ translocates from the non-raft fraction to the raft fraction and is tyrosine-phosphorylated; i.e., activated [51]. Raft localization of PLC␥ is transient and cannot be detected 20 min after insemination. The molecular mechanism of the translocation of PLC␥ to rafts is unknown. It may involve PLC binding of PA, but not an SH2 domain-dependent mechanism as discussed above (Section 3). Direct interaction of Src and PLC␥ is also possible, as their co-immunoprecipitation from the extract of fertilized eggs was shown [30]. Another mechanism for the translocation may involve the pleckstrin homology (PH) domain of PLC␥. This domain has a high affinity for PIP2 and PI 3,4,5-trisphosphate (PIP3 ) [75]. When eggs were preincubated with LY294002, which inhibits PI 3-kinase and formation of PIP3 , sperm cannot promote Src activation and Ca2+ release, and eggs do not activate (Sato et al., unpublished results). Moreover, the 85-kDa regulatory subunit of PI 3-kinase undergoes raft translocation on a time course similar to that of PLC␥ (Sato et al., unpublished results). Further study should evaluate whether PIP3 plays a role in fertilization. Another important aspect of the study on the egg rafts is the potential for identification of components of the egg membrane rafts capable of interacting with sperm membranes, which would suggest a role for this molecule(s) in sperm–egg fusion and/or in egg activation. Although previous studies have suggested that egg-associated molecules serve as a sperm receptor (e.g., gp69/64 [45,46]), these egg molecules localize to the egg vitelline membrane. This layer is external to the plasma membrane and thus could not be directly involved in signaling events of the egg cytoplasm. Thus, gp69/64 may be involved in sperm binding but not signal transduction. A surprising result obtained using egg rafts from unfertilized eggs was that the addition of sperm to the raft preparation could promote activation of Src that was associated with rafts [51,74]. Furthermore, in combination with the extract prepared from unfertilized eggs (an extract, referred to the “cytostatic factorarrested” or “CSF” extract), sperm-treated egg rafts can reproduce several events of egg activation including PLC␥ activation, IP3 production, and transient Ca2+ release [51,76]. These results support the possibility that egg rafts contain sperm-interacting machinery that may be a complex of several components. A strong candidate for a putative sperm receptor protein in the egg rafts is uroplakin III (UPIII). We have shown that this

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Fig. 1. A model for the signaling network that triggers intracellular Ca2+ release in fertilized Xenopus eggs. Transient Ca2+ release, depicted as ‘Ca2+ ’ in the figure, in the fertilized Xenopus egg involves two major processes: IP3 -induced Ca2+ release (IICR) (1) and Ca2+ -induced Ca2+ release (CICR) (2). IP3 and DG (3) are produced by the hydrolysis of PIP2 that is catalyzed at least in part by PLC␥, a member of the PLC family of enzymes. Minutes after sperm–egg interaction, PLC␥ undergoes translocation from the cytoplasm to lipid/membrane microdomains called ‘rafts’. Src then phosphorylates PLC␥ on tyrosine and activates the lipase (4). Some portion of tyrosine-phosphorylated PLC␥ reenters the egg cytoplasm (5) and may catalyze IP3 production in membranes within the cytoplasm of the fertilized egg. Increased concentration of intracellular-free Ca2+ ions, as promoted by IICR and CICR, may also be responsible for further activation of PLC in the egg cytoplasm (6). It has been demonstrated that the SH2 domain of PLC␥, which can play a major role in PLC␥ membrane-translocation and activation by tyrosine phosphorylation, is not required in Xenopus egg fertilization. Thus, raft-translocation of PLC␥ should utilize an alternative mechanism: the PH domain of PLC␥ may bind to PIP2 and/or PIP3 in the membrane (7). PIP3 is an enzymatic product of PI 3-kinase. The 85-kDa subunit of PI 3-kinase is shown to translocate from the non-raft egg membrane to the raft membranes within minutes of sperm–egg interaction (8) and a pharmacological PI 3-kinase inhibitor, LY294002, inhibits sperm-induced PLC␥ activation and the Ca2+ transient. As discussed in the text, it is unlikely that PLC␨-like sperm-derived factors are responsible for the Ca2+ transient (9), although we suggest that another contribution from sperm may play a role (i.e., PA and protease; see below). Sperm-induced activation of Src, an upstream event of PLC␥ activation, occurs in the raft membranes (10). Src is concentrated in the raft membranes of unfertilized eggs and its activation can be reconstituted in vitro by the addition of sperm to the isolated rafts. Sperm-induced Src activation may involve three pathways: activation of heterotrimeric GTP-binding protein(s) (G␣␤␥) (11) that would connect the extracellular proteolysis of UPIII by the sperm-derived protease activity (9) and the intracellular enzymatic up-regulation of Src. Secondly, PIP3 , a possible trigger for raft-translocation of PLC␥, could also be a trigger for Src activation (12). Third, involvement of PLD activity and its enzymatic product, PA, both of which increase after sperm–egg interaction, may activate Src (PA may also bind and directly activate PLC␥) (13). The origin of stimulatory PA (from sperm or zygote or both?) is now under investigation. One candidate molecule for a sperm receptor is UPIII (14), a single-transmembrane protein that is predominantly tyrosine-phosphorylated (possibly by Src) in the rafts of fertilized eggs (15). The function of the tyrosine-phosphorylated form of UPIII is unknown. A specific antibody against the extracellular domain of UPIII can inhibit normal fertilization and sperm-derived tryptic protease(s), which are thought to be important in initiating egg activation and partial proteolysis of UPIII (9). Sperm–egg interaction and egg activation signaling may also involve a sperm disintegrin-egg integrin interaction (9). UPIII interacts with a tetraspanin partner, UPIb (16), to provide proper molecular organization in the egg rafts. Note that the thick grey arrows in the figure represent activation events and that the thin black arrows represent translocation events.

