A hypothesis on p34cdc2 sequestration based on the existence of Ca(2+)-coordinated changes in H+ and MPF activities during Xenopus egg activation [corrected]

The entry into, and exit from, mitosis are controlled by a universal M-phase promoting factor (MPF) composed of at least p34cdc2 and a cyclin. Embryonic systems are convenient for studying the association and dissociation of the active MPF complex because oocytes and eggs are naturally arrested at a specific point of the cell cycle until progression to the next point is triggered by a hormonal signal or sperm. In amphibians, eggs prior to fertilization are arrested at metaphase 2 of meiosis due to the presence of a stabilized MPF complex. Fertilization (egg activation) produces a transient increase in intracellular free Ca2+, a propagating Ca2+ wave, that specifically triggers the destruction of cyclin, leading to MPF inactivation and entry into the first embryonic inter-phase. We have recently shown that intracellular pH (pHi) variations in amphibian eggs, a large increase at fertilization and small oscillations during the embryonic cell cycle, were temporally and functionally related to the corresponding changes in MPF activity. In addition, the recent finding that the pHi increase at fertilization in Xenopus eggs is a propagating, Ca(2+)-dependent pH wave which closely follows the Ca2+ wave, together with the absence in the egg plasma membrane of pHi-regulating systems responsible for that pHi increase, suggest the existence of cortical or subcortical vesicles acidifying in the wake of the Ca2+ wave, thus producing the pH wave.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of frog (Xenopus laevis) eggs by inositol trisphosphate. I. Characterization of Ca2+ release from intracellular stores

Iontophoresis of inositol 1, 4, 5-triphosphate into frog (Xenopus laevis) eggs activated early developmental events such as membrane depolarization, cortical contraction, cortical granule exocytosis, and abortive cleavage furrow formation (pseudocleavage). Inositol 1, 4-bisphosphate also triggered these events, but only at doses approximately 100-fold higher, whereas no level of fructose-1, 6-bisphosphate tested activated eggs. Using Ca2+-selective microelectrodes, we observed that activating doses of inositol 1, 4, 5-trisphosphate triggered a Ca2+ release from intracellular stores that was indistinguishable from that previously observed at fertilization (Busa, W. B., and R. Nuccitelli, 1985, J. Cell Biol., 100:1325-1329), whereas subthreshold doses triggered only a localized Ca2+ release at the site of injection. The subthreshold IP3 response could be distinguished from the major Ca2+ release at activation with respect to their dose-response characteristics, relative timing, sensitivity to external Ca2+ levels, additivity, and behavior in the activated egg, suggesting that the Xenopus egg may possess two functionally distinct Ca2+ pools mobilized by different effectors. In light of these differences, we suggest a model for intracellular Ca2+ mobilization by sperm-egg interaction.     

A comparison of intracellular changes in porcine eggs after fertilization and electroactivation.

The experiments compare intracellular changes in porcine eggs induced by electrical activation with those induced by sperm penetration. Adequate electrostimulation induces changes in both cortical granule exocytosis and protein synthesis similar to those induced by sperm during fertilization. However, ionic changes induced by electrostimulation differ markedly from those initiated at fertilization. Thus, dynamic video imaging using Fura-2 as a Ca2+ probe provides evidence that parthenogenetic activation induced by electrostimulation is initiated by a single sharp rise in the concentration of intracellular free calcium ([Ca2+]i) in the egg. The intracellular Ca2+ transient increase is triggered by an influx of extracellular Ca2+ immediately after electrostimulation. The amplitude of the intracellular Ca2+ transient increase is a function both of the extracellular Ca2+ concentration and of electric field parameters (field strength and pulse duration). Imaging demonstrates further that a single electrical pulse can only induce a single Ca2+ transient which usually lasts three to five minutes; no further Ca2+ transients are observed unless additional electrical stimuli are applied. By contrast, sperm-induced activation is characterised by a series of Ca2+ spikes which continue for at least 3 hours after sperm-egg fusion. The pattern of Ca2+ spiking after fertilization is not consistent during this period but changes both in frequency and amplitude. Overall, the results demonstrate that, although electrostimulation induces both cortical granule exocytosis and protein reprogramming in porcine eggs, it does not reproduce the pattern of [Ca2+]i changes induced by sperm entry at fertilization.     

