The Drosophila calcipressin sarah is required for several aspects of egg activation

Activation of mature oocytes initiates development by releasing the prior arrest of female meiosis, degrading certain maternal mRNAs while initiating the translation of others, and modifying egg coverings. In vertebrates and marine invertebrates, the fertilizing sperm triggers activation events through a rise in free calcium within the egg. In insects, egg activation occurs independently of sperm and is instead triggered by passage of the egg through the female reproductive tract ; it is unknown whether calcium signaling is involved. We report here that mutations in sarah, which encodes an inhibitor of the calcium-dependent phosphatase calcineurin, disrupt several aspects of egg activation in Drosophila. Eggs laid by sarah mutant females arrest in anaphase of meiosis I and fail to fully polyadenylate and translate bicoid mRNA. Furthermore, sarah mutant eggs show elevated cyclin B levels, indicating a failure to inactivate M-phase promoting factor (MPF). Taken together, these results demonstrate that calcium signaling is involved in Drosophila egg activation and suggest a molecular mechanism for the sarah phenotype. We also find the conversion of the sperm nucleus into a functional male pronucleus is compromised in sarah mutant eggs, indicating that the Drosophila egg's competence to support male pronuclear maturation is acquired during activation.     

Fusion of membranes during fertilization. Increases of the sea urchin egg''s membrane capacitance and membrane conductance at the site of contact with the sperm

The early events of fertilization that precede and cause activation of an egg have not been fully elucidated. The earliest electrophysiological change in the sea urchin egg is a sperm-evoked increase of the egg's membrane conductance. The resulting depolarization facilitates entry of the fertilizing sperm and precludes the entry of supernumerary sperm. The sequence of the increase in the egg's membrane conductance, gamete membrane fusion, egg activation, and sperm entry, including causal relationships between these events, are not known. This study reports the use of whole egg voltage clamp and loose patch clamp to monitor simultaneously changes of membrane conductance and capacitance at the site of sperm-egg contact. Measurements were made during sperm-egg interactions where sperm entry readily proceeded or was precluded by maintaining the egg's membrane potential either at large, negative values or at positive values. Whenever the sperm evoked an increase of the egg's membrane conductance, that increase initiated abruptly, was localized to the site of sperm attachment, and was accompanied by a simultaneous abrupt increase of the membrane capacitance. This increase of capacitance indicated the establishment of electrical continuity between gametes (possibly fusion of the gametes' plasma membranes). If sperm entry was blocked by large negative membrane potentials, the capacitance cut off rapidly and simultaneously with a decrease of the membrane conductance, indicating that electrical continuity between gametes was disrupted. When sperm entry was precluded by positive membrane potentials, neither conductance nor capacitance increased, indicating that sperm entry was halted before the fusion of membranes. A second, smooth increase of capacitance was associated with the exocytosis of cortical granules near the sperm in eggs that were activated. Electrical continuity between the gametes always preceded activation of the egg, but transient electrical continuity between the gametes alone was not always sufficient to induce activation.     

Guanosine 5''-thiotriphosphate may stimulate phosphoinositide messenger production in sea urchin eggs by a different route than the fertilizing sperm.

We show that microinjecting guanosine-5'-thiotriphosphate (GTP gamma S) into unfertilized sea urchin eggs generates an intracellular free calcium concentration [( Ca]i) transient apparently identical in magnitude and duration to the calcium transient that activates the egg at fertilization. The GTP gamma S-induced transient is blocked by prior microinjection of the inositol trisphosphate (InsP3) antagonist heparin. GTP gamma S injection also causes stimulation of the egg's Na+/H+ antiporter via protein kinase C, even in the absence of a [Ca]i increase. These data suggest that GTP gamma S acts by stimulating the calcium-independent production of the phosphoinositide messengers InsP3 and diacylglycerol (DAG). However, the fertilization [Ca]i transient is not affected by heparin, nor can the sperm cause calcium-independent stimulation of protein kinase C. It seems that the bulk of InsP3 and DAG production at fertilization is triggered by the [Ca]i transient, not by the sperm itself. GDP beta S, a G-protein antagonist, does not affect the fertilization [Ca]i transient. Our findings do not support the idea that signal transduction at fertilization operates via a G-protein linked directly to a plasma membrane sperm receptor.     

