Early ionic events in activation of the mammalian egg

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1 Reviews of Reproduction (1998) 3, Early ionic events in activation of the mammalian egg Dalit Ben-Yosef 1 and Ruth Shalgi 2 1 Sara Racine IVF Unit, LIS Maternity Hospital, Tel Aviv Sourasky Medical Center, Tel Aviv and 2 Department of Embryology and Teratology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel At fertilization of the mammalian egg, the spermatozoon initially binds to and then fuses with the egg plasma membrane. This critical event activates specific biochemical pathways within the egg. Activation of the egg induces resumption of meiosis and the start of rapid embryonic mitotic divisions on the one hand, and cortical granule exocytosis leading to modification of the zona pellucida and a block to polyspermy on the other. It has been shown in different systems that changes in intracellular ion concentrations can serve as second messengers of signal transduction mechanisms. The use of specific fluorescence probes, combined with the image analysis technique, facilitates the measurement of their dynamics in real time in the living cell and, thereby, assessment of their role in activation of the mammalian egg. This review focuses on the dynamics of intracellular and ph and their role in transducing the sperm signal to downstream cell cycle regulators. The mammalian oocyte enters meiosis during embryonic life. The meiotic division proceeds to the first prophase, and is then arrested at the diplotene stage, when the germinal vesicle (GV), a characteristic nuclear structure, can be observed. Resumption of meiosis, also known as oocyte maturation, occurs in response to the preovulatory surge of LH, and is followed by ovulation (Tsafriri and Dekel, 1994). The mature oocyte, commonly referred to as the egg, is again arrested at the second metaphase (MII), and considered a relatively quiet cell, with a pre-set developmental programme. At fertilization, the spermatozoon overcomes this second cell cycle arrest, and initiates the sequence of biochemical events that leads to the rapid embryonic mitotic divisions. These events, triggered within the egg at fertilization, are collectively referred to as egg activation. Cortical granule exocytosis (CGE) is an early event characteristic of egg activation. Completion of CGE leads to modification of the zona pellucida and the block to polyspermy (Ducibella, 1991, Kline and Stewart-Savage, 1994; T. Raz and R. Shalgi, unpublished). Activation by the spermatozoon also leads to later events including resumption of meiosis, pronuclei formation, initiation of DNA synthesis and cleavage (Xu et al., 1994). This review focuses on the dynamics of early ionic changes leading to mammalian egg activation. How does a spermatazoon activate an egg? Fertilization in eutherian mammals takes place in the ampullary region of the oviduct. The first interaction between the spermatozoon and egg is at the zona pellucida, an extracellular coat that surrounds all mammalian eggs. The acrosome-reacted spermatozoon penetrates the zona pellucida, passes the perivitelline space and interacts with the plasma membrane of the egg (reviewed by Schultz and Kopf, 1995). This initial interaction involves specific recognition between components on the postacrosomal region over the equatorial segment of the acrosome-reacted sperm head, and the oolemma (Shalgi and 1998 Journals of Reproduction and Fertility /98 $12.50 Phillips, 1980; Myles et al., 1994; reviewed by Yanagimachi 1994). The interaction between spermatazoon and egg membranes initiates a cascade of biochemical events that trigger the egg to proliferate and develop into a new organism. Mechanisms by which a spermatazoon activates an egg The sperm factor theory This hypothesis proposes that the cellular messenger is generated as a result of fusion between the spermatazoon and egg membranes and delivery of an activating component from the sperm cytosol to the ooplasm. The hypothesis was introduced for mammalian spermatozoa by Swann (1990), after microinjection of soluble sperm extracts that were capable of inducing early events of egg activation (that is, an increase in intracellular concentration ([ ] i ) followed by oscillations). The cytosolic sperm factor triggers oscillations in several mammalian species, and is not species specific (Homa and Swann, 1994; Wu et al., 1997). A widespread efficacy of the factor was demonstrated by its ability also to induce oscillations in neurones and hepatocytes (Berrie et al., 1996). A soluble sperm protein correlated with oscillation-inducing activity in eggs has been identified, and termed oscillin. Oscillin is an oligomer with a subunit of 33 kda that exhibits a specific intracellular localization at the equatorial segment of the sperm head, an appropriate region for egg activation during fusion (Parrington et al., 1996). The hypothesis requires fusion between the spermatozoon and egg before any increase in cellular second messengers occurs. Using two independent fluorescence methods and confocal microscopy, Lawrence et al. (1997) demonstrated that sperm egg fusion is the prelude to the initial increase at fertilization. They estimated the time interval between sperm egg fusion and the onset of the [ ] i oscillations as 1 3 min. The clinical intracytoplasmic sperm injection

