Calcium ionophore-induced egg activation and apoptosis are associated with the generation of intracellular hydrogen peroxide

The present study was designed to investigate whether calcium ionophore-induced activation and apoptosis are associated with the generation of hydrogen peroxide (H₂O₂) in rat eggs cultured in vitro. Culture of metaphase-II (M-II) arrested eggs in Ca²⁺/Mg²⁺-deficient medium did not induce egg activation, while a second polar body was observed in 20% of eggs when cultured in Ca²⁺/Mg²⁺-supplemented medium. In Ca²⁺/Mg²⁺-deficient medium, lower concentrations of calcium ionophore (0.2,0.4 and 0.8 µm) not only induced egg activation in a dose-dependent manner but also generation of intracellular H₂O₂ (84.40±0.50 ng/egg) when compared to control eggs (80.46±1.34 ng/egg). The higher concentration of calcium ionophore (1.6 µm) induced apoptosis and pronounced generation of intracellular H₂O₂ (92.43±0.93 ng/egg) in treated eggs. Conversely, cell-permeant antioxidant such as 2(3)-tert-butyl-4-hydroxyanisole (BHA) reduced intracellular H₂O₂ level (81.20±1.42 ng/egg) and protected against calcium ionophore-induced morphological changes characteristics of egg activation and apoptosis. These results clearly suggest that calcium ionophore-induced activation and apoptosis are associated with the generation of intracellular H₂O₂ in rat eggs.     

Effect of Ca2+ on deformation, polar body extrusion and pronucleus formation in the egg of the ascidian, Ciona savignyi

Cortical deformation and polar body extrusion are the principal events that occur at fertilization in the ascidian egg. We demonstrated that the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the fertilized egg of Ciona savignyi increased at egg deformation (main peak) and then several small Ca²⁺ spikes (1st spikes) appeared before the first polar body extrusion. Brief Ca²⁺ spikes (2nd spikes), then appeared in the period between the first and second polar body extrusion. When eggs were fertilized in Ca²⁺-free artificial seawater, the main peak and 1st spikes appeared, but the 2nd spikes did not, suggesting that the Ca²⁺ required for the main peak and 1st spikes is released from the intracellular store in this species and that extracellular Ca²⁺ is required for the 2nd spikes. When [Ca²⁺]_i was clamped at a low level (0.03–0.13 μmol/L) by injecting the egg with low-Ca²⁺ buffers and the egg was then inseminated, deformation, polar body extrusion and pronucleus formation were suppressed. In contrast, egg deformation and first polar body extrusion were induced without insemination when [Ca²⁺]_i was 0.9 μmol/L. A higher Ca²⁺ concentration of 1.2–10.1 μmol/L was required for extrusion of the second polar body and pronucleus formation. These data suggest that sequential Ca²⁺ increases (i.e. main peak and 1st and 2nd spikes) are prerequisite for the deformation and polar body extrusion of the egg. Furthermore, in eggs arrested at the second meiotic metaphase after first polar body extrusion by the injection of Ca²⁺ buffer, subsequent injection of excess Ca²⁺ caused formation of an irregular second polar body-like protrusion, suggesting latent arrest at the second meiotic metaphase in the ascidian egg.     

Effects of Calcium-BAPTA Buffers and the Calmodulin Antagonist W-7 on Mouse Egg Activation

