An electrical block is required to prevent polyspermy in eggs fertilized by natural mating of Xenopus laevis

In 27% DeBoer's saline (DBS), which yields maximum fertility rates, Xenopus eggs fertilized in vitro are monospermic, regardless of sperm concentration. One block to polyspermy (the “slow” block), described previously, occurs at the fertilization envelope that is elevated in response to the cortical reaction. This paper describes properties of an earlier, “fast” block at the plasma membrane and evaluates the functional significance of the two blocks at physiological sperm concentrations in natural mating conditions. Unfertilized eggs have a resting membrane potential of ?19 mV in 27% DBS. Fertilization triggers a rapid depolarization to +8 mV (the fertilization potential, FP); the potential remains positive for ca. 15 min. Activation of eggs with the ionophore, A23187, produces a slower but similar depolarization (the activation potential, AP). As in other amphibian eggs, the FP appears to result from a net efflux of Cl^?, since the peak of the FP (or the AP in ionophore-activated eggs) decreases as the concentration of chloride salts in the medium is increased. In 67% DBS no FP or AP is observed; eggs fertilized in 67% DBS become polyspermic and average 2 sperm entry sites per egg. In the 5–37 mM range, I^? and Br^?, but not F^?, are more effective than Cl^? in producing polyspermy. In 20 mM NaI the plasma membrane hyperpolarizes in response to sperm or ionophore; 100% levels of polyspermy and an average of 14 sperm entry sites per egg are observed. NaI does not inhibit or retard elevation of the fertilization envelope; the cortical reaction and fertilization envelope are normal in transmission electron micrographs. In 67% DBS, which also inhibits the fast block, the slow block was estimated to become functional 6–8 min after insemination. Eggs fertilized by natural mating in 20 mM NaI exhibit polyspermy levels of 50–90% and average 5 sperm entry sites per egg. Since eggs become polyspermic when fertilized by natural mating under conditions that inhibit the fast, but not the slow, block to polyspermy, we conclude that the fast block is essential to the prevention of polyspermy at the sperm concentrations normally encountered by the egg.     

The block to sperm penetration in zonal-free mouse eggs

The rate of sperm penetration and the number of sperm penetrating zona-free mouse eggs were found to be dependent on sperm concentration. At the lowest sperm concentrations examined (10² cells/ml, sperm-egg ratios of approximately 1:1), most eggs were penetrated (75%), and polyspermy was low (19%) following 3 hr of incubation. The number of sperm penetrating the egg was logarithmically related to sperm concentration. All eggs showed a delay of at least 20 min between insemination and penetration, and penetration was complete in approximately 2 hr at 10⁴ sperm/ml; this penetration block was attributed to egg-related changes. The existence and timing of the egg plasma membrane block to polyspermy were evaluated by reinsemination experiments. In this approach, the block was triggered in zona-free eggs with a low concentration of capacitated epididymal sperm at time 0, and the eggs were subsequently challenged with high sperm concentrations. The presence or absence of a block was inferred from the degree of polyspermy observed in these eggs after 3 hr of incubation. Adjusting for sperm concentration-dependent delays between insemination and sperm penetration, a blocking time of approximately 40 min was obtained.     

Fast polyspermy block and activation potential : Correlated changes during oocyte maturation of a starfish

Sperm entry into the oocyte of the starfish, Asterina pectinifera, was prevented when the membrane potential of the oocyte was held more positive than −10 to −5 mV, and multiple sperm entries were induced when the potential was held more negative. Based on this potential-dependent fertilization block mechanism, it was demonstrated that an activation potential (AVP) which is induced immediately after the attachment of the first sperm to the egg surface plays the role of a fast polyspermy block. The AVP-mediated polyspermy block mechanism develops as the oocyte matures and deteriorates as it ages. AVPs of mature oocytes exceeded −5 mV (the critical potential level for fertilization block) within 1 sec, and the potential stayed at +12 mV even after the initiation of fertilization membrane elevation. Consequently, the entry of a second sperm is prevented. In contrast, AVPs of overripe oocytes took about 15 sec to attain −5 mV, or they did not attain −5 mV at all. In overripe oocytes multiple sperm entries were associated with “step depolarization(s)” in the rising phase of the AVPs before membrane elevation took place. Immature oocytes generated AVPs associated with sperm entries, but without membrane elevation. AVPs in immature oocytes were characterized by the step depolarization(s) in the rising phase, and an AVP could be evoked again by a second insemination 20 min after the first insemination. These findings indicate that immature oocytes lack both fast and slow polyspermy block mechanisms.     