2 Early ionic events in mammalian egg activation 97 procedure (ICSI) further supports the concept of the spermatozoon containing an activating molecule in its cytosol. The ICSI procedure results in full egg activation, as well as normal embryonic development, although no contact between spermatozoon and egg membranes occurs. The receptor theory This hypothesis proposes that recognition between spermatozoon and egg receptors, analogous to hormone action in somatic cells, is sufficient to induce activation. The model suggests that the egg receptor for the spermatozoon transduces a signal that mediates a cascade of subsequent cellular events. It is suggested that the spermatozoon may use a G-proteincoupled receptor, or a tyrosine (Tyr) kinase receptor. Although the receptor itself has not yet been identified, studies using specific activators or inhibitors support either of the two receptor-mediated models. It has been demonstrated that complete activation of the mouse egg in the absence of spermatozoa can be achieved by stimulation of an exogenous heterotrimeric and monomeric G-protein-coupled receptor (Williams et al., 1992; Moore et al., 1993, 1994). Strong support for the G-protein model has been received from inhibition studies. Microinjection of the G-protein antagonist, GDPβS, into hamster or mouse eggs blocked several aspects of sperm-induced egg activation (Miyazaki, 1988; Moore et al., 1994). Some aspects of egg activation can occur through the Tyr kinase receptor pathway (starfish: Shilling et al., 1994; Xenopus: Yim et al., 1994; sea urchin: Abassi and Foltz, 1994), yet the Tyr kinase receptor in mammals has not been examined. Second messengers that mediate egg activation In the last decade, progress has been made in the development of fluorescence probes specific for different ions. These probes, combined with the image analysis technique, provide a valuable tool with which to follow changes in intracellular ion concentrations in living cells, and to assess their role in mammalian egg activation. Some of these ions were tested initially in lower species and were shown to transduce the sperm signal to downstream cell cycle regulators. Calcium The primary signal transduction event observed at fertilization in all mammalian eggs studied, including those of humans, is an increase in [ ] i followed by oscillations (mouse: Cuthbertson and Cobbold, 1985; hamster: Miyazaki, 1991; pig: Sun et al., 1992; cow: Fissore et al., 1992; rat: Ben-Yosef et al., 1993; rabbit: Fissore and Robl, 1993; human: Taylor et al., 1993). A representative pattern is shown (Fig. 1). Dynamic measurements of [ ] i revealed a marked change accompanying fertilization of the mammalian egg. This first fertilization transient takes the form of a wave originating at the point of sperm entry (Miyazaki, 1988). A similar wave was described in Xenopus and sea urchin eggs, in which fertilization produces only a single transient. However, in mammals, the first distinctive transient is followed by a series of oscillations of high amplitude and short duration that persist for several hours. The basal [ ] i in ovulated MII Fig. 1. Intracellular oscillations in fertilized rat eggs. A representative tracing of repetitive transients in rat eggs inseminated at the time indicated by the arrow. Eggs were isolated and loaded with Fura 2. Changes in intracellular were recorded in individual fertilized eggs from insemination until 70 min after insemination. An example of an in vitro fertilized zona-free egg that responded to sperm attachment by repetitive transients is shown. arrested mammalian eggs is approximately 100 nmol l 1, a characteristic value within many different inactive somatic cells. The initial transient starts within 1 3 min of sperm fusion. This displays variability in its amplitude and duration, although it is usually lower and lasts longer than the subsequent transients (duration 3 6 min, with an amplitude of approximately 3 4 times the basal level). This first transient is followed by a series of narrower transients (duration min) of high amplitude (up to 6 8 times the basal level). Rodent eggs, as well as those of humans, exhibit high frequency oscillations (regular peak-to-peak intervals ranging between 2 and 4 min), whereas in bovine and porcine eggs, min elapse between two transients. Thus, it appears that the response during fertilization is characterized by a major difference between the first transient and those that follow, as well as a major difference between the kinetics of intracellular increase and its subsequent decrease in a single transient. The first transient is, as a rule much wider (fourfold) than the subsequent transient in a series in the same egg. When analysed by video rate kinetics (25 frames s 1 ), the first transient is often biphasic (Fig. 2a). When the duration of the first transient was compared with the duration of the subsequent transients, it was suggestive of a fusion of two consecutive transients forming the first transient. This implies that the mechanisms of intracellular sequestration and/ or extrusion are not yet coordinated during the first event. Whatever the correct interpretation may be, it is clear from the phenomenology that the first transient differs markedly from the subsequent transients. This pattern may be a result of the initial rise in [ ] i itself, or other parallel cellular events initiated by fertilization. The shape of the subsequent transients is similar in practically all eggs that responded (Figs 1 and 3). The increase in [ ] i is very slow, and is followed by a rapid increase once a certain threshold concentration is attained (Fig. 2b). This corresponds well to the biphasic sensitivity of IP 3 - mediated -release described by Bezprozvanny et al. (1991).