Protein kinase C acts downstream of calcium at entry into the first mitotic interphase of Xenopus laevis.

Transit into interphase of the first mitotic cell cycle in amphibian eggs is a process referred to as activation and is accompanied by an increase in intracellular free calcium [( Ca2+]i), which may be transduced into cytoplasmic events characteristic of interphase by protein kinase C (PKC). To investigate the respective roles of [Ca2+]i and PKC in Xenopus laevis egg activation, the calcium signal was blocked by microinjection of the calcium chelator BAPTA, or the activity of PKC was blocked by PKC inhibitors sphingosine or H7. Eggs were then challenged for activation by treatment with either calcium ionophore A23187 or the PKC activator PMA. BAPTA prevented cortical contraction, cortical granule exocytosis, and cleavage furrow formation in eggs challenged with A23187 but not with PMA. In contrast, sphingosine and H7 inhibited cortical granule exocytosis, cortical contraction, and cleavage furrow formation in eggs challenged with either A23187 or PMA. Measurement of egg [Ca2+]i with calcium-sensitive electrodes demonstrated that PMA treatment does not increase egg [Ca2+]i in BAPTA-injected eggs. Further, PMA does not increase [Ca2+]i in eggs that have not been injected with BAPTA. These results show that PKC acts downstream of the [Ca2+]i increase to induce cytoplasmic events of the first Xenopus mitotic cell cycle.     

Role of calcium in regulating intracellular pH following the stepwise release of the metabolic blocks at first-meiotic prophase and second-meiotic metaphase in amphibian oocytes

31P-NMR has been used to monitor changes in intracellular pH following the sequential release of the block at first-meiotic prophase by hormones and the block at second-meiotic metaphase by fertilization in Rana eggs and oocytes. The broad phosphoprotein signal was eliminated by a combination of spin-echo and deconvolution techniques. pHi was determined from the pH-dependent separation of intracellular Pi and phosphocreatine resonances. Agents that release the prophase block (progesterone, insulin, D-600, La3+) increased pHi from 7.38 to 7.7-7.8 within 1-3 h. Noninducers such as 17 beta-estradiol were without effect. By second-metaphase arrest (ovulated, unfertilized) the pHi had fallen to 7.1-7.2. pHi underwent a transient increase to about 7.7 within the first 30 min at fertilization, with a slow 0.1-0.2 pH unit oscillation during early cleavage. The progesterone-induced elevation of intracellular pH is not blocked by amiloride and occurs in Na+-free medium. A transient rise in pHi occurs when the prophase-arrested oocyte is transferred to Ca2+-free medium or when ionophore A23187 is added to the Ca2+-containing medium. Agents that inhibit the resumption of the first meiotic division either block the rise in pHi (procaine, PMSF) or shorten the time-course of the rise in pHi (ionophore A23187). Conditions that elevate intracellular Ca2+ levels and/or increase Ca2+ exchange produce an increase in pHi, whereas those conditions that decrease intracellular Ca2+ levels and/or exchange produce a fall in pHi within 1 h. The time-course of the increase in pHi both following release of the prophase block and at fertilization coincide with a fall in intracellular cAMP and release of surface and/or intracellular Ca2+. These results suggest that: (1) pHi is a function of cytosolic free Ca2+ levels and/or Ca2+ exchange across the oocyte plasma membrane, and (2) meiotic agonists (progesterone, insulin, D-600) and mitogens (sperm, ionophore A23187) modulate intracellular and/or membrane Ca2+ with the resulting changes in pHi and cAMP and resumption of the meiotic divisions.     

Changes in intracellular free calcium activity in Xenopus eggs following imposed intracellular pH changes using weak acids and weak bases.