Fast polyspermy block and activation potential : Electrophysiological bases for their changes during oocyte maturation of a starfish

In the starfish oocyte, the activation potential (AVP) upon fertilization establishes a fast polyspermy block. In the present paper, factors affecting the peak level of the AVP were analyzed by comparing current-voltage relations of the oocyte membrane just before and after insemination at various maturation stages. These factors were: (1) conductance of the oocyte membrane before insemination; (2) magnitude of the conductance increase induced by sperm; and (3) equilibrium potential of the AVP. Mature oocytes showed extremely low membrane conductance before insemination, and this provided a more positive-going AVP establishing monospermy. Overmature oocytes before fertilization showed higher conductance than mature oocytes and usually became polyspermic upon insemination. Application of Ba²⁺ ions reduced the conductance of the unfertilized, overmature oocyte to a state similar to that of the mature oocyte. Correspondingly, Ba²⁺ reduced the probability of polyspermy in overmature oocytes. Old oocytes showed even higher conductance before insemination than overmature oocytes and, in addition, apparently showed a large negative value for the equilibrium potential of the AVP. In old oocytes, these two factors account for the small amplitude of the AVP, which allows multiple sperm entries. Furthermore, the magnitude of the conductance increase induced by sperm seemed to change during the maturation process.     

Initiation of the activation potential by an increase in intracellular calcium in eggs of the frog,Rana pipiens

The membrane potential of the frog egg undergoes a transient positive shift at fertilization which is a block to polyspermy. This paper addresses the question of how a sperm elicits this “fertilization potential.” Iontophoretic injection of Ca²⁺ activates Rana pipiens eggs to develop and initiates a transient, positive-going shift in the membrane potential (the activation potential) which is like the sperm-induced fertilization potential in amplitude, duration, and Cl^? dependence. Activation potentials are elicited by Ca² injection into both animal and vegetal regions of the egg, but the rate of the initial depolarization is much less when Ca²⁺ is injected into the vegetal region. Injections of K⁺, Na⁺, Cl^?, or Mg²⁺ do not result in activation potentials, but the Ca²⁺ analogs, Sr²⁺ and Ba²⁺, can substitute for Ca²⁺. Treatment of eggs with the divalent cation ionophore, A23187, also initiates a transient, positive-going depolarization. Because injection of Ca²⁺ is sufficient to elicit a response almost identical to a fertilization potential, the ion transport mechanisms necessary to produce a fertilization potential must preexist in the unfertilized eggs; the sperm contributes only the stimulus to activate these mechanisms. The results reported here suggest that the stimulus may be a rise in free Ca²⁺.     

Polyspermy block in jellyfish eggs: Collaborative controls by Ca and MAPK

Jellyfish eggs neither undergo apparent cortical reaction nor show any significant change in the membrane potential at fertilization, but nevertheless show monospermy. Utilizing the perfectly transparent eggs of the hydrozoan jellyfish Cytaeis uchidae, here we show that the polyspermy block is accomplished via a novel mechanism: a collaboration between Ca²⁺ and mitogen-activated protein kinase (MAPK). In Cytaeis, adhesion of a sperm to the animal pole surface of an egg was immediately followed by sperm–egg fusion and initiation of an intracellular Ca²⁺ rise from this site. The elevated Ca²⁺ levels lasted for several minutes following the sperm–egg fusion. The Ca²⁺ rise proved to be necessary and sufficient for a polyspermy block, as inhibiting a Ca²⁺ rise with EGTA promoted polyspermy, and conversely, triggering a Ca²⁺ rise by inositol 1,4,5-trisphosphate (IP₃) or excess K⁺ immediately abolished the egg’s capacity for sperm–egg fusion. A Ca²⁺ rise at fertilization or by artificial stimulations evoked dephosphorylation of MAPK in eggs. The eggs in which phosphorylated MAPK was maintained by injection of mRNA for MAPK kinase kinase (Mos), like intact eggs, exhibited a Ca²⁺ rise at fertilization or by IP₃ injection, and shut down the subsequent sperm–egg fusion. However, the Mos-expressing eggs became capable of accepting sperm following the arrest of Ca²⁺ rise. In contrast, addition of inhibitors of MAPK kinase (MEK) to unfertilized eggs caused MAPK dephosphorylation without elevating Ca²⁺ levels, and prevented sperm–egg fusion. Rephosphorylation of MAPK by injecting Mos mRNA after fertilization recovered sperm attraction, which is known to be another MAPK-dependent event, but did not permit subsequent sperm–egg fusion. Thus, it is possible that MAPK dephosphorylation irreversibly blocks sperm–egg fusion and reversibly suppresses sperm attraction. Collectively, our data suggest that both the fast and late mechanisms dependent on Ca²⁺ and MAPK, respectively, ensure a polyspermy block in jellyfish eggs.     

A rapid sodium-dependent block to polyspermy in sea urchin eggs

The rapid electrical depolarization of the egg's plasma membrane which protects sea urchin ova against polyspermy in the interval between stimulation by the fertilizing spermatozoon and completion of the cortical reaction is believed to be mediated by the influx of sodium (Na⁺) ions. This hypothesis was tested in Arbacia punctulata and Strongylocentrotus purpuratus by inhibiting the rapid block to polyspermy with low-Na⁺ (choline-substituted) seawater, and the cortical granule secretion-mediated block with soybean trypsin inhibitor (SBTI). Eggs inseminated in low-Na⁺ seawater or SBTI became heavily polyspermic. Polyspermy elicited by low Na⁺ or SBTI was increased in dejellied Strongylocentrotus eggs. However, the severity of polyspermy was not enhanced in low Na⁺ plus SBTI because the fertilizing capacity of sperm and gamete binding were reduced in low-Na⁺ media. Since SBTI completely suppresses the cortical granule secretion-mediated block to polyspermy in Arbacia for about 3 min postinsemination, the rate at which SBTI-treated eggs became polyspermic was used to measure the duration and efficacy of the rapid block. The half-time for SBTI-treated Arbacia eggs to become polyspermic in natural (425 mM Na⁺) seawater was 89.9 ± 4.7 sec (N = 4). The plot of incidence of polyspermy vs time was essentially an inverse mirror image of electrophysiologic data on repolarization of the oolemma during fertilization. The rapid block is also Na⁺ dependent, since SBTI-treated eggs became polyspermic more rapidly in 26 mM Na⁺ seawater (half-time, 15.8 ± 1.6 sec; N = 3, P < 0.01).     

Timing the early events during sea urchin fertilization

To determine precisely the timing, duration, and sequences of the earliest events during sea urchin (Lytechinus variegatus) fertilization, the bioelectric recordings of microelectrode-impaled eggs were electronically superimposed, by video mixing, over the microscopic differential interference contrast image of the same egg at insemination. Videotape analysis, utilizing a slow-motion analyzer, demonstrates that the successful sperm triggers the bioelectric membrane potential reversal within 3.36 +/- 3.02 sec (0.72-9.76 sec range; sigma = 23 eggs) of sperm-egg attachment. This sperm, actively gyrating about its attachment site, is indistinguishable from the other, unsuccessful sperm until 12.66 +/- 2.72 sec (6.72-16.60 sec range; sigma = 15) later when the sperm tail ceases its beating and sperm incorporation ensues. The cortical granules begin to discharge, and the fertilization coat starts to elevate at the fusion site at 20.79 +/- 3.18 sec (13.62-26.08 sec range; sigma = 12) after the onset of the fertilization potential, i.e., an average of about 8 sec after the cessation of sperm-tail motility during incorporation. In most cases, the bioelectric responses starts within 7 sec of sperm adhesions; if the data are analyzed excluding the few slow cases, the fertilization potential is found to start 1.93 sec (+/- 1.28 sec) after sperm attachment. These results indicate that the first successful sperm triggers the fast block to polyspermy within 3.4 sec, perhaps as quickly as 1.9 sec, of sperm-egg adhesion, about 13 sec before the first morphological indication of fertilization, and about 21 sec before the characteristic elevation of the fertilization coat responsible for the late block to polyspermy.     

Ion channels and signaling pathways used in the fast polyspermy block

Fertilization of an egg by multiple sperms, polyspermy, is lethal to most sexually reproducing species. To combat the entry of additional sperm into already fertilized eggs, organisms have developed various polyspermy blocks. One such barrier, the fast polyspermy block, uses a fertilization‐activated depolarization of the egg membrane to electrically inhibit supernumerary sperm from entering the egg. The fast block is commonly used by eggs of oviparous animals with external fertilization. In this review, we discuss the history of the fast block discovery, as well as general features shared by all organisms that use this polyspermy block. Given the diversity of habitats of external fertilizers, the fine details of the fast block‐signaling pathways differ drastically between species, including the identity of the depolarizing ions. We highlight the known molecular mediators of these signaling pathways in amphibians and echinoderms, with a fine focus on ion channels that signal these fertilization‐evoked depolarizations. We also discuss the investigation for a fast polyspermy block in mammals and teleost fish, and we outline potential fast block triggers. Since the first electrical recordings made on eggs in the 1950s, the fields of developmental biology and electrophysiology have substantially matured, and yet we are only now beginning to discern the intricate molecular mechanisms regulating the fast block to polyspermy.     

U.V. Irradiation Inhibits The Electrical Block to Polyspermy in Echinoderms*

Oocytes of the sea urchin Sphaerechinus granularis and the startish Marthasterias glacialis have been submitted to U.V. irradiation before fertilization. This treatment significantly increased the incidence and severity of polyspermy in the sea urchin and was also found effective on starfish oocytes. These were found more resistant to damage than sea urchin eggs and U.V. irradiation did not affect either their response to the hormone l-methyladenine or the rate of elevation of the fertilization envelope, which assures the late and definitive block to polyspermy. Electrophysiological measurements performed on M. glacialis oocytes definitively demonstrate that U.V. irradiation completely inactivates voltage-dependent sodium channels, without altering the other main conductances, Cl^?, K⁺ or Ca²⁺. After such a treatment, the relative permeability of the membrane to Na⁺ as compared to K⁺ shifted from 0.019±0.003 to 0.003±0.002 and only the calcium component of the action potentials could be observed. However, a fertilization potential, preceded by small sperm induced steps, is still present in these conditions, although its peak and plateau level are greatly reduced. These new findings are discussed, which confirm the electrical nature of the fast block to polyspermy but question about the specificity of those sperm-gated channels which are supposed to trigger the fertilization potential.     

Bioelectric responses at fertilization: Separation of the events associated with insemination from those due to the cortical reaction in sea urchin,Lytechinus variegatus

The bioelectric responses at fertilization of the sea urchin Lytechinus variegatus are a complex series of membrane potential and resistance changes that occur concomitant with gamete fusion, ionic fluxes, and the cortical granule discharge. This work attempts to separate the electrical effects of sperm-egg interactions from those of the cortical reactions. Two approaches were taken to discern the electrical events associated with insemination, distinct from cortical granule discharge: (1) fertilization of eggs treated with 3% urethane, 10 mM procaine, or 10 mM nicotine, to prevent the cortical reaction and (2) refertilization of fertilized eggs (denuded with 1 mM aminotriazole containing 1 mg/ml soybean trypsin inhibitor). Cortical granule discharge in the absence of sperm incorporation was investigated by artificial activation with 5 μM A23187 or by fertilization in the presence of 10 μM cytochalasin D, which prevents incorporation. These results are consistent with a model in which the sperm-egg interaction triggers both a rapid (50–400 msec), but minor (?10 mV), electrical transient that leads to an action potential and then both the Na⁺-dependent fast block to polyspermy and the late block resulting from the secretion of the cortical granules.     

Membrane depolarization facilitates sperm entry, large fertilization cone formation, and prolonged current responses in sea urchin oocytes

Depolarization of the sea urchin egg's membrane is required for two processes during fertilization: the entry of the fertilizing sperm and the block to polyspermy which prevents the entry of supernumerary sperm. In an immature sea urchin oocyte, the depolarization is very small in response to the attachment of a sperm. The purpose of this study was to determine whether the depolarization evoked by sperm attaching to an oocyte can facilitate sperm entry or induce the block to polyspermy. Individual oocytes of the sea urchin with diameters which ranged from 86 to 102% that of the average diameter for mature eggs from the same female were examined. The oocytes have a membrane potential of -73 +/- 6 mV (SD, n = 80) and a very low input resistance compared to that of mature eggs. Single sperm, following attachment to an oocyte, elicit a brief, small depolarization with a maximum amplitude of 8 +/- 1.4 mV (SE, n = 15), frequently followed by the formation of tiny filament-like fertilization cones, but the sperm fail to enter. If oocytes are voltage-clamped at membrane potentials more negative than -20 mV, following attachment of the sperm small transient inward currents occur, similar filament-like cones form, and the sperm do not enter. When many sperm attach to an oocyte which is not voltage clamped, the depolarizations sum to create a large depolarization with an amplitude of 60 to 80 mV, which shifts the oocyte's membrane potential to a value between -10 and +5 mV; more positive values are not attained. At such membrane potentials, whether the potential is maintained by the summed depolarizations of many attached sperm or by voltage clamp, large fertilization cones form, the sperm enter, and the oocytes can become highly polyspermic. In oocytes voltage clamped at +20 mV, however, both sperm entry and fertilization cone formation are suppressed. Therefore, both types of voltage-dependence for sperm entry are present in oocytes, although the depolarization caused by a single sperm is not large enough to permit its entry, nor is the depolarization caused by many sperm sufficient to prevent the entry of supernumerary sperm.     

Absence of a complete block to polyspermy after fertilization of Mytilus galloprovincialis (Mollusca,Pelecypoda) oocytes

Mytilus galloprovincialis oocytes undergo monospermic fertilizations (1 sperm nucleus/oocyte) over a wide range of sperm-oocyte ratios beyond which the number of penetrating sperm increases either linearly or exponentially over 10 min. Artificial activation of oocytes by KCl or the ionophore A 23187, up to the polar body extrusion stage, allows successful fertilizations upon a subsequent insemination. No organized and complete detachment of supernumerary oocyte-bound sperm is detected after fertilization. Reducing the external Na⁺ concentration promotes a higher rate of fertilizations. These results suggest that no complete block to polyspermy is established in this species but that a partial block, Na⁺ dependent, might be sufficient to ensure monospermic fertilizations under natural conditions.     

A fast block to polyspermy in frogs mediated by changes in the membrane potential

The role of the egg membrane potential in the prevention of polyspermy in Rana pipiens was studied with intracellular microelectrodes and ion-substituted media. At fertilization, the egg membrane potential shifts from a resting value of ?28 to +8 mV in a single step of less than 1 sec. A second, slower shift reaches a maximum amplitude of +17 mV; the membrane potential is positive for a total of 21 min. When the membrane potential of unfertilized eggs exposed to sperm was held at +1 to +22 mV for 30 min by injecting current through a second intracellular electrode, the initiation of the first cleavage furrow was delayed about 20 min, suggesting that the eggs were not fertilized while the membrane potential was positive. Injection of a similar amount of current after fertilization did not delay cleavage. Furthermore, fertilization in ion-substituted media suggests a correlation between the maximum amplitude of the positive-going shift and the incidence of polyspermy. Up to 25% of eggs were polyspermic when inseminated in the presence of NaI, and the maximum amplitude was reduced to ?20 mV when eggs were fertilized in 40 mM NaI. In contrast, fertilization in 40 mM NaCl reduced the maximum amplitude only to +6 mV, and produced no polyspermy. In solutions of NaBr, intermediate effects on the membrane potential and polyspermy were seen. Comparable results were obtained with the toad, Bufo americanus. We conclude that the membrane potential shift prevents polyspermy.     

A cell surface block to polyspermy occurs in golden hamster eggs

We have examined the frequency and fate of supernumerary sperm in the perivitelline space (PVS) of in vitro fertilized hamster eggs to determine if there is a cell surface block to polyspermy. The zona pellucida block to polyspermy is very effective since only one sperm penetrated the zona pellucida in 72.8% of the 876 fertilized eggs examined. Of the polypenetrated eggs, 41.6% had a supernumerary sperm within the PVS. The proportion of polypenetrated eggs with PVS sperm did not change when the duration of coincubation was increased from 3 to 6 hr. PVS sperm were found in 67% of the inseminations. From these data we conclude that there is a cell surface block to polyspermy in the hamster. To investigate the mechanism of the cell surface block, we used the Hoechst-transfer technique (R. Hinkley, B. Wright, and J. Lynn, 1986, Dev. Biol. 118, 148-154) to monitor sperm-egg fusion. We first demonstrated that dye transfer from zona pellucida-free eggs to sperm only occurred when fusion was possible, i.e., in the presence of calcium, and that dye was transferred to all fused sperm. When cumulus-free, zona-intact eggs were preloaded with Hoechst dye and viewed 3 hr postinsemination, three classes of eggs with supernumerary sperm in the PVS were observed: eggs with only Hoechst-positive sperm (62%), eggs with only Hoechst-negative sperm (27%), and eggs with both a Hoechst-positive and a Hoechst-negative sperm (11%). Because of the limited time resolution of the Hoechst-transfer technique, the cell surface block could operate by preventing sperm fusion (Hoechst-negative), by the failure of the eggs to incorporate fused sperm (Hoechst-positive), and/or by the "unfusing" of fused sperm (Hoechst-positive and Hoechst-negative). We are unable at this time to differentiate between these mechanisms.     

Electrically mediated fast polyspermy block in eggs of the marine worm, Urechis caupo

Previous work has established that the polyspermy block in Urechis acts at the level of sperm-egg membrane fusion. (J. Exp. Zool. 196:105). Present results indicate that during the first 5--10 min after insemination the block is mediated by a positive shift in membrane potential (the fertilization potential) elicited by the penetrating sperm, since holding the membrane potential of the unfertilized egg positive by passing current reduces the probability of sperm entry, while progressively reducing the amplitude of the fertilization potential by decreasing external Na+ progressively enhances multiple sperm penetrations. Also, a normal fertilization potential is correlated with a polyspermy block even under conditions (pH 7) in which eggs do not develop. We have investigated the mechanism of the electrical polyspermy block by quantifying the relationship between sperm incorporation, membrane potential and ion fluxes. Results indicate that the polyspermy block is mediated by the electrial change per se and not by the associated fluxes of Na+, Ca++, and H+.     

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.     

Ascidian eggs release glycosidase activity which aids in the block against polyspermy

To ensure normal development, most animals have evolved a number of mechanisms to block polyspermy including prevention of binding to surface coats as well as sperm-egg fusion. Ascidian sperm bind to vitelline coat (VC) glycosides. In the genus Ascidia, N-acetylglucosamine (GlcNAc) is the ligand to which sperm bind. The number of sperm bound to the VC is biphasic following fertilization; sperm binding increases through the first minute or so, then abruptly declines. At fertilization, the eggs of Ascidia callosa, A. ceratodes, A. mentula, A. nigra and Phallusia mammillata release N-acetylglucosaminidase into the sea water (SW). This has been shown to inactivate VC GlcNAc groups, blocking the binding of supernumerary sperm and polyspermy in A. nigra. This block to polyspermy is inactivated by GlcNAc (2mM) or 150 mM-Na+ (choline substituted) SW. These treatments are not additive and therefore probably affect the same process. In A. callosa, fertilization in low Na+ SW causes a 60% decline in enzyme release and a similar increase in the number of sperm remaining on the VC at 4 min as well as a great increase in polyspermy. Thus the principal block to polyspermy in ascidian eggs involves the release of N-acetylglucosaminidase which appears to be Na+ dependent. Enzyme activity is found in the supernatant SW by 15 s after fertilization, suggesting that it is stored very near the egg surface. Histochemical staining of whole eggs and embryos shows loss of surface-associated enzyme activity following fertilization. Like other lysosomal enzymes this N-acetylglucosaminidase is mannosylated and has an acidic pH optimum.     

Polyspermic fertilization of sea urchin eggs treated with protease inhibitors: Localization of sperm receptor sites at the egg surface

The normal elevation of the fertilization membrane and the establishment of the block to polyspermy are retarded in Arbacia punctulata eggs by specific protease inhibitors, soybean trypsin inhibitor (SBTI), leupeptin, and antipain. Ultrastructural observations show that the vitelline layer remains attached to the plasma membrane of fertilized SBTI treated eggs at numerous sites (cortical projections). Quantitive morphometric analysis indicates that the vitelline layer elevates from about 65% of the surface of SBTI treated eggs during the first 3 min post insemination. However, the vulnerability of SBTI treated eggs to refertilization (polyspermy) only declined during the subsequent gradual detachment of the vitelline layer from the cortical projections over the next 15 min. Antipain and leupeptin (10^?5 to 10^?3M) also promoted polyspermy in Arbacia eggs by a process of refertilization extending for a 10- to 15-min period after the initial monospermic insemination. Normal cleavage and development was obtained when eggs were placed in leupeptin and antipain (10^?3M) after the fertilization membrane had elevated. The data indicate that the normal secretory function (or functions) of the cortical granule protease in establishing the block to polyspermy is retarded by these protease inhibitors, and that the vitelline layer is transformed into a mechanical barrier to prevent penetration by supernumerary sperm during its detachment from the plasma membrane of the egg. Furthermore, the vitelline layer in unfertilized eggs appears to be a mosaic structure, with sperm receptor sites localized in regions of the egg's surface, which give rise to cortical projections in the presence of SBTI.     

NAADP triggers the fertilization potential in starfish oocytes

In invertebrates oocytes or eggs, the fertilization or activation potential establishes the fast electrical block to polyspermy and, in some species, provides the Ca2+ influx which contributes to the following intracellular Ca2+ wave. In echinoderms, the molecule triggering the activation potential is still unknown. The aim of this study was to assess whether nicotinic acid-adenine dinucleotide phosphate (NAADP) elicited the fertilization potential in starfish oocytes. The changes in membrane potential induced by the sperm were measured in oocytes held at a low resting potential, so that the Ca2+-action potential was inactivated and only the initial slower depolarization caused by the sperm could be studied. Decreasing extracellular Na+ concentration did not prevent the onset of the fertilization potential, while removal of external Ca2+ abolished it. The pre-incubation with SK&F 96365 and verapamil and the pre-injection of BAPTA inhibited the fertilization potential, while the injection of heparin only reduced its duration. The biophysical and pharmacological properties of the sperm-elicited depolarization were similar to those displayed by the NAADP-activated Ca2+-mediated current recently described in starfish oocytes. Indeed, the desensitization of NAADP-receptors prevented the onset of the fertilization potential. Taken together, these data suggest that NAADP could trigger the fertilization potential in starfish oocytes.