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 frequency of calcium oscillations in mouse eggs at fertilization is modulated by the number of fused sperm.

In a variety of calcium signaling systems, the frequency of intracellular calcium oscillations is physiologically important. Probably multiple factors control the frequency of calcium oscillations in the egg after fertilization and many of these remain to be identified. In this study, we present the first rigorous set of data showing that monospermic fertilization is important for setting the physiological calcium oscillation frequency. Recordings in 152 zona-free eggs show that the general pattern of the calcium oscillations is identical in monospermic and polyspermic eggs; however, the oscillation frequency is higher in polyspermic eggs (P < 10(-6)). The frequency of the late oscillations increases with the number of sperm heads incorporated: 5.2 +/- 0.3 spikes per hour (mean +/- SEM; n = 55) in monospermic eggs, 6.6 +/- 0.3 (n = 62) in dispermic eggs, 8.7 +/- 0.7 (n = 23) in trispermic eggs, and 8.9 +/- 0.9 (n = 12) in eggs with four or more sperm heads. The frequency of the early oscillations is also increased in polyspermic eggs. Seventy-eight additional eggs were divided into two groups and inseminated with two different sperm concentrations ("low" and "high") to obtain one group mainly monospermic and the other mainly polyspermic. The two groups of eggs oscillated at different frequencies (P < 10(-5)). These data rule out the possibility of an egg effect in which some eggs would have the dual properties of oscillating faster and of being able to fuse with several sperm cells. These data instead suggest that the sperm modulates the frequency of the oscillations in a dose-dependent manner.     

Time Sequence of Early Events in Fertilization in the Medaka Egg

The time sequence of early events in fertilization was examined in eggs of the medaka Oryzias latipes . The mean time after insemination required for sperm attachment to the egg surface through the micropyle depended on sperm concentrations. It was 3 ± 1 sec with a range from 1 to 6 sec after insemination when concentration of spermatozoa was high (about 2 × 10⁸/ml at 23°–25°C). The mean time from sperm attachment until cessation of its movement on the egg surface was 4 ± 1 sec with a range from 1 to 9 sec. Small cortical alveoli at the animal pole region within 15 μm of the sperm attachment point began to undergo exocytosis 9 ± 0.3 sec (range 5–16 sec) after sperm attachment. The velocity at which the exocytosis wave propagated increased from the earliest initiation point of exocytosis up to the 100 μm area, and became constant at about 12 μm/sec from 100 μm to 500 μm from the sperm attachment point. The present results suggest that at the time of fertilization in the fish egg, exocytosis of small cortical alveoli in the area about 15 μm away from the sperm attachment point occurs simultaneously.     

The Ca2+ 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 Ca(2+) increase occurs as a Ca(2+) wave at each sperm entry site in the polyspermic egg. Some Ca(2+) waves are preceded by a transient spike-like Ca(2+) increase, probably caused by a tryptic protease in the sperm acrosome at the contact of sperm on the egg surface. The following Ca(2+) wave was induced by a sperm factor derived from