Results of numerous experiments indicate that the transient rise in intracellular Ca²⁺following sperm–egg fusion is essential for the subsequent events that constitute egg activation. Some events of egg activation, e.g., cortical granule exocytosis, however, appear more sensitive to intracellular Ca²⁺than other events, e.g., cell cycle resumption. To examine if specific events of egg activation have different thresholds for Ca²⁺, we manipulated buffered intracellular Ca²⁺concentrations by microinjecting Ca²⁺-BAPTA buffers and then examined the effect on the cortical granule exocytosis, recruitment of maternal mRNAs, and cell cycle resumption. We find that whereas cortical granule exocytosis occurs over a narrow threshold range of injected free Ca²⁺concentrations between 0.5 and 1.0 μM,recruitment of maternal mRNAs is only partially stimulated at injected free Ca²⁺concentrations of 2.5 μM,and no evidence for cell cycle resumption was observed (up to 2.5 μMCa²⁺). Although the Ca²⁺- and phospholipid-dependent protein kinase, protein kinase C, is implicated in aspects of egg activation, calmodulin is also a potential target for the transient increase in Ca²⁺that occurs following fertilization. Whereas incubation of eggs in the presence of the calmodulin antagonist W-7 followed by insemination does not block cortical granule exocytosis, cell cycle resumption, as assessed by the metaphase-to-anaphase transition, a decrease in histone H1 kinase activity and the time course for the emission of the second polar body are significantly delayed/inhibited.     

Osmotrophy in fossil protoctists and early animals

Summary

The role of Ca²⁺ in activation and early development of locust eggs was examined through measurement of ooplasmic Ca²⁺ levels before and after fertilization, and through experimental activation of unfertilized eggs. Ooplasmic pCa (i.e. the negative logarithm of Ca²⁺ activity) measured in intact eggs decreased from 5.35 before fertilization, to 4.77 and 3.00 by 1 day and 3 days after fertilization, respectively. pCa was also determined for samples of ooplasm collected by rupturing eggs under paraffin oil. The pCa was 5.10 in ooplasm isolated from unfertilized eggs, and 3.84 in ooplasm collected from eggs within 4 h of fertilization. Ooplasmic pCa remained between 3.97 and 3.12 from 1–6 days after fertilization. Since a decline in pCa indicates an increase in ooplasmic Ca²⁺ activity, the data suggest that regulation of ooplasmic Ca²⁺ during post-fertilization development involves release of Ca²⁺ from internal stores. Experimental egg activation was examined in eggs dissected from the oviducts before fertilization and incubated on moist filter paper. Some eggs were first immersed in experimental solutions for 30–60 minutes before incubation. The presence of an embryo 2 or 4 days after fertilization or experimental treatment was used as an indicator of egg activation. Activation occurred in 92% and 12% of fertilized and untreated eggs, respectively. The percentage of unfertilized eggs which activated increased to 47% if eggs were soaked 30–60 minutes in physiological saline, and to as much as 65%-68% if eggs were injected with Ca²⁺ buffers or if a Ca²⁺ action potential was evoked. Up to 36% and 42% of unfertilized eggs activated after incubation in Ca²⁺-free salines or in the presence of the Ca²⁺-channel blocker Cd²⁺, respectively. Taken together, the results suggest that entry of external Ca²⁺ through voltage dependent channels increases the proportion of eggs which activate, but is not an absolute requirement for activation.     

The Ca increase by the sperm factor in physiologically polyspermic newt fertilization: Its signaling mechanism in egg cytoplasm and the species-specificity

The newt, Cynops pyrrhogaster, exhibits physiological polyspermic fertilization, in which several sperm enter an egg before egg activation. An intracellular Ca²⁺ increase occurs as a Ca²⁺ wave at each sperm entry site in the polyspermic egg. Some Ca²⁺ waves are preceded by a transient spike-like Ca²⁺ increase, probably caused by a tryptic protease in the sperm acrosome at the contact of sperm on the egg surface. The following Ca²⁺ wave was induced by a sperm factor derived from sperm cytoplasm after sperm–egg membrane fusion. The Ca²⁺ increase in the isolated, cell-free cytoplasm indicates that the endoplasmic reticulum is the major Ca²⁺ store for the Ca²⁺ wave. We previously demonstrated that citrate synthase in the sperm cytoplasm is a major sperm factor for egg activation in newt fertilization. In the present study, we found that the activation by the sperm factor as well as by fertilizing sperm was prevented by an inhibitor of citrate synthase, palmitoyl CoA, and that an injection of acetyl-CoA or oxaloacetate caused egg activation, indicating that the citrate synthase activity is necessary for egg activation at fertilization. In the frog, Xenopus laevis, which exhibits monospermic fertilization, we were unable to activate the eggs with either the homologous sperm extract or the Cynops sperm extract, indicating that Xenopus sperm lack the sperm factor for egg activation and that their eggs are insensitive to the newt sperm factor. The mechanism of egg activation in the monospermy of frog eggs is quite different from that in the physiological polyspermy of newt eggs.     