Involvement of calcium signaling and the actin cytoskeleton in the membrane block to polyspermy in mouse eggs

This study examines the effects of actin microfilament-disrupting drugs on events of fertilization, with emphasis on gamete membrane interactions. Mouse eggs, freed of their zonae pellucidae, were treated with drugs that perturb the actin cytoskeleton by different mechanisms (cytochalasin B, cytochalasin D, jasplakinolide, latrunculin B) and then inseminated. Cytochalasin B, jasplakinolide, and latrunculin B treatments resulted in a decrease in the percentage of eggs fertilized and the average number of sperm fused per egg. However, cytochalasin D treatment resulted in an increase in the average number of sperm fused per egg and the percentage of polyspermic eggs. This increase in polyspermy occurred despite the observation that cytochalasin D treatment caused a decrease in sperm-egg binding and did not affect spontaneous acrosome reactions or sperm motility. This suggested that cytochalasin D-treated eggs had an impaired ability to establish a block to polyspermy at the level of the plasma membrane. The effect of cytochalasin D on the block to polyspermy was not due to a general disruption of egg activation because sperm-induced calcium oscillations and cortical granule exocytosis were similar in cytochalasin D-treated and control eggs. However, buffering of intracellular calcium levels with the calcium chelator BAPTA-AM resulted in an increase in polyspermy. Together, these data suggest that a postfertilization decrease in egg membrane receptivity to sperm requires functions of the egg actin cytoskeleton that are disrupted by cytochalasin D. Furthermore, egg activation-associated increased intracellular calcium levels are necessary but not sufficient to affect postfertilization membrane dynamics that contribute to a membrane block to polyspermy.     

Getting sperm and egg together: things conserved and things diverged

Sperm-egg interactions occur at multiple levels on the egg surface, first with the egg's extracellular matrix and then with the egg's plasma membrane. The BioPore minisymposium on "The Egg Surface" at the 1999 annual meeting of the Society for the Study of Reproduction highlighted a series of events underlying successful interactions of the sperm with the egg: 1) composition, synthesis, and assembly of the mouse egg's extracellular matrix, the zona pellucida, during oogenesis; 2) oocyte maturation and development of the sperm-binding domain of mouse eggs; and 3) characterization of functional domains in different sperm ligands (fertilin-alpha and fertilin-beta in the mouse and lysin in the abalone) that recognize cognate binding sites on the egg surface. Data that were presented are reviewed here and discussed with respect to conserved and divergent features of gamete functions.     

Mouse gamete interactions during fertilization in vitro. Chlortetracycline as a fluorescent probe for the mouse sperm acrosome reaction

We have developed an assay for detecting the acrosome reaction in mouse sperm using chlortetracycline (CTC) as a fluorescent probe. Sperm known to be intact with nonreacted acrosomes show CTC fluorescence in the presence of Ca2+ over the anterior portion of the sperm head on the plasma membrane covering the acrosome. Sperm which have undergone the acrosome reaction do not show fluorescence on the sperm head. Mouse sperm bind to zonae pellucidae of cumulus-free eggs in vitro in a Ca2+-dependent reaction; these sperm are intact by the CTC assay. Intact sperm bind to mechanically isolated zonae under the same conditions: the egg is apparently unnecessary for this inital reaction. Sperm suspensions, in which greater than 50% of the motile population had completed the acrosome reaction, were prepared by incubation in hyperosmolal medium followed by treatment with the divalent cation ionophore, A23187. Cumulus-free eggs challenged with such sperm suspensions preferentially bind intact sperm; acrosome-reacted sperm do not bind. We conclude that the plasma membrane of the mouse sperm is responsible for recognition of the egg's zona pellucida and that the obligatory sequence of reactions leading to fusion of mouse gametes is binding of the intact sperm to the zona pellucida, followed by the acrosome reaction at the zona surface, followed in turn by sperm penetration of the zona.     