The aim of this work was to determine the potential relationships between rises in intracellular pH (pHi) and intracellular free calcium activity (Cai2+) during cell activation in Xenopus eggs. To do this, we used two weak bases, NH4Cl and procaine, and a weak acid, CO2, and measured Cai2+ variations in response to these imposed pHi variations. NH4Cl and procaine increased Cai2+ in both unactivated and activated eggs. Procaine was found to alkalinize the egg cytoplasm, whereas the other weak base, NH4Cl, acidified the egg cytoplasm. On the other hand, CO2 was found to acidify the cytoplasm and to substantially decrease Cai2+, also in unactivated and activated eggs. In addition, CO2 triggered an increase in the conductance of the plasma membrane to Cl- ions, similarly to what had been found previously with weak bases (Charbonneau, M. (1989) Cell Differ. Develop. 26, 39-52). These Cl- channels, similarly to the sperm-triggered Cl- channels during the fertilization potential, are supposed to be Ca2(+)-sensitive. Therefore, the changes in Ca2+ observed in response to CO2 do not seem to be responsible for the opening of these Cl- channels, which would rather be triggered by an increase in Cai2+ localized near the plasma membrane. We conclude therefore that weak acids and bases represent appropriate tools for studying cytosolic Ca2+ homeostasis, but not for dissecting the complex pathways involved in signal transduction.     

Molecular dissection of egg fertilization signaling with the aid of tyrosine kinase-specific inhibitor and activator strategies

Fertilization is triggered by sperm-egg interaction and fusion that initiate a transient rise(s) in the free intracellular calcium ([Ca(2+)](i)) that is responsible for a series of biochemical and cell biological events, so-called "egg activation". Calcium-dependent egg activation leads to the initiation of developmental program that culminates in the birth of individuals. A growing body of knowledge has uncovered the molecular mechanisms underlying sperm-induced transient [Ca(2+)](i) increase(s) to some extent; namely, in most animals so far studied, a second messenger inositol 1,4,5-trisphosphate (IP(3)) seems to play a pivotal role in inducing [Ca(2+)](i) transient(s) at fertilization. However, signaling mechanisms used by sperm to initiate IP(3)-[Ca(2+)](i) transient pathway have not been elucidated. To approach this problem, we have employed African clawed frog, Xenopus laevis, as a model animal and conducted experiments designed specifically to determine the role of the Src family protein-tyrosine kinases (SFKs or Src family PTKs) in the sperm-induced egg activation. This review compiles information about the use of PTK-specific inhibitors and activators for analyzing signal transduction events in egg fertilization. Specifically, we focus on molecular identification of Xenopus Src and the signaling mechanism of the Src-dependent egg activation that has been established recently. We also summarize recent advances in understanding the role of the Src family kinases in egg fertilization of other model organisms, and discuss future directions of the field.     

A depolarization can trigger Ca2+ uptake and the acrosome reaction when preceded by a hyperpolarization in L. pictus sea urchin sperm.

The acrosome reaction (AR) is an exocytotic event that allows sperm to recognize and fuse with the egg. In the sea urchin sperm this reaction is triggered by the outer investment of the egg, the jelly, which induces ionic movements leading to increases in intracellular Ca2+ ([Ca2+]i) and intracellular pH (pHi), a K(+)-dependent transient hyperpolarization which may involve K+ channels, and a depolarization which depends on external Ca2+. The present paper explores the role of the hyperpolarization in the triggering of the acrosome reaction. The artificial hyperpolarization of Lytechinus pictus sperm with valinomycin in K(+)-free seawater raised the pHi, caused a small increase in 45Ca2+ uptake, and triggered some AR. When the cells were depolarized with KCl (30 mM) 40-60 sec after the induced hyperpolarization, the pHi decreased and there was a significant increase in 45Ca2+ uptake, [Ca2+]i, and the AR. This waiting time was necessary in order to allow the pHi change required for the AR to occur. Thus, the jelly-induced hyperpolarization may lead to the intracellular alkalinization required to trigger the AR, and, on its own or via pHi, may regulate Ca2+ transport systems involved in this process. Because of the key role played by K+ in the triggering of the AR, the presence and characteristics of ion channels in L. pictus isolated sperm plasma membranes are being explored. Planar lipid bilayers into which these membranes were incorporated by fusion displayed 85 pS single channel transitions which were cation selective.     