Injection of a porcine sperm factor induces activation of mouse eggs

Injection of sperm preparations into mammalian oocytes and eggs has been shown to elicit persistent [Ca²⁺]i oscillations that closely resemble fertilization-associated Ca²⁺ release. However, the ability of these sperm fractions to initiate egg activation has not been clearly demonstrated. In the present experiments, mouse eggs injected with a porcine sperm preparation were evaluated for early and late events of activation. Events monitored included, among early events, the generation of [Ca²⁺]i oscillations and cortical granule exocytosis and, among late events, the decrease in histone H1 and myelin basic protein kinase activities, polar body extrusion, pronuclear formation, and cleavage to the two-cell stage. Injection of sperm fractions consistently evoked [Ca²⁺]i oscillations that, in turn, initiated all events of activation. Uninjected control eggs or eggs injected with buffer or heat-treated sperm fractions failed to show Ca²⁺ responses or activation. In addition, injection of sperm fractions into recently ovulated eggs (experiments were concluded within 15 hr after human chorionic gonadotropin administration) induced high rates of activation, while similarly aged eggs exposed to 7% ethanol for 5 min, a known parthenogenetic treatment, failed to activate. Together these results indicate that injection of sperm fractions elicits [Ca²⁺]i oscillations that are capable of initiating normal egg activation. These results support the hypothesis that a sperm component participates in the generation of fertilization-associated [Ca²⁺]i oscillations. Mol. Reprod. Dev. 49:37–47, 1998. © 1998 Wiley-Liss, Inc.     

Evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory granule membrane: Comparison of exocytosis of injected bovine chromaffin granule membranes and endogenous cortical granules inXenopus laevis oocytes

Summary 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin granule membranes and of endogenous cortical granules to undergo exocytosis inXenopus laevis oocytes and eggs in response to cytosolic Ca²⁺. Exocytosis of chromaffin granule membranes was detected by the appearance of dopamine--hydroxylase of the chromaffin granule membrane in the oocyte or egg plasma membrane. Cortical granule exocytosis was detected by release of cortical granule lectin, a soluble constituent of cortical granules, from individual cells.2. Injected chromaffin granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca²⁺ induced by incubation with ionomycin.3. Elevated Ca²⁺ triggered cortical granule exocytosis in eggs but not in oocytes.4. Injected chromaffin granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous cortical granules.5. Protein kinase C activation by phorbol esters stimulates cortical granule exocytosis in bothXenopus laevis oocytes andX. laevis eggs (Bement, W. M., and Capco, D. G.,J. Cell Biol. 108, 885–892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin granule membrane exocytosis in oocytes, indicating that although cortical granules and chromaffin granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both.6. These results suggest that structural or biochemical characteristics of the chromaffin granule membrane result in its ability to respond to a Ca²⁺ stimulus. In the oocytes, cortical granule components necessary for Ca²⁺-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca²⁺ stimulus and downstream events.     

Release of mouse eggs from metaphase arrest by protein synthesis inhibition in the absence of a calcium signal or microtubule assembly

Mouse egg activation, which includes release from meiotic metaphase II arrest, results from fertilization-induced increase in intracellular calcium concentration ([Ca²⁺]_i). However, during egg activation caused by exposure to the protein synthesis inhibitor, cycloheximide, [Ca²⁺]_i did not change. Although eggs fertilized in the presence of microtubule inhibitors remain arrested at metaphase, eggs treated for 32 hr with cycloheximide and the microtubule inhibitor, colcemid, formed nuclei. In untreated eggs aged in culture for 24 hr, the microtubule spindles became deformed. These eggs formed nuclei after exposure to cycloheximide, but not the calcium ionophore A23187. Our results indicate that eggs in which protein synthesis is inhibited are released from metaphase without an increase in [Ca²⁺]_i, and despite disruption of the Spindle. © 1995 Wiley-Liss, Inc.     

