Fertilization potential and polyspermy prevention in the egg of the nemertean, Cerebratulus lacteus

We investigated the electrical properties of the egg of the nemertean worm Cerebratulus, and found evidence that an electrically-mediated polyspermy block operates for a period of about 1 hr after fertilization. At fertilization, in natural or artificial sea water, the membrane potential shifts from its resting level of about -66 mV to a peak of about +43 mV, and in most cases remains greater than 0 mV for more than 1 hr. The average potential during the first 30 min is +22 +/- 8 mV (SD, n = 12). When the external Na+ concentration is reduced from 486 to 51 mM (choline substituted) the fertilization potential amplitude is reduced; the average potential during the first 30 min is -27 +/- 21 mV (SD, n = 5). Eggs inseminated in 51 mM Na+ sea water become polyspermic, indicating that polyspermy prevention depends on an electrically-mediated mechanism. The electrical block is required for about 60 min, since transfer to 51 mM Na+ sea water during this period results in polyspermy. During the first hour following fertilization, the egg is also developing a permanent, nonelectrical block; the degree of polyspermy which results upon transfer to low Na+ sea water decreases progressively with time. The permanent block appears to be at the level of the egg plasma membrane or glycocalyx, since the egg envelope is not a barrier to sperm penetration, nor does its removal induce polyspermy. Electron micrographs show no obvious changes in the morphology of the extracellular layers, plasma membrane or cortex of the egg after fertilization.     

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.     

An electrically mediated block to polyspermy in the primitive urodele Hynobius nebulosus and phylogenetic comparison with other amphibians

At fertilization, the egg of the primitive urodele, Hynobius nebulosus, produced a fertilization potential which rose from -12 to +47 mV. A similar activation potential was elicited by pricking with a needle, by applying A23187, or by electric shock. The potential change was mediated by an increased permeability to Cl-. Clamping the egg's membrane potential at +40 mV blocked fertilization, while clamping at +20 mV induced polyspermy. These results indicated the occurrence of an electrical polyspermy block, typical of anurans, but atypical of urodeles. Furthermore, Hynobius eggs fertilized by natural mating incorporated only one sperm nucleus, and experimentally polyspermic eggs underwent multipolar division. Accessory sperm did not degenerate in the egg cytoplasm, indicating lack of an intracellular polyspermy block. By comparison, fertilization of Bufo japonicus (anuran) was also voltage dependent, whereas that of Cynops pyrrhogaster (urodele) was voltage independent. Thus polyspermy prevention mechanisms in Hynobius closely resemble those of anuran amphibians and differ from those of higher urodeles.     

Preventing polyspermy in mammalian eggs—Contributions of the membrane block and other mechanisms

The egg's blocks to polyspermy (fertilization of an egg by more than one sperm) were originally identified in marine and aquatic species with external fertilization, but polyspermy matters in mammalian reproduction too. Embryonic triploidy is a noteworthy event associated with pregnancy complications and loss. Polyspermy is a major cause of triploidy with up to 80% of triploid conceptuses being the result of dispermic fertilization. The mammalian female reproductive tract regulates the number of sperm that reach the site of fertilization, but mammals also utilize egg‐based blocks to polyspermy. The egg‐based blocks occur on the mammalian egg coat (the zona pellucida) and the egg plasma membrane, with apparent variation between different mammalian species regarding the extent to which one or both are used. The zona pellucida block to polyspermy has some similarities to the slow block in water‐dwelling species, but the mammalian membrane block to polyspermy differs substantially from the fast electrical block that has been characterized in marine and aquatic species. This review discusses what is known about the incidence of polyspermy in mammals and about the mammalian membrane block to polyspermy, as well as notes some lesser‐characterized potential mechanisms contributing to polyspermy prevention in mammals.     

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.     

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.     

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.     

Studies of the mechanism of the electrical polyspermy block using voltage clamp during cross-species fertilization

Prevention of polyspermic fertilization in sea urchins (Jaffe, 1976, Nature (Lond.). 261:68-71) and the worm Urechis (Gould-Somero, Jaffe, and Holland, 1979, J. Cell Biol. 82:426-440) involves an electrically mediated fast block. The fertilizing sperm causes a positive shift in the egg's membrane potential; this fertilization potential prevents additional sperm entries. Since in Urechis the egg membrane potential required to prevent fertilization is more positive than in the sea urchin, we tested whether in a cross-species fertilization the blocking voltage is determined by the species of the egg or by the species of the sperm. With some sea urchin (Strongylocentrotus purpuratus) females, greater than or equal to 90% of the eggs were fertilized by Urechis sperm; a fertilization potential occurred, the fertilization envelope elevated, and sometimes decondensing Urechis sperm nuclei were found in the egg cytoplasm. After insemination of sea urchin eggs with Urechis sperm during voltage clamp at +50 mV, fertilization (fertilization envelope elevation) occurred in only nine of twenty trials, whereas, at +20 mV, fertilization occurred in ten of ten trials. With the same concentration of sea urchin sperm, fertilization of sea urchin eggs occurred, in only two of ten trials at +20 mV. These results indicate that the blocking voltage for fertilization in these crosses is determined by the sperm species, consistent with the hypothesis that the fertilization potential may block the translocation within the egg membrane of a positively charged component of the sperm.     