3 98 D. Ben-Yosef and R. Shalgi Fig. 3. oscillations in fertilized human eggs. Aged human oocytes (48 h after pickup) that failed to be fertilized after insemination in vitro were loaded with Fura 2. Changes in cellular were recorded and a representative pattern of two fertilized eggs is shown (O. Rufas, D. Ben-Yosef, B. Fisch and R. Shalgi, unpublished). Fig. 2. Video rate kinetics of the first distinctive transient, compared with the subsequent oscillations. Rat eggs loaded with Fura 2 were inseminated and changes in cellular were recorded (25 frames s 1 ). (a) Video rate kinetics of a representative pattern of the first distinctive transient. The transient is often biphasic with an amplitude of about 3 4 times the basal concentration and a duration of 3 6 min. (b) Video rate kinetics of a representative transient from a series of oscillations. These transients last min each, and increase to 6 8-fold of the basal concentration. The regular peak-to-peak intervals range between 2 and 4 min. The transient starts with a very slow increase in [ ] i, followed by a rapid increase once a certain threshold [ ] i is attained. However, the decrease of [ ] i to the basal concentration is biphasic with the slow phase followed by a rapid phase. The increasing concentrations of [ ] i promote faster release, forming a feed-forward loop. Moreover, the decrease of [ ] i to basal concentrations is, in many eggs, clearly biphasic (slow phase followed by a rapid phase), suggesting two distinct mechanisms of intracellular removal acting in tandem. The repetitive transients continue for several hours after sperm entry (Cuthbertson and Cobbold, 1985). As fertilization progresses, the amplitude and frequency of the transients decreases and the duration increases (Ben-Yosef et al., 1993; Fissore and Robl, 1993) until an absolute cessation during entry into interphase, at the time when pronuclei are forming (Jones et al., 1995). Continuous measurements of intracellular changes at fertilization have been performed systematically on zonafree eggs, which are practical for this purpose although susceptible to polyspermy. The phenomenon of cyclical transients in in vivo fertilized eggs has been shown to reflect the physiological events that occur during fertilization in mammals (Ben-Yosef et al., 1993). It has been shown by studying the role of early changes in egg during fertilization that a single transient is sufficient to cause exocytosis of most of the cortical granules, leading to an effective block to polyspermy as well as inducing resumption of the second meiotic division (Jaffe, 1985; Tombes et al., 1992; Vincent et al., 1992, Kline and Stewart-Savage, 1994). Moreover, blocking the fertilization-induced [ ] i increase inhibits egg activation (Kline and Kline, 1992). However, microinjecting mouse eggs with physiological concentrations of inositol 1,4,5-tris-phosphate (IP 3 ), resulted in CGE but not in emission of the second polar body or pronuclei formation (Kurasawa et al., 1989). Fine-tuning of the single transient demonstrates that low concentrations allow CGE, while higher concentrations are required for cell cycle resumption (Raz et al., 1998). Therefore, it is conceivable that cell cycle progression is dependent upon the temporal and spatial aspects of the transient. Most activators of release induce a larger increase than the initial response at fertilization, thus leading to complete activation of the egg. It is possible that the first physiological transient is not adequate for complete egg activation, and additional transients are required. In two studies, Ozil and coworkers simulated the normal sperm-induced transients by repeated electric field pulses, evoking increases at a frequency similar to that seen at fertilization (Ozil, 1990; Vitullo and Ozil, 1992). They suggested that the oscillations may somehow be coupled to events occurring during the first cell cycle, and come into play when a prolonged signal is required after a single stimulus, as in fertilization. A deeper understanding of the mechanism associated with physiological egg activation in mammals is important especially in the field of human IVF. Therefore, it is of interest to verify whether fertilization-associated events observed in rodents resemble those characterized by activation of human eggs. The first evidence of the existence of oscillations in human eggs was provided by Taylor et al. (1993) who found