High cytosolic free calcium level signals apoptosis through mitochondria-caspase mediated pathway in rat eggs cultured in vitro

The present study was aimed to find out whether an increase of cytosolic free calcium level induces egg apoptosis through mitochondria-caspase mediated pathway. To increase cytosolic free calcium level and morphological apoptotic changes, ovulated eggs were cultured in Ca²⁺/Mg²⁺ free media-199 with or without various concentrations of calcium ionophore (0.5, 1, 2, 3, 4 μM) for 3 h in vitro. The morphological apoptotic changes, cytosolic free calcium level, hydrogen peroxide (H₂O₂) concentration, catalase activity, cytochrome c concentration, caspase-9 and caspase-3 activities and DNA fragmentation were analyzed. Calcium ionophore induced morphological apoptotic features in a concentration-dependent manner followed by degeneration at higher concentrations (3 and 4 μM). Calcium ionophore increased cytosolic free calcium level, induced generation of hydrogen peroxide (H₂O₂) and inhibited catalase activity in treated eggs. The increased H₂O₂ concentration was associated with increased cytochrome c concentration, caspase-9 and caspase-3 activities that resulted in the induction of morphological features characteristic of egg apoptosis. The increased caspase-3 activity finally induced DNA fragmentation as evidenced by TUNEL positive staining in calcium ionophore-treated eggs. These findings suggest that high cytosolic free calcium level induces generation of H₂O₂ that leads to egg apoptosis through mitochondria-caspase mediated pathway.     

Protein kinase C is required for the disappearance of MPF upon artificial activation in mouse eggs

The aim of the present study was to investigate the implication of protein kinase C (PKC) in the mouse egg activation process. We used OAG (1-oleoyl-2-acetyl-sn-glycerol) as a PKC activator, calphostin C as a specific PKC inhibitor, and the calcium ionophore A23187 as a standard parthenogenetic agent. The exposure of zona-free eggs to 150 μM or 50 μM OAG for 10 min resulted in meiosis II completion in ∼80% of instances. By contrast, at a lower concentration (25 μM), the PKC stimulator was ineffective as parthenogenetic agent. Shortly after the application of 150 μM OAG, the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) increased transiently in all the eggs examined, whereas after the addition of 50 μM OAG, [Ca²⁺]_i remained unchanged for at least 20 min. During this period, the activity of M-phase promoting factor (MPF) dramatically decreased and most of the eggs entered anaphase except when the PKC was inhibited by calphostin C. Similarly, MPF inactivation and meiosis resumption were prevented in calphostin C-loaded eggs following treatment with A23187, even though the ionophore-induced Ca²⁺ signalling was not affected. Taken together, our results indicate that stimulation of PKC is a sufficient and necessary event to induce meiosis resumption in mouse eggs and strongly suggest that, in this species, the mechanism by which a transient calcium burst triggers MPF inactivation involves a PKC-dependent pathway. Mol. Reprod. Dev. 48:292–299, 1997. © 1997 Wiley-Liss, Inc.     

Regulation of diacylglycerol production and protein kinase C stimulation during sperm- and PLCzeta-mediated mouse egg activation