Sperm-egg interactions in the mouse: sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein

In this investigation, the interaction of mouse sperm with unfertilized eggs and embryos, solubilized zonae pellucidae isolated from eggs and embryos, and purified zona pellucida glycoproteins ZP1, 2, and 3 (J. D. Bleil, and P. M. Wassarman, (1980b) Dev. Biol. 76, 185-202) has been examined in vitro by light and electron microscopy. The experiments described were carried out in order to determine the temporal sequence of events during sperm-egg interaction in vitro and to identify the component(s) of zonae pellucidae responsible for inducing mouse sperm to undergo the acrosome reaction. "Pulse-chase" analysis of the sequence of sperm-egg interactions revealed that mouse sperm first "attach" loosely and then "bind" tightly to the unfertilized egg's zona pellucida. Binding of sperm to egg zonae pellucidae is followed by induction of the acrosome reaction. Induction of the acrosome reaction can be mediated by the zona pellucida, since solubilized zonae pellucidae isolated from unfertilized eggs were found to be just as effective as the calcium ionophore A23187 in inducing the reaction in vitro. Furthermore, ZP3 purified from zonae pellucidae isolated from unfertilized eggs, but not from two-cell embryos, was also just as effective as either solubilized zonae pellucidae from eggs or ionophore A23187 in inducing the acrosome reaction. ZP1 and 2 from both eggs and embryos, and ZP3 from embryos, had little effect on the extent of the acrosome reaction as compared to control samples. The results of these and other experiments (J. D. Bleil, and P. M. Wassarman, (1980b) Cell 20, 873-882) strongly suggest that, at least in vitro, mouse sperm recognize and bind to ZP3 of egg zonae pellucidae, and that such binding leads to the induction of the acrosome reaction. Modification of ZP3 following fertilization eliminates sperm binding to zonae pellucidae and, consequently, induction of the acrosome reaction is precluded.     

The dynamics of calcium oscillations that activate mammalian eggs

It has been known for some time that mammalian eggs are activated by a series of intracellular calcium oscillations that occur shortly after sperm egg membrane fusion. Recent work has identified a novel sperm specific phospholipase C zeta as the likely agent that stimulates the calcium oscillations in eggs after sperm-egg membrane fusion. PLCzeta is stimulated by low intracellular calcium levels in a manner which suggests that there is a regenerative feedback of calcium release and PLCzeta induced inositol 1,4,5-trisphophate (InsP(3)) production in eggs. This implies calcium oscillations in fertilizing mammalian eggs are driven by underlying oscillations of InsP(3). This model of oscillations is supported by the response of mouse eggs to sudden increases in InsP(3). The cellular targets of calcium oscillations include calmodulin-dependent protein kinases, protein kinase C and mitochondria. There is evidence that eggs might be best activated by multiple calcium increases rather than a single calcium rise. As yet we do not fully understand how the target of calcium in a mammalian egg might decode the patterns of calcium changes that can occur during egg activation.     

Kinetics of actin assembly attending fertilization or artificial activation of sea urchin eggs

Changes in the state of actin assembly triggered by fertilization or by artificial activation of sea urchin eggs were quantified using the DNase I inhibition assay. Insemination of Lytechinus pictus or Strongylocentrotus purpuratus eggs induces a cyclic variation in the level of G-actin as follows: between 0 and 30 s after insemination, the G-actin content decreases. This is followed by an increase in the amount of monomeric actin between 30 and 60 s, and then from 60 s to 5 min postinsemination there is a progressive decrease in the egg's level of G-actin. This latter decrease is more pronounced in S. purpuratus eggs than in L. pictus eggs. Using sperm mimetics that trigger an increase in intracellular calcium concentration (A23187 in sodium-free seawater), a cytoplasmic alkalinization (NH4Cl), a plasma membrane depolarization (seawater enriched with potassium ions), or all three of these phenomena (A23187 in normal seawater), each phase depicted at fertilization correlates with the following metabolic events accompanying egg awakening: phase 1, of uncertain origin (possibly related to plasma membrane depolarization); phase 2, elevation of intracellular calcium concentration; phase 3, alkalinization of the intracellular milieu but only if the transient intracellular calcium rise has taken place.     

Sperm motility in the horseshoe crab,Limulus polyphemus L: I. Sperm behavior near eggs and motility initiation by egg extracts

Limulus spermatozoa are nonmotile when spawned and become motile only after encountering a sperm motility initiating factor (SMI) exuded by the egg. SMI extracts (produced by washing intact eggs with distilled water, lyophilizing the supernatant to dryness, and redissolving the dried extract in artificial seawater, ASW) initiate sperm motility in the absence of eggs. Utilizing such SMI extracts, sperm motility initiation was found to be unaffected by changes in temperature from 16 to 30°C, pH from 6.3 to 8.6, and salinity from 85 to 125% ASW. Within these ranges, sperm motility initiation was an “all-or-nothing” response, with greater than 99% of the spermatozoa becoming motile. Also, each sperm swam with apparently the same speed (at a given temperature) until spontaneously stopping within 10 min after the addition of SMI extracts. Evidence was found that SMI may bind irreversibly to a receptor, which is inactivated within a few seconds or minutes, leading to the observed cessation of motility. Observations of sperm behavior near intact eggs showed no evidence of chemotaxis. Spermatozoa observed to swim toward intact eggs progressed with a uniform speed and were motile less than 5 sec from initiation of motility until attaching to the egg. The presence of an all-or-nothing response to SMI, the independence of sperm motility to experimental parameters, and several other characteristics of the animal and its spermatozoa make Limulus a potentially excellent model animal for examination of sperm motility control mechanisms.     