protein, a 30-kDa raft-associated single-transmembrane protein, is tyrosine phosphorylated upon fertilization [77]. The phosphorylation of UPIII could be detected as early as 5 min post-insemination of dejellied Xenopus eggs [78]; it is slower than the phosphorylation of Src and PLC␥ (as early as 1–2 min post-insemination) [78]. It is thought that the carboxyl-terminal cytoplasmic tail of UPIII may be involved in intracellular signaling via tyrosine phosphorylation. Moreover, the relatively larger extracellular region of UPIII is likely to be involved in sperm–egg interaction because: (1) a specific antibody against the extracellular domain inhibits fertilization [77]; (2) the extracellular domain of UPIII is a target of sperm-derived tryptic protease and inhibition of this proteolysis by several inhibitors blocks fertilization [78]. In addition, proteolysis of egg membrane components (including UPIII) is important for sperminduced Src activation. Thus, the molecules for signal transduction from sperm–egg interaction to [Ca2+ ]i release can be envisioned to occur as follows: binding of sperm membrane components to UPIII and its associated partner(s) results in partial proteolysis of UPIII by the sperm protease, leading to activation of PLD and production of PA, which induces Src activation, stimulation of PLC␥, IP3 production and [Ca2+ ]i release

(Fig. 1). Although involvement of heterotrimeric/monomeric G proteins is still in dispute, it is interesting to note that sperminduced Src activation could be mimicked by GTP␥S, a potent activator for G proteins [51]. The relationship between these steps and sperm–egg membrane fusion is unknown. In mammalian bladder border cells, UPIII is known to associate with a tetraspanin protein uroplakin Ib (UPIb) to form a rigid membrane structure called the asymmetric unit membrane [79–81]. UPIb has a similar structural architecture to CD9, which is known to be essential for sperm–egg fusion in the mouse [82–85] and immunochemical studies suggest that both CD9 and UPIb are present in Xenopus eggs [74,78]. Therefore, future experiments will center in on the analysis of UPIII/UPIb and on its possible involvement in sperm–egg fusion. 6. Lessons from Xenopus system: species-specific or universal? On may suggest that the Xenopus system is a good model organism for studying signal transduction of fertilization and egg activation. Like Xenopus fertilization, sea urchin, ascidians, starfish and zebrafish fertilization may also involve tyrosine

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kinase activation [48,86–89]. More specifically, a Src inhibitor blocks the sea urchin fertilization, Src is activated at fertilization and binds PLC␥, whereas elevation of Src activity can induce fertilization events [86]. In opposition to the idea that sperm act through Src tyrosine kinase, mammalian sperm are believed to act through a “sperm factor” that diffuses from the sperm into the egg [90]. Mammalian sperm factor has been identified as constitutively active PLC␨ as immunodepletion of PLC␨ from crude sperm factor preparation inhibits its ability to release [Ca2+ ]i , and injection of recombinant PLC␨ induces [Ca2+ ]i oscillations similar to those found in mammalian fertilization [90–92]. More recently, others and we have shown evidence against a role for a tyrosine kinase in mammalian (porcine) sperm factor action and fertilization [93,94]. These results are similar to those found for boar sperm factor: its ability to release calcium was not inhibited by tyrosine kinase inhibitors (genistein or tyrphostin B42 [90]). Taken together, these data suggest that, in spite of the fact that both Xenopus and mammalian eggs are arrested in metaphase II, signal transduction pathways leading to PLC activation at fertilization may be quite different. On the other hand, the state of arrest of the egg from sea urchins is different from Xenopus and mammals (e.g., in sea urchin, the egg is arrested after completion of meiosis) but the system used for egg activation may be the Src/PLC␥ path. Due to these apparently contradictory circumstances, the study of Xenopus fertilization may lead to clarification of species-specificity as well as general scheme of fertilization at molecular level. 7. Perspectives Recent advancements in the biology of Xenopus fertilization have allowed investigators to explore the molecular network of sperm–egg interaction/fusion and sperm-induced egg activation events. The methods effectively employed so far include application of pharmacological reagents that activate or inhibit molecules of interest, monitoring of biochemical responses with use of specific probes such as antibodies and affinity tags, and reconstitution of signaling events in vitro using cell-free extracts such as the cytostatic factor-arrested unfertilized egg and cycling extracts. Although the Xenopus system has limitations in the use of some genetic approaches (e.g., gene knockout and gene knockdown), it offers certain advantages in biochemical and cell biological studies that make it a valuable system in which to study fertilization. To fulfill this expectation, the need to integrate two methodologies becomes necessary: one is the systematic characterization of the signaling components in gametes and embryos during fertilization (utilizing proteomics, lipidomics and glycomics) and the other is the visualization of signaling events in living gametes and embryos. Accomplishing these tasks in the Xenopus egg system may lead to novel information in the biology of fertilization and facilitate studies very difficult to perform in other vertebrates such as mouse and fish. More specifically, we believe that further integration of identified signaling molecules with visualization of their cellular distribution in egg lipid/membrane microdomains (rafts) is likely to make important contributions not easily achieved in other vertebrate species.

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