Background information. At fertilization in mammalian eggs, the sperm induces a series of Ca²⁺ oscillations via the production of inositol 1,4,5‐trisphosphate. Increased inositol 1,4,5‐trisphosphate production appears to be triggered by a sperm‐derived PLCζ (phospholipase C‐ζ) that enters the egg after gamete fusion. The specific phosphatidylinositol 4,5‐bisphosphate hydrolytic activity of PLCζ implies that DAG (diacylglycerol) production, and hence PKC (protein kinase C) stimulation, also occurs during mammalian egg fertilization. Fertilization‐mediated increase in PKC activity has been demonstrated; however, its precise role is unclear. Results. We investigated PLCζ‐ and fertilization‐mediated generation of DAG in mouse eggs by monitoring plasma‐membrane translocation of a fluorescent DAG‐specific reporter. Consistent plasma‐membrane DAG formation at fertilization, or after injection of physiological concentrations of PLCζ, was barely detectable. However, when PLCζ is overexpressed in eggs, significant plasma‐membrane DAG production occurs in concert with a series of unexpected secondary high‐frequency Ca²⁺ oscillations. We show that these secondary Ca²⁺ oscillations can be mimicked in a variety of situations by the stimulation of PKC and that they can be prevented by PKC inhibition. The way PKC leads to secondary Ca²⁺ oscillations appears to involve Ca²⁺ influx and the loading of thapsigargin‐sensitive Ca²⁺ stores. Conclusions. Our results suggest that overproduction of DAG in PLCζ‐injected eggs can lead to PKC‐mediated Ca²⁺ influx and subsequent overloading of Ca²⁺ stores. These results suggest that DAG generation in the plasma membrane of fertilizing mouse eggs is minimized since it can perturb egg Ca²⁺ homoeostasis via excessive Ca²⁺ influx.     

Extracellular calcium protects against verapamil-induced metaphase-II arrest and initiation of apoptosis in aged rat eggs

Non-specific L-type calcium channel blockers, such as verapamil (≥50 μM), induce metaphase-II (M-II) arrest and apoptosis in aged rat eggs cultured in Ca²⁺-deficient medium. However, the effects of extracellular Ca²⁺ on verapamil-induced M-II arrest and apoptosis have not yet been reported. We have demonstrated that postovulatory aging induced exit from M-II arrest by extruding a second polar body, a morphological sign of spontaneous egg activation (SEA). Verapamil inhibited SEA and induced egg apoptosis in a dose-dependent manner in Ca²⁺-deficient medium. The initiation of apoptotic features was observed at 50 μM of verapamil. Extracellular Ca²⁺ (1.80 mM) reduced intracellular H₂O₂ level, bax protein expression, caspase-3 activity, DNA fragmentation and protected against 50 μM, but not higher concentrations of ≥100 μM in verapamil-induced egg apoptosis. These results suggest that extracellular Ca²⁺ ions have a role during SEA and protect against verapamil-induced apoptosis in aged rat eggs.     

Free intracellular cations in echinoderm oocytes and eggs

The concentrations of Ca²⁺, Na⁺ and H⁺ in echinoderm oocytes and eggs were measured during maturation and activation using ion-selective microelectrodes. In both oocytes and eggs, from three species of starfish and two species of sea urchin, the resting level of cytosolic Ca²⁺ was about 10^-7 M. We did not detect any change in Ca²⁺ concentration either during hormone-induced oocyte maturation (starfish) or during egg activation (starfish and sea urchin) induced by spermatozoa or chemical agents. During 1-methyl-adenine induced maturation of starfish oocytes the intracellular level of Na⁺ increased from 12–35 mM to 40–90 mM, while the pH changed from 6.6–6.8 to 7.0–7.2 Aged oocytes, with intact germinal vesicles, also had elevated levels of Na⁺ and pH.     

Fertilization Induces a Transient Exposure of Phosphatidylserine in Mouse Eggs

Phosphatidylserine (PS) is normally localized to the inner leaflet of the plasma membrane and the requirement of PS translocation to the outer leaflet in cellular processes other than apoptosis has been demonstrated recently. In this work we investigated the occurrence of PS mobilization in mouse eggs, which express flippase Atp8a1 and scramblases Plscr1 and 3, as determined by RT-PCR; these enzyme are responsible for PS distribution in cell membranes. We find a dramatic increase in binding of flouresceinated-Annexin-V, which specifically binds to PS, following fertilization or parthenogenetic activation induced by SrCl₂ treatment. This increase was not observed when eggs were first treated with BAPTA-AM, indicating that an increase in intracellular Ca²⁺ concentration was required for PS exposure. Fluorescence was observed over the entire egg surface with the exception of the regions overlying the meiotic spindle and sperm entry site. PS exposure was also observed in activated eggs obtained from CaMKIIγ null females, which are unable to exit metaphase II arrest despite displaying Ca²⁺ spikes. In contrast, PS exposure was not observed in TPEN-activated eggs, which exit metaphase II arrest in the absence of Ca²⁺ release. PS exposure was also observed when eggs were activated with ethanol but not with a Ca²⁺ ionophore, suggesting that the Ca²⁺ source and concentration are relevant for PS exposure. Last, treatment with cytochalasin D, which disrupts microfilaments, or jasplakinolide, which stabilizes microfilaments, prior to egg activation showed that PS externalization is an actin-dependent process. Thus, the Ca²⁺ rise during egg activation results in a transient exposure of PS in fertilized eggs that is not associated with apoptosis.     