Mechanism of univalent antisperm antibody inhibition of fertilization in the sea urchin, Arbacia punctulata

Univalent antisperm antibodies (IFab) markedly inhibited the fertilizing capacity of sperm when tested on intact, dejellied, and "demembranated" Arbacia punctulata eggs. Sperm motility and egg jelly penetration were not affected by IFab. Antifertilizin was excluded as the essential sperm antigen involved in the fertilization-inhibiting action. Sperm pretreated with IFab did not bind to the surfaces of either dejellied or demembranated eggs, whereas control globulin (CFab) and seawater-pretreated sperm bound to such eggs in high numbers. Electron microscopy showed that IFab-treated sperm failed to undergo the acrosome reaction. This excluded "bindin" as the essential antigen. Inhibition of fertilization by IFab was reversed or bypassed by artificial induction of the acrosome reaction with ionophore A23187. It is concluded that univalent antisperm antibody treatment inhibits the fertilizing capacity of sperm by preventing a sperm-egg interaction that results in the acrosome reaction; consequently, attachment of the sperm to the egg is prevented.     

A localized zone of increased conductance progresses over the surface of the sea urchin egg during fertilization

Although activation of a sea urchin egg by sperm leads to three phases of membrane conductance increase in the egg, the mechanism by which the sperm causes these conductance changes is not known. We used the loose patch clamp technique to localize the conductance changes in voltage clamped eggs. A patch of the egg's membrane was isolated from the bath by pressing the loose patch clamp pipette against the egg surface. Sperm added to the bath attached to the surface of the egg in a region other than at the isolated membrane patch. During phase 1 of the activation current, no changes of the membrane conductance were detected. At the time of, and subsequent to the onset of phase 2, large currents recorded between the interior of the patch pipette and the bath were attributed to changes of the seal resistance between the surface of the egg and the pipette. A local change of membrane conductance was observed during phase 2 despite the changes of seal resistance. During phase 2, the large amplitude and short duration of the local membrane conductance increase relative to the membrane, conductance increase for the whole egg during phase 2 indicated that the conductance increase occurred over the entire surface of the egg, but not simultaneously. The time when the peak conductance for the membrane patch occurred, relative to the time of onset for phase 2 in the whole egg, depended on the distance, measured in a straight line, between the site of sperm attachment and the tip of the pipette. These data indicate that the localized conductance increase progressed over the surface of the egg from the site of sperm attachment to the opposite pole of the egg. It is proposed that the local conductance increase, the cortical reaction, and the change of seal resistance are all evoked by a common cytoplasmic message that progresses throughout the cytoplasm of the egg from the site of sperm attachment to the opposite pole of the egg.     

Cyclic ADPR and calcium signaling in sea bream (Sparus aurata) egg fertilization.

The cell egg is in a state of quiescence and only after its fusion with the sperm, a series of pre-programmed metabolic processes will be activated, culminating with embryonic development. The egg/sperm fusion induces a transitory increase of Ca(2+) in the cytoplasm, which is responsible for the activation of both precocious and late reactions. The release of Ca(2+) occurs by stimulation of the ionic specific channels. In addition to IP(3), a new Ca-release inducer was recently evidenced, cyclic ADP ribose (cADPR), in some invertebrates and mammals. Here, we report the first evidence of the cADPR presence in fish. Our data also demonstrate that in the sea bream egg, cADPR is involved in the fertilization process; in fact, its level increases after the entrance of the sperm. By in vitro experiments, it was shown that cADPR induces a release of Ca(2+) in the egg homogenate, indicating that in sea bream, the increase of cADPR can induce an intracellular Ca(2+) release. Since cADPR is a product of NAD(+) metabolism, the activity of several enzymes involved in the NAD(+) metabolism was investigated. Sea bream eggs are pelagic and only floating eggs after insemination develop into viable embryos. In the present work, NAD(+) metabolism was studied in both types of egg. All the tested enzymes showed similar specific activity in both floating and sinking eggs. In the latter, cADPR was not detectable and the nucleotides content was significantly lower, evidencing a scarce energetic charge in sinking eggs.     