A role for protein kinase C during rat egg activation

Upon sperm-egg interaction, an increase in intracellular calcium concentration ([Ca(2+)](i)) is observed. Several studies reported that cortical reaction (CR) can be triggered not only by a [Ca(2+)](i) rise but also by protein kinase C (PKC) activation. Because the CR is regarded as a Ca(2+)-dependent exocytotic process and because the calcium-dependent conventional PKCs (cPKC) alpha and beta II are considered as exocytosis mediators in various cell systems, we chose to study activation of the cPKC in the rat egg during in vivo fertilization and parthenogenetic activation. By using immunohistochemistry and confocal microscopy techniques, we demonstrated, for the first time, the activation of the cPKC alpha, beta I, and beta II during in vivo fertilization. All three isozymes examined presented translocation to the egg's plasma membrane as early as the sperm-binding stage. However, the kinetics of their translocation was not identical. Activation of cPKC alpha was obtained by the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) or by 1-oleoyl-2-acetylglycerol (OAG) but not by the calcium ionophore ionomycin. PKC alpha translocation was first detected 5-10 min after exposure to TPA and reached a maximum at 20 min, whereas in eggs activated by OAG, translocation of PKC alpha was observed almost immediately and reached a maximum within 5 min. These results suggest that, although [Ca(2+)](i) elevation on its own does not activate PKC alpha, it may accelerate OAG-induced PKC alpha activation. We also demonstrate a successful inhibition of the CR by a myristoylated PKC pseudosubstrate (myrPKCPsi), a specific PKC inhibitor. Our study suggests that exocytosis can be triggered independently either by a [Ca(2+)](i) rise or by PKC.     

Activators of protein kinase C trigger cortical granule exocytosis, cortical contraction, and cleavage furrow formation in Xenopus laevis oocytes and eggs

Prophase I oocytes, free of follicle cells, and metaphase II eggs of the amphibian Xenopus laevis were subjected to transient treatments with the protein kinase C activators, phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate, and 1-olyeoyl-2-acetyl-sn-glycerol. In both oocytes and eggs, these treatments triggered early events of amphibian development: cortical granule exocytosis, cortical contraction, and cleavage furrow formation. Surprisingly, activation of oocytes occurred in the absence of meiotic resumption, resulting in cells with an oocytelike nucleus and interior cytoplasm, but with a zygotelike cortex. PMA-induced activation of oocytes and eggs did not require external calcium, a prerequisite for normal activation of eggs. PMA-induced activation of eggs was inhibited by retinoic acid, a known inhibitor of protein kinase C. In addition, pretreatment of eggs with retinoic acid prevented activation by mechanical stimulation and inhibited activation by calcium ionophore A23187. The results suggest that protein kinase C activation is an integral component of the Xenopus fertilization pathway.     

小鼠卵受精和人工激活过程中胞质游离Ca^2＋变化及其在卵激活中的作用

休止于第二次成熟分裂中期（ＭＩ）的小鼠卵母细胞分别乙醇,钙离子载体Ａ２３１８７、电刺激或精子激活并用Ｃａ＾２＋特异荧光探针－Ｆｕｒａ２／ＡＭ测定细胞内游离Ｃａ＾２＋的变化。结果表明,受精诱导ＭⅡ卵内游离Ｃａ＾２＋浓度多次跃升（ｏｓｃｉｌｌａｔｉｏｎ）乙醇,钙离子载体及１次电刺激仅诱导胞内Ｃａ＾２＋１次升高,人工诱导激活的卵可象正常受精卵一样卵裂并发育至囊胚,用ＥＧＴＡ阻止受精和人工激活过程中卵内游     