Changes in microtubule structures during the first cell cycle of physiologically polyspermic newt eggs

The unfertilized egg of the newt, Cynops pyrrhogaster, has a second meiotic spindle at the animal pole and numerous cortical cytasters. After physiologically polyspermic fertilization, all sperm nuclei incorporated into the egg develop sperm asters, and the cortical cytasters change into bundles of cortical microtubules. The size of the sperm asters in the animal hemisphere is ∼5.6-fold larger than that in the vegetal hemisphere. Only one sperm nucleus moves toward the center of the animal hemisphere to form a zygote nucleus with the egg nucleus. This movement is inhibited by nocodazole, but not by cytochalasin B. The centrosome in the zygote nucleus divides into two parts to form a bipolar spindle for the first cleavage synchronously with the nuclear cycle, but centrosomes of accessory sperm nuclei in the vegetal hemisphere remained to form monopolar interphase asters and subsequently degenerate around the first cleavage stage. The size of sperm asters in monospermically fertilized Xenopus eggs was ∼37-fold larger than those in Cynops eggs. Since sperm asters that formed in polyspermically fertilized Xenopus eggs exclude each other, the formation of a zygote nucleus is inhibited. Cynops sperm nuclei form larger asters in Xenopus eggs, whereas Xenopus sperm nuclei form smaller asters in Cynops eggs compared with those in homologous eggs. Since there was no significant difference in the concentration of monomeric tubulin between those eggs, the size of sperm asters is probably regulated by a component(s) in egg cytoplasm. Smaller asters in physiologically polyspermic newt eggs might be useful for selecting only one sperm nucleus to move toward the egg nucleus. Mol. Reprod. Dev. 47:210–221, 1997. © 1997 Wiley-Liss, Inc.     

Reduced external calcium or sodium stimulates calcium influx in Pelvetia eggs

The effect of external calcium and sodium ion concentrations on the calcium fluxes on the Pelvetia fastigiata De Toni egg was measured. Decreasing external [Ca²⁺] greatly increased the permeability of the eggs to Ca²⁺; at 1 mM external Ca²⁺ this permeability was 60 times as great as it was at the normal [Ca²⁺] of 10 mM. Lowering the external [Na⁺] also increased Ca²⁺ influx; at 2 mM Na⁺, the Ca²⁺ influx was 2–3 times as great as it was at the normal [Na⁺] if choline was used as a Na⁺ substitute. Lithium was less effective as a Na⁺ substitute in increasing Ca²⁺ influx. The extra Ca²⁺ influx in low [Na⁺] seemed to be dependent on internal [Na⁺]. The Ca²⁺ efflux increased transiently and then declined in low Na⁺ media.     

A fluorescence dequenching method for monitoring exocytotic membrane fusion in fertilization of single sea urchin eggs

A method for monitoring exocytotic membrane fusion by using a fluorescent membrane probe is presented. The method is based on the relief from concentration-dependent self-quenching (dequenching) of fluorescence of 5-N-(octadecanoyl)aminofluorescein (AF18), an amphiphilic derivative of fluorescein. The validity and usefulness of this method were shown by the following results: 1) self-quenching of AF18 fluorescence occurred in the plasma membrane of unfertilized eggs of a sea urchin, Pseudocentrotus depressus, which were heavily stained with the fluorescent dye; 2) dequenching of AF18 fluorescence occurred upon fertilization in normal eggs but not in EGTA-injected eggs; 3) Ca²⁺ induced both AF18 fluorescence dequenching and cortical granule disappearance in the isolated sea urchin egg cortex; and 4) simultaneous measurements of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and dequenching of AF18 fluorescence by using a simple one-excitation and two-emission wavelength system.     