4 Early ionic events in mammalian egg activation 99 that insemination of zona-free, as well as zona-enclosed, human oocytes resulted in cyclical transients (every min) of short duration (2 min) and high amplitude. Rufas et al. (1994) demonstrated that aged human oocytes (48 h old) that failed to fertilize after insemination in vitro could fuse with and decondense human spermatozoa. Using this model, insemination of aged human oocytes was shown to result in cyclical transients with regular peak-to-peak intervals of 1.5 min and fairly short duration (1.2 min; Fig. 3). Collectively, these findings demonstrate that aged human oocytes may serve as a model for the study of early events in human egg activation. The mechanism by which the fertilizing spermatozoon triggers the intracellular increase is not completely determined. The most recent evidence points to sperm-initiated hydrolysis of the egg phosphatidylinositol 4,5-biphosphate, generating IP 3, which is known to release from intracellular stores. G-protein-coupled receptors undergo a conformational change, which activates its coupled enzyme phospholipase C (PLCβ) to induce IP 3 production. Tyrosine kinase receptors undergo ligand-mediated dimerization, allowing the kinase domains to phosphorylate each other on specific Tyrs. These phosphotyrosines (P-Tyrs) provide docking sites for cytosolic PLCγ1 (a cytosolic Tyr kinase), which are thereby translocated to the plasma membrane to catalyse IP 3 and diacylglycerol (DAG) production. Dupont et al. (1996) have demonstrated that mouse oocytes have mrna encoding PLCβ1, PLCβ3 and PLCγ. However, when protein expression was studied, only PLCγ could be immunodetected. A specific PLC inhibitor (U73122) exerted an inhibitory effect on oocytes activated by spermatozoa, as well as on oocytes activated by acetylcholine, which stimulates spiking through PLC activation. Collectively, the above data suggest that the activation of PLC to generate IP 3 plays a critical role in fertilization. The site of release and sequestration is generally accepted as the endoplasmic reticulum. Mehlmann et al. (1995) demonstrated that competence to undergo normal activation at fertilization is associated with a marked reorganization of the egg endoplasmic reticulum, and the development of the IP 3 - induced release system. Nevertheless, two types of mechanism can mediate release from intracellular stores: IP 3 - induced release (IICR) mediated by the IP 3 receptor, and -induced release (CICR) mediated either by the ryanodine receptor or by IP 3 receptor. Studies on eggs of different classes (sea urchin compared with Xenopus and mammals) have claimed that different mechanism(s) operate at fertilization. By microinjection of a functional blocking monoclonal antibody to the IP 3 receptor of the release channel, Miyazaki et al. (1992) demonstrated that IICR from intracellular stores operates at fertilization in the hamster egg, and that it is essential for the initiation, propagation, and oscillation of the sperm-induced transients. When the same antibody was injected into the mouse egg before insemination, it prevented CGE and pronuclear formation, as well as some other activation-related events (Xu et al., 1994). Likewise, in rabbit eggs, IICR has been shown to participate in the generation of fertilization-associated intracellular increases, and it has been suggested that the time to reach threshold concentrations of IP 3 dictates the periodicity of the oscillations (Fissore (a) 490 Fluorescence intensity (b) 340:380 Fluorescence intensity ratio 5 min Fig. 4. ph i and [ ] i of the same egg during fertilization. Rat eggs were double loaded with Fura-2-acetoxymethyl (AM) ester and 2,7 -biscarboxyethyl-5(6) carboxyfluorescein (BCECF)-AM. Simultaneous recording at four wavelengths (340, 380, 440, 490 nm), for [ ] i and ph i in the same egg, was performed. (a) 490 fluorescence intensity of two representative fertilized eggs (doubleloaded). The emission in 490 nm, the ph-sensitive wavelength is unchanged. Therefore, it can be concluded that no change in intracellular ph accompanies fertilization. (b) 340:380 fluorescence intensity ratio of the same two fertilized eggs as in (a). The exact time of the first [ ] i transient reflects sperm attachment to the oolemma and confirms fertilization of the specific monitored egg. and Robl, 1994). Therefore, a single-pool model for oscillations, assuming a continuous influx of from outside and -sensitized IICR, was proposed to operate at fertilization of mammalian eggs. Although ryanodine receptors are present and functional in mouse and bovine eggs, release of mediated by this receptor is not essential for sperm-induced egg activation (Ayabe et al., 1995; Yu et al., 1995). Preincubating eggs with ryanodine, or its addition to oscillating oocytes, indicates that the ryanodine receptor may be involved in the modulation of oscillations, rather than in their initiation (Wu et al., 1997). Swann and Lai (1997) postulated that the sperm protein oscillin may affect the intracellular stores directly by altering the intracellular channel. Once modified, the release channels would display an increased sensitivity to CICR, which may involve IP 3 or ryanodine receptor or both. Either the sperm factor, or a sperm-receptor signal transduction mechanism, could lead to IP 3 production and initiation of intracellular oscillations. At present, there is no direct evidence to support either of the sperm-mediated models, and a combination of both theories appears more conceivable. It is