Mechanisms leading to cortical reaction in the mammalian egg

Activation of the mammalian egg results in cortical reaction (CR), which is correlated with an increase in intracellular Ca²⁺ concentration and PKC activation. The CR is a gradual rather then an “all or none” response, and can be regulated by different concentrations of parthenogenetic activators. To evaluate the biological significance of parthenogenetic induced CR, rat eggs were fertilized or activated by different concentrations of ionomycin and TPA. Cortical granules (CG) were monitored by electron microscopy, while the CG exudate was visualized by Lens culinaris lectin and Texas Red, using light and confocal microscopy. The ability of the CR to trigger a full block to polyspermy was examined in an IVF system. Our study demonstrates the existence of light and dark CG, which differ by number, distribution in the egg cortex, and sensitivity to parthenogenetic activators. Sperm penetration or high concentration of activators, trigger depletion of both light and dark CG, leading to a full CR. Low concentration of activators altered the CG density, the ratio of dark/light CG, and induced partial CR that was sufficient to cause a block to polyspermy. The results imply that Ca²⁺ rise or PKC activation have different effects on light and dark CG. In recently fertilized or parthenogenetically activated eggs, CG exudate appeared as evenly distributed spots, whereas in more advanced stages of fertilization the exudate was scattered as patchy aggregates. This observation suggests a difference in the dispersion of CG exudate after fertilization as compared to parthenogenetic activation. Mol. Reprod. Dev. 51:295–303, 1998. © 1998 Wiley-Liss, Inc.     

Maitotoxin triggers the cortical reaction and phosphatidylinositol-4,5-bisphosphate breakdown in amphibian oocytes

Maitotoxin, a potent marine toxin extracted from peredinians, was found to mimic fertilization in Xenopus oocytes and to trigger the breakdown of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2, the precursor of inositol 1,4,5-trisphosphate], an increase of intracellular pCa and the cortical reaction, including the exocytosis of cortical granules and a wave-like propagation of contraction in the animal hemisphere. All these effects of maitotoxin required the presence of external calcium. Moreover, the toxin considerably increased Ca2+ influx in amphibian oocytes arrested at first meiotic prophase, due to the permanent activation of voltage-dependent Ca2+ channels. Nevertheless it is doubtful that maitotoxin acts primarily as a Ca2+ ionophore or at the level of Ca2+ channels. Indeed no stimulation of Ca2+ uptake was observed in metaphase-II-arrested oocytes, although maitotoxin readily triggered the breakdown of PtdIns(4,5)P2 as well as the cortical reaction in such cells. On the other hand, PtdIns(4,5)P2 breakdown was not reduced in oocytes microinjected with EGTA, although the calcium chelator prevented the oocytes from undergoing the cortical reaction. Taken together, these findings support the view that the toxin might act primarily by increasing PtdIns(4,5)P2 phosphodiesterase activity.     

Injection of a sperm extract triggers egg activation in the newt Cynops pyrrhogaster

Unfertilized eggs of the newt Cynops pyrrhogaster are arrested at the second meiotic metaphase. The primary signal for egg activation is a transient increase in [Ca2+](i), which is triggered by the fertilizing sperm and propagates over the egg cortex as a Ca2+ wave. We injected an extract of Cynops sperm (SE) into unfertilized eggs and induced a wave-like [Ca2+](i) increase which resulted in activation and resumption of meiosis. The SE-injected eggs showed degradation of cyclin B1 and DNA replication. When SE was boiled or treated with proteinase K before injection, it was unable to cause egg activation. Preinjection of Ca2+ -chelator BAPTA before SE injection inhibited egg activation. These results indicate that a heat-labile and proteinaceous factor in the sperm cytoplasm induces a transient increase in [Ca2+](i) which is required for egg activation. Injection of IP3 into unfertilized eggs caused an increase in [Ca2+](i) and egg activation, but injection of cADP-ribose did not. These results support the hypothesis that Ca2+ release at fertilization occurs via IP3 receptors.     

Inositol lipid hydrolysis contributes to the Ca2+ wave in the activating egg of Xenopus laevis.