Comparison of Ca uptake characteristics of microsomal fractions isolated from unfertilized and fertilized sea urchin eggs

The microsomal fraction isolated from sea urchin H. pulcherrimus eggs has the ability to actively accumulate Ca²⁺ in the presence of ATP. The Ca²⁺ uptake was sustained by addition of oxalate and was apparently insensitive to sodium azide. The sequestered microsomal Ca was readily released by the divalent cation ionophore A23187. The microsomal fraction obtained from fertilized eggs accumulated Ca²⁺ about five times more quickly than did that from unfertilized eggs. The increased Ca²⁺ uptake by microsomal fraction obtained from fertilized eggs was due to an increase in the maximum velocity of Ca²⁺ uptake and there was no difference in K_m for calcium between the two fractions.     

ANIMAL/VEGETAL DIFFERENCES IN CORTICAL GRANULE EXOCYTOSIS DURING ACTIVATION OF THE FROG EGG*

One of the more striking morphological events during egg activation is exocytosis of the cortical granules. In the frog egg, the wave of cortical granule exocytosis takes about 100 sec to traverse the animal half, and travels slower in the vegetal half. We examined cortical granule exoctyosis during activation with respect to this animal/vegetal difference. In eggs which were acquiring the ability to be activated (recovering from CO₂-intoxication or undergoing meiotic maturation), animal half cortical granules became capable of responding to activating stimuli prior to vegetal half ones. Since Ca²⁺ is involved in exocytosis, we examined the effect of Ca²⁺ on cortical granule breakdown in vitro. There was no difference in sensitivity to Ca²⁺ of cortical granules from immature vs. mature eggs, but animal half cortical granules were more sensistive to Ca²⁺ than vegetal half ones. Finally, we found that prick-activation of eggs at the vegetal pole was frequently unsuccessful but would occur when external Ca²⁺ was raised. These experiments show that there are regional differences in the frog egg with respect to cortical granule responsiveness, and they suggest that the differences are due to Ca²⁺ sensitivity.     

The electrical block to polyspermy induced by an intracellular Ca2+ increase at fertilization of the clawed frogs,Xenopus laevis and Xenopus tropicalis

Polyspermy blocking, to ensure monospermic fertilization, is necessary for normal diploid development in most animals. We have demonstrated here that monospermy in the clawed frog, Xenopus tropicalis, as well as in X. laevis, is ensured by a fast, electrical block to polyspermy on the egg plasma membrane after the entry of the first sperm, which is mediated by the positive‐going fertilization potential. An intracellular Ca²⁺ concentration ([Ca²⁺]_i) at the sperm entry site was propagated as a Ca²⁺ wave over the whole egg cytoplasm. In the X. tropicalis eggs fertilized in 10% Steinberg's solution, the positive‐going fertilization potential of +27 mV was generated by opening of Ca²⁺‐activated Cl^?‐channels (CaCCs). The fertilization was completely inhibited when the egg's membrane potential was clamped at +10 mV and 0 mV in X. tropicalis and X. laevis, respectively. In X. tropicalis, a small number of eggs were fertilized at 0 mV. In the eggs whose membrane potential was clamped below ?10 mV, a large increase in inward current, the fertilization current, was recorded and allowed polyspermy to occur. A small initial step‐like current (IS current) was observed at the beginning of the increase in the fertilization current. As the IS current was elicited soon after a small increase in [Ca²⁺]_i, this is probably mediated by the opening of CaCCs. This study not only characterized the fast and electrical polyspermy in X. tropicalis, but also explained that the initial phase of [Ca²⁺]_i increase causes IS current during the early phase of egg activation of Xenopus fertilization.