5 100 D. Ben-Yosef and R. Shalgi channel Egg plasma membrane Sperm protein oscillin R PTK G protein IP 3 PLC PIP 2 PKC DAG Alkaline environment ER, store Protein phosphorylation? -dependent enzymes Cortical reaction? PTK/PTP Protein tyrosine phosphorylation Block to polyspermy Cell cycle activation? Protein tyrosine dephosphorylation Fig. 5. Suggested mechanism of sperm-induced egg activation. Binding of a spermatozoon to a G-protein-coupled receptor (G-protein) or, alternatively, to a tyrosine-phosphorylated (PTK) receptor (R) induces an increase in intracellular concentration ([ ] i ). A soluble sperm protein (oscillin) can also be responsible for this response. The source of for the transients originates from the endoplasmic reticulum (ER). Moreover, the continuous oscillations also require an influx of from outside to refill the previously emptied stores. The [ ] i increase induces resumption of meiosis and activation of the cell cycle in one direction, and cortical granule exocytosis leading to the block to polyspermy, in another direction. The increase in [ ] i activates -dependent enzymes, which can facilitate activation of non-receptor protein tyrosine kinases (PTK) or inactivation of protein tyrosine phosphatases (PTPs). Phosphorylation of specific substrate proteins further transduces the sperm signal to downstream cell cycle regulators. The naturally high ph i within the ovulated egg provides a suitable environment in which the enzymes activated by the transient can function. Cortical reaction can be triggered by the initial increase, or by a -dependent protein kinase C (PKC). DAG, diacylglycerol; IP 3, inositol 1,4,5-tris-phosphate; PIP 2, phosphatidylinositol 4,5-biphosphate; PLC, phospholipase C. also possible that the sperm receptor is composed of multiple molecules that activate more than one signalling pathway (reviewed by Whitaker and Swann, 1993; Schultz and Kopf, 1995; Kline, 1996). ph Although the role of as an intracellular regulator in mammalian egg activation has been studied extensively, very little is

6 Early ionic events in mammalian egg activation 101 known about intracellular ph (ph i ) changes or function during this stage of mammalian egg development. In different systems, growth factors may act, at least in part, by increasing ph i. Events participating in cell division are also controlled by changes in ph i. In studies of the involvement of ph i in mediating egg activation, it was found that, at least in several lower species, alkalinization of units accompanies fertilization (sea urchin eggs, Urechis caupo and Xenopus; reviewed by Freeman and Ridgway, 1993). In these species, it was suggested initially that alkalinization is involved in regulating later events of egg activation, such as the migration of the sperm or egg pronuclei, or the initiation of protein and DNA synthesis (reviewed by Whitaker and Steinhardt, 1985; Epel, 1990). However, Rees et al. (1995) showed that ph i is only one of many signals involved in the activation of protein synthesis at fertilization. The milieu of the egg at the time of fertilization differs between species that undergo external fertilization (such as sea urchins) and mammals, which are fertilized internally. Therefore, an experimental system was designed in which the ph of the ampullar fluid of the oviduct, the millieu in which the spermatozoon fertilizes the egg, could be measured using bicarbonate-buffered medium under controlled CO 2 conditions (Ben-Yosef et al., 1996). The ph of the rat ampullar fluid was determined as being alkaline (ph ). In contrast with the ampullar fluid, the ph of the follicular fluid is similar to that of serum (ph ; reviewed by Fisch et al., 1990). In accordance with these results, Maas et al. (1977) reported that monkey ampullar fluid is more alkaline after ovulation (ph 7.7) than during the follicular phase (ph ~ 7.2). Under physiological conditions (ph out = 7.5), the ph i of rat eggs at MII is relatively high (ph = 7.75). However, under