We have used fluorescence ratio-imaging of fura-2 in the activating egg of Xenopus laevis to study the wave of increased intracellular free Ca2+ concentration ([Ca2+]i) while monitoring that of cortical granule exocytosis. Naturally matured eggs were dejellied, injected with fura-2, and activated by the iontophoresis of 1-30 nCoul of inositol-1,4,5-trisphosphate which triggers an immediate increase in free [Ca2+]i at the injection site. The Ca2+ rise spreads throughout the egg, reaching the opposite side in 5-8 min, and is followed by elevation of the fertilization envelope about 20-30 sec behind the [Ca2+]i wave. [Ca2+]i returns to preactivation levels within about 20 min after activation. We further studied the role of phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis by microinjecting antibodies to PIP2 into the egg. PIP2 antibodies did not alter the propagation velocity of the wave but greatly reduced the amount of Ca2+ released in the egg cortex. These data suggest that PIP2 hydrolysis plays a role in the release of [Ca2+]i in the outer regions of the egg following activation.     

Ca2+ and pH determine the interaction of chromaffin cell scinderin with phosphatidylserine and phosphatidylinositol 4,5,-biphosphate and its cellular distribution during nicotinic-receptor stimulation and protein kinase C activation

Nicotinic stimulation and high K(+)-depolarization of chromaffin cells cause disassembly of cortical filamentous actin networks and redistribution of scinderin, a Ca(2+)-dependent actin filament-severing protein. These events which are Ca(2+)-dependent precede exocytosis. Activation of scinderin by Ca2+ may cause disassembly of actin filaments leaving cortical areas of low cytoplasmic viscosity which are the sites of exocytosis (Vitale, M. L., A. Rodríguez Del Castillo, L. Tchakarov, and J.-M. Trifaró. 1991. J. Cell. Biol. 113:1057-1067). It has been suggested that protein kinase C (PKC) regulates secretion. Therefore, the possibility that PKC activation might modulate scinderin redistribution was investigated. Here we report that PMA, a PKC activator, caused scinderin redistribution, although with a slower onset than that induced by nicotine. PMA effects were independent of either extra or intracellular Ca2+ as indicated by measurements of Ca2+ transients, and they were likely to be mediated through direct activation of PKC because inhibitors of the enzyme completely blocked the response to PMA. Scinderin was not phosphorylated by the kinase and further experiments using the Na+/H+ antiport inhibitors and intracellular pH determinations, demonstrated that PKC-mediated scinderin redistribution was a consequence of an increase in intracellular pH. Moreover, it was shown that scinderin binds to phosphatidylserine and phosphatidylinositol 4,5-biphosphate liposomes in a Ca(2+)-dependent manner, an effect which was modulated by the pH. The results suggest that under resting conditions, cortical scinderin is bound to plasma membrane phospholipids. The results also show that during nicotinic receptor stimulation both a rise in intracellular Ca2+ and pH are observed. The rise in intracellular pH might be the result of the translocation and activation of PKC produced by Ca2+ entry. This also would explain why scinderin redistribution induced by nicotine is partially (26-40%) inhibited by inhibitors of either PKC or the Na+/H+ antiport. In view of these findings, a model which can explain how scinderin redistribution and activity may be regulated by pH and Ca2+ in resting and stimulated conditions is proposed.     

Structural modifications induced by TPA (12-O-tetradecanoyl phorbol-13-acetate) in sea urchin eggs

We investigated the effect of the phorbol ester TPA (12-O-tetradecanoyl phorbol 13-acetate) on the egg morphology of the sea urchin Arbacia lixula. Our study indicates that TPA alters the cortical region of the egg: the pigment granules migrate toward the surface, while cortical granules detach from the plasma membrane. Cortical granule exocytosis did not occur but the endocytosis process was turned on. Prolonged treatment of the eggs by TPA partially inhibits the cortical granule exocytosis normally triggered by fertilization. We discuss the effects of TPA in terms of its interaction with the Ca2+ pool and cytoskeletal structures. In order to discern the respective roles of pHi and protein kinase C activity in endocytosis process activation, we compared the ultrastructural effects of TPA and ammonia. Finally, the role of pigment vesicles in egg metabolism activation is discussed.     