Protein kinase C activity in mouse eggs regulates gamete membrane interaction

Gamete membrane interaction is critical to initiate the development of a new organism. The signaling pathways governing this event, however, are poorly understood. In this report, we provide the first evidence that protein kinase C activity in mouse eggs plays a crucial role in the regulation of this process. Stimulating PKC activity in mouse eggs by phorbol 12-myristate 13-acetate (PMA) drastically inhibited the egg's membrane ability to bind and fuse with sperm. Surprisingly, this significant reduction of gamete membrane interaction was also observed in eggs treated with the PKC inhibitors staurosporine and calphostin c. In further analysis, we found that while no change of egg actin cytoskeleton was detected after either PMA or calphostin c treatment, the structural morphology of egg surface microvilli was severely altered in the PMA-treated eggs, but not in the calphostin c-treated eggs. Moreover, sperm, which bound but did not fuse with the eggs treated with the anti-CD9 antibody KMC8, were liberated from the egg membrane after PMA, but not calphostin c, treatment. Taken together, these results suggest that egg PKC may be precisely balanced to regulate gamete membrane interaction in a biphasic mode, and this biphasic regulation is executed through two different mechanisms.     

Reduction of the fertilizing capacity of sea urchin sperm by cannabinoids derived from marihuana. I. Inhibition of the acrosome reaction induced by egg jelly.

delta 9-Tetrahydrocannabinol (THC) and two other major cannabinoids derived from marihuana--cannabidiol (CBD) and cannabinol (CBN)--inhibit fertilization in the sea urchin Strongylocentrotus purpuratus by reducing the fertilizing capacity of sperm (Schuel et al., 1987). Sperm fertility depends on their motility and on their ability to undergo the acrosome reaction upon encountering the egg's jelly coat. Pretreatment of S. purpuratus sperm with THC prevents triggering of the acrosome reaction by solubilized egg jelly in a dose (0.1-100 microM) and time (0-5 min)-dependent manner. Induction of the acrosome reaction is inhibited in 88.9 +/- 2.3% of sperm pretreated with 100 microM THC for 5 min, while motility of THC-treated sperm is not reduced compared to solvent (vehicle) and seawater-treated controls. The acrosome reaction is inhibited 50% by pretreatment with 6.6 microM THC for 5 min and with 100 microM THC after 20.8 sec. CBN and CBD at comparable concentrations inhibit the acrosome reaction by egg jelly in a manner similar to THC. THC does not inhibit the acrosome reaction artificially induced by ionomycin, which promotes Ca2+ influx, and nigericin, which promotes K+ efflux. THC partially inhibits (20-30%) the acrosome reaction induced by A23187, which promotes Ca2+ influx, and NH4OH, which raises the internal pH of the sperm. Addition of monensin, which promotes Na+ influx to egg jelly or to A23187, does not overcome the THC inhibition. Inhibition of the egg jelly-induced acrosome reaction by THC produces a corresponding reduction in the fertilizing capacity of the sperm. The adverse effects of THC on the acrosome reaction and sperm fertility are reversible.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Latrunculin inhibits the microfilament-mediated processes during fertilization, cleavage and early development in sea urchins and mice