the same conditions, the ph i of the GV oocyte is 0.2 ph units lower. These data correlate well with the values obtained for the ampullary fluid and with the assumption that the high concentrations of bicarbonate and K + in the oviductal fluid would tend to increase the ph i of the egg (Baltz et al., 1990). The ph i of the rat egg does not change during fertilization, and fertilization-induced changes do not affect ph i in rat eggs (Ben-Yosef et al., 1996). Similar results have been observed in mouse eggs (Kline and Zagray, 1995; Phillips and Baltz, 1996). In accordance with the assumption of Freeman and Ridgway, (1993), it is expected that, in species such as rats in which the ph i of the egg is relatively high, no further alkalinization upon fertilization is required to facilitate subsequent activation events. A representative pattern of the intracellular ph and in two fertilized eggs is shown (Fig. 4a,b). Several studies have attempted to explore the mechanism of the increase in ph i in those species in which it is known to accompany fertilization. Initially, it was reported that the ph i increase in sea urchin eggs is caused by stimulation of an amiloride sensitive Na + exchanger, which was found to be -calmodulin-dependent (Johnson and Epel, 1976; Shen and Sui, 1989; Kuhtreiber et al., 1993). However, some of the fertilization-triggered ph i increase may also be attributed to CO 2 release from the egg (Gillies et al., 1981). In Spisula oocytes, the Na + -dependent acid release is regulated by protein kinase C (PKC; Dube, 1988). Xenopus oocytes also express the Na + / exchanger, yet alkalinization at fertilization in this species is independent of external Na + (Webb and Nuccitelli, 1981; Sasaki et al., 1992). This could be explained by the fact that, in Xenopus, the eggs develop in fresh water where the [Na + ] is usually much lower than the intracellular [Na + ] of the egg. The Na + / exchanger is the only ph regulation mechanism known to act in the absence of bicarbonate. Nevertheless, when studying ph i regulation in mammals it is essential to work under physiological HCO 3 conditions, where additional acid transporter ph-regulating mechanisms can be identified. The limited information available regarding ph regulation mechanisms in mammalian eggs suggests that mouse eggs and embryos have only a bicarbonate chloride exchanger and are capable of recovery from an alkaline load only in the presence of HCO 3 (Baltz et al., 1990; House, 1994). Our findings of the ability of rat eggs to maintain their ph i only in the presence of bicarbonate is in accordance with these studies. On the basis of the aforementioned data, we suggest the following sequence of events leading to mammalian egg activation (Fig. 5). Binding of a spermatozoon to a G-proteincoupled receptor, or alternatively to a Tyr-phosphorylated receptor, induces an increase in [ ] i. A soluble sperm protein that is delivered to the egg cytoplasm upon fusion can also be responsible for this response. The source of for the initial transient, as well as for the subsequent oscillations, originates from the endoplasmic reticulum. Moreover, the continuous oscillations also require an influx of from outside to refill the previously emptied stores, and are produced by -sensitized IICR. Calcium-induced release mediated by the ryanodine receptor may be involved in the modulation of oscillations. The intracellular increase induces resumption of meiosis and activation of the cell cycle in one direction, and cortical granule exocytosis, which leads to block to polyspermy, in another direction (reviewed by Ducibella, 1996). The increase in [ ] i activates -dependent enzymes (Watanabe et al., 1989; Lorca et al., 1994; Malcov et al., 1997) which, through an unknown cascade of biochemical events, facilitate activation of non-receptor protein tyrosine kinases (PTKs) or, alternatively, inactivation of protein tyrosine phosphatases (PTPs; Ben-Yosef et al., 1998; Talmor et al., 1998). Phosphorylation of specific substrate proteins further transduces the sperm signal to downstream cell cycle regulators. The naturally high ph i within the ovulated egg provides a suitable environment in which the enzymes activated by the transient at fertilization can function, and no further increase in ph i is necessary to initiate changes in egg metabolism during fertilization. The cortical reaction can be triggered by the initial increase, or by a -dependent PKC. The authors thank Yoram Oron for his thoughtful suggestions and the Desktop Publishing Unit in Tel-Aviv University for technical assistance in graphics. 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