Characterization of a sperm factor for egg activation at fertilization of the newt Cynops pyrrhogaster

Eggs of the newt, Cynops pyrrhogaster, arrested at the second meiotic metaphase are activated by sperm at fertilization and then complete meiosis to initiate development. We highly purified a sperm factor for egg activation from a sperm extract with several chromatographies. The purified fraction containing only a 45 kDa protein induced egg activation accompanied by an intracellular Ca2+ increase when injected into unfertilized eggs. Although injection of mouse phospholipase C (PLC) zeta-mRNA caused a Ca2+ increase and egg activation, partial amino acid sequences of the 45 kDa protein were homologous to those of Xenopus citrate synthase, but not to PLCs. An anti-porcine citrate synthase antibody recognized the 45 kDa protein both in the purified fraction and in the sperm extract. Treatment with the anti-citrate synthase antibody reduced the egg-activation activity in the sperm extract. Injection of porcine citrate synthase or mRNA of Xenopus citrate synthase induced a Ca2+ increase and caused egg activation. A large amount of the 45 kDa protein was localized in two lines elongated from the neck to the middle piece of sperm. These results indicate that the 45 kDa protein is a major component of the sperm factor for egg activation at newt fertilization.     

An elevated free cytosolic Ca2+ wave follows fertilization in eggs of the frog, Xenopus laevis

The eggs of most or all animals are thought to be activated after fertilization by a transient increase in free cytosolic Ca2+ concentration ([Ca2+]i). We have applied Ca2+-selective microelectrodes to detect such an increase in fertilized eggs of the frog, Xenopus laevis. As observed with an electrode in the animal hemisphere, [Ca2+]i increased from 0.4 to 1.2 microM over the course of 2 min after fertilization, and returned to its original value during the next 10 min. No further changes in [Ca2+]i were detected through the first cleavage division. In eggs impaled with two Ca2+ electrodes, the Ca2+ pulse was observed to travel as a wave from the animal to the vegetal hemisphere, propagating at a rate of approximately 10 microns/s across the animal hemisphere. The apparent delay between the start of the fertilization potential and initiation of the Ca2+ wave at the sperm entry site as approximately 1 min. Through these observations describe only the behavior of subcortical [Ca2+]i, we suggest that our data represent the subcortical extension of the cortical Ca2+ wave thought to trigger cortical granule exocytosis, and we present evidence that both the timing and magnitude of the Ca2+ pulse we observed are consistent with this identity. This first quantification of subcortical [Ca2+]i during fertilization indicates that the Ca2+ transient is available to regulate processes (e.g., protein synthesis) in the subcortical cytosol.     

Ca2+-independent protein kinase C signalling in mouse eggs during the early phases of fertilization

Protein kinase C (PKC), an enzyme playing a central role in signal transduction pathways, is activated in fertilized mouse eggs downstream of the fertilization Ca2+ signal, to regulate different aspects of egg activation. Given the presence of Ca2+-independent PKC isoforms within the egg, we investigated whether fertilization triggers PKC stimulation in mouse eggs by activating Ca2+-independent signalling pathways. An increase in PKC activity was detected as early as 10 min after the beginning of insemination, when about 90% of eggs had fused with sperm and the first Ca2+ rise was evident in most of the eggs. A similar level of activity was found 20 min later, when about 60% of eggs had resumed meiosis. When the Ca2+ increase was buffered by an intracellular Ca2+ chelating agent, PKC stimulation was not blocked but only slightly reduced. Confocal microscopy analysis revealed that the increase in PKC activity at fertilization coincided with the translocation of PKCdelta, a Ca2+-independent and diacylglycerol-dependent PKC isoform, to the meiotic spindle. When, in the absence of the Ca2+ signal, metaphase-anaphase transition was inhibited, PKCdelta moved to the meiotic spindle but still maintained a sustained cytoplasmic distribution. In summary, our results indicate that: 1) PKC activation is an early event of egg activation; 2) both Ca2+-dependent and Ca2+-independent pathways contribute to increased PKC activity at fertilization; 3) PKCdelta is one of the isoforms participating in this signalling process.