Latrunculin A, a marine toxin from a Red Sea sponge, is a potent inhibitor of the microfilament-mediated processes of fertilization and early development in sea urchins and in mice. Sperm from sea urchins, but not those from Limulus or mice, were affected by latrunculin, and fertilization in both sea urchins and in mice was arrested but at different stages. Sea urchin sperm treated with 2.6 microM latrunculin are unable to assemble acrosomal processes and their ability to fertilize eggs is impaired. The unwinding of the Limulus sperm acrosomal process occurs in the presence of latrunculin. Treated mouse sperm are able to fertilize mouse oocytes in vitro, suggesting that microfilaments may not be required in this mammalian sperm. In sea urchin eggs, sperm incorporation, microvillar elongation and cytokinesis are inhibited. Microtubule-mediated motility occurs normally. 20 nM latrunculin prevents the morphogenetic movements during gastrulation. It reduces the viscosity of actin gels from sea urchin egg homogenates. In unfertilized mouse oocytes, it prevents the colcemid-induced dispersion of the meiotic chromosomes; accumulations of cortical actin are noted adjacent to the scattered chromosomes. Sperm incorporation during mouse fertilization in vitro is unaffected suggesting that sperm entry may occur independent of microfilament activity in mammals. However, the apposition of the pronuclei at the center of the egg cytoplasm does not occur, providing evidence that cytoplasmic microfilaments may be required for the motions leading to pronuclear union during mouse fertilization. It inhibits the second polar body formation and cytokinesis. These results indicate that latrunculin is a potent inhibitor of microfilament-mediated processes in sperm, eggs and embryos, and that it may prove to be a powerful new drug for exploring the cellular behavior of microfilaments in the maintenance of cell shape and during motility.     

Identification of a translocation deficiency in cortical granule secretion in preovulatory mouse oocytes.

Preovulatory, germinal vesicle (GV)-stage mouse oocytes are unable to undergo normal cortical granule (CG) secretion. Full secretory competence is observed by metaphase II (MII) of meiosis and involves the development of calcium response mechanisms. To identify the deficient or inhibited step in CG secretion, preovulatory GV-stage oocytes were stimulated and tested for their ability to undergo translocation, docking, and/or fusion. The mean CG distance to the plasma membrane was not reduced in fertilized or sperm fraction-injected, GV-stage oocytes relative to that in control GV-stage oocytes. In addition, analysis of individual CG distances to the plasma membrane indicated no subpopulation of CGs competent to translocate. Further analysis demonstrated that secretory incompetence likely is not due to a lack of proximity of CGs to the egg's primary calcium store, the endoplasmic reticulum. Calcium/calmodulin-dependent protein kinase II (CaMKII), which is reportedly involved in secretory granule translocation and secretion in many cells, including eggs, was investigated. A 60-kDa CaMKII isoform detected by Western blot analysis increased 150% during oocyte maturation. The CaMKII activity assays indicated that MII-stage eggs correspondingly have 110% more maximal activity than GV-stage oocytes. These data demonstrate that the primary secretory deficiency is due to a failure of CG translocation, and that a maturation-associated increase in CaMKII correlates with the acquisition of secretory competence and the ability of the egg to undergo normal activation.     

Signal transduction at fertilization: the Ca2+ release pathway in echinoderms and other invertebrate deuterostomes

Gamete interaction and fusion triggers a number of events that lead to egg activation and development of a new organism. A key event at fertilization is the rise in intracellular calcium. In deuterostomes, this calcium is released from the egg's endoplasmic reticulum and is necessary for proper activation. This article reviews recent data regarding how gamete interaction triggers the initial calcium release, focusing on the echinoderms (invertebrate deuterostomes) as model systems. In eggs of these animals, Src-type kinases and phospholipase C-gamma are required components of the initial calcium trigger pathway in eggs.     

The activation wave of calcium in the ascidian egg and its role in ooplasmic segregation

We have studied egg activation and ooplasmic segregation in the ascidian Phallusia mammillata using an imaging system that let us simultaneously monitor egg morphology and calcium-dependent aequorin luminescence. After insemination, a wave of highly elevated free calcium crosses the egg with a peak velocity of 8-9 microns/s. A similar wave is seen in egg fertilized in the absence of external calcium. Artificial activation via incubation with WGA also results in a calcium wave, albeit with different temporal and spatial characteristics than in sperm-activated eggs. In eggs in which movement of the sperm nucleus after entry is blocked with cytochalasin D, the sperm aster is formed at the site where the calcium wave had previously started. This indicates that the calcium wave starts where the sperm enters. In 70% of the eggs, the calcium wave starts in the animal hemisphere, which confirms previous observations that there is a preference for sperm to enter this part of the egg (Speksnijder, J. E., L. F. Jaffe, and C. Sardet. 1989. Dev. Biol. 133:180-184). About 30-40 s after the calcium wave starts, a slower (1.4 microns/s) wave of cortical contraction starts near the animal pole. It carries the subcortical cytoplasm to a contraction pole, which forms away from the side of sperm entry and up to 50 degrees away from the vegetal pole. We propose that the point of sperm entry may affect the direction of ooplasmic segregation by causing it to tilt away from the vegetal pole, presumably via some action of the calcium wave.