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.     

The fast block against polyspermy in fucoid algae is an electrical block

Fertilization potentials in Pelvetia fastigiata, Fucus vesiculosus, and Fucus ceranoides were studied to examine whether eggs of fucoid algae have an electrical block against polyspermy. The resting potential of eggs of all species was about -60 mV, depolarizing, respectively, to -24 +/- 5 mV (SD, n = 9) for 7.5 +/- 2.1 (n = 8) min, -26 +/- 5 (n = 9) mV for 6.4 +/- 2.3 (n = 9) min, and -24 +/- 6 (n = 5) mV for 6.7 +/- 1.9 (n = 4) min. The depolarization was slower, and the fertilization potential was about 10 mV more negative in eggs of both F. vesiculosus and Pelvetia fertilized in 45-mM Na+ ASW; many of these eggs were polyspermic. Steady current was passed through unfertilized eggs of F. vesiculosus prior to insemination to test the potential dependence of fertilization. Eggs (n = 10) bound sperm at all potentials tested (-45 to -23 mV), but fertilization was prevented if eggs were held at potentials more positive than -45 to -37 mV. Eggs underwent a second depolarization if artificially hyperpolarized to potentials more negative than -50 mV immediately after the rise of a normal fertilization potential. Thus, fucoid eggs have an electrical fast block against polyspermy. Only in F. ceranoides does the formation of the cell wall after fertilization appear to be fast enough (i.e., 3-6 min postfertilization versus at 10-15 min in F. vesiculosus and P. fastigiata) to replace the fertilization potential as a polyspermy block. Nonfertilizing fucoid sperm swim away from the egg surface by 1-3 min after rise of the fertilization potential. This suggests that there is another "intermediate block" against polyspermy.     

Mechanistic studies of the plasma membrane block to polyspermy in mouse eggs

The mechanisms responsible for the plasma membrane associated block to polyspermy in mouse eggs were studied. Reinsemination experiments using zona-free eggs indicated that, after fertilization, the egg plasma membrane is altered such that sperm binding to the egg plasma membrane is blocked, except in the region of the second polar body. Activation of the egg with either ethanol or strontium chloride did not result in a block to polyspermic penetration, as artificially activated eggs displayed identical penetration levels as to nonactivated control eggs. The penetrability of activated eggs was not altered by the presence or absence of the zona pellucida during activation. Lectin staining for egg cortical granule material indicated that activation did cause cortical granule exocytosis; however, activated eggs remained penetrable. These data support the following conclusions: (1) an alteration in the ability of the egg plasma membrane to allow sperm adherence accounts for the block to polyspermy; (2) establishment of the plasma membrane block to polyspermy is sperm dependent, since artificial egg activation does not result in a block response; (3) the contents of the egg's cortical granules do not play a role in the establishment of the plasmalemma block response. © 1993 Wiley-Liss, Inc.     

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.     

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.     

The spike component of the fertilization potential in the toad, Bufo japonicus: changes during meiotic maturation and absence during cross-fertilization

Immature oocytes of the toad, Bufo japonicus, inseminated between first- and second-meiotic metaphase, exhibited polyspermy. Monospermy occurred when the oocytes had reached second-meiotic metaphase. Electrical recording during insemination of the immature oocyte showed fast-rising and slow-rising spikes followed by a gradual shift to a positive membrane potential. The number of fast spikes in each oocyte corresponded well with the number of sperm observed in cytological sections. Mature oocytes elicited one fast spike followed by a rapid rise to a positive plateau. Ion-substitution experiments indicated that, like the plateau, the initial fast spike is mediated mainly by increased permeability of the oocyte plasma membrane to halides such as Cl- or I-. When inseminated with sperm of the newt, Cynops pyrrhogaster, mature Bufo oocytes exhibited polyspermy accompanied by a gradual hyperpolarization and a slowly developing positive plateau, without the fast spike that occurs in self-species fertilization. These results indicated that the spike component of the fertilization potential can be dissociated from the plateau component, and may be elicited by different mechanisms.     

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.     

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.     

Calcium influx mediates the voltage-dependence of sperm entry into sea urchin eggs

Sperm entry was monitored in voltage-clamped sea urchin eggs following insemination in a variety of artificial seawaters. In regular seawater, maintaining the membrane potential at increasingly negative values progressively inhibits sperm entry. Reducing [Ca(2+)](o) relieves the inhibition, shifting the sperm entry vs voltage relationship toward more negative potentials. Raising [Ca(2+)](o) shifts the relationship in the other direction. Large changes in [Na(+)](o) or [Mg(2+)](o) do not affect sperm entry although changing [Na(+)](o) dramatically changes the currents following sperm attachment. Applying one of seven different calcium channel blockers or replacing Ca(2+) with Ba(2+) or Sr(2+) or microinjecting calcium chelators into the cytoplasm relieves the block to sperm entry at negative potentials. We conclude that the block to sperm entry at negative potentials is mediated by calcium which crosses the membrane and acts at an intracellular site.     

The ascidian egg envelope in fertilization: structural and molecular features

In this report, unpublished and recent findings concerning the structure and function of the ascidian egg coat are compiled in context with fertilization. In the initial stage of ascidian fertilization, sperm interact with a complex egg investment that consists of a layer of follicle cells attached to an acellular vitelline coat. Increasing evidence exists that ascidian sperm are activated at their encounter with the follicle cells. The molecular basis of sperm-follicle cell interactions is discussed in context with sperm binding, membrane proteins and sperm bound glycosidase. The model that suggests a block to polyspermy established by glycosidase released from the follicle cells on fertilization is evaluated and compared with assured facts. Although a number of questions remain to be answered, our recent findings that a cloned beta-hexosaminidase from P. mammillata binds exclusively to the follicle cells of unfertilized but not fertilized eggs, indicates that the follicle cells participate in the block to polyspermy. A dual function, mediating sperm activation and a block to polyspermy attributes to the ascidian follicle cells a key position in fertilization.     

Insemination of rabbit eggs is associated with slow depolarization and repetitive diphasic membrane potentials

The plasma membrane of the rabbit egg allows only one sperm to enter the egg during fertilization, but the mechanism of this block to polyspermy is unknown. Electrophysiology and in vitro fertilization techniques were employed in this study to investigate the possibility that a voltage block to polyspermy exists in rabbit eggs. Ovulated zona-intact eggs had a mean membrane potential of -71 +/- 2.1 mV (interior negative). A stereotypic response occurred 12-135 min following in vitro insemination in 19 of 40 eggs. Association of this stereotypic response with the appearance of pronuclei suggested that the electrical response was related to some interaction of gametes. This response consisted of a slow transient 8 +/- 1.5 mV depolarization upon which were superimposed up to 36 repetitive diphasic insemination potentials. Each potential consisted of a brief 2.0 +/- 0.44 mV hyperpolarization followed by a slow 2.5 +/- 0.45 mV depolarization. The small amplitude of the stereotypic response when compared with the large variation of resting potentials suggested that the response was insufficient to block polyspermy by a mechanism dependent upon the magnitude of the rabbit egg membrane potential.     

Ionic mechanism of the fertilization potential of the marine worm, Urechis caupo (Echiura)

Microelectrode and tracer flux studies of the Urechis egg during fertilization have shown: (a) insemination causes a fertilization potential; the membrane potential rises from an initial level of -33 +/- 6 mV to a peak at +51 +/- 6 mV (n = 16), falls to a plateau of about +30 mV, then returns to the original resting potential 9 +/- 1 min (n - 10) later; (b) the fertilization potential results from an increase in Na+ permeability, which is amplified during the first 15 s by a Ca++ action potential; (c) the maximum amplitude of the fertilization potential, excluding the first 15 s, changes by 51 mV for a 10-fold change in external [Na+]; (d) in the 10 min period after insemination, both Na+ and Ca++ influxes increase relative to unfertilized egg values by factors of 17 +/- 7 (n = 6) and 34 +/- 14 (n = 4), respectively; the absolute magnitude of the Na+ influx is 16 +/- 6 times larger than that of Ca++; (e) in the absence of sperm these same electrical and ionic events are elicited by trypsin; thus, the ion channels responsible must preexist in the unfertilized egg membrane; (f) increased Na+ influx under conditions of experimentally induced polyspermy indicates that during normal monospermic fertilization, only a fraction of available Na+ channels are opened; we conclude that these channels are sperm-gated; (g) Ca++ influx at fertilization is primarily via the membrane potential-gated channel, because kinetics are appropriate, and influx depends on potential in solutions of varying [Na+], but is independent of number of sperm incorporations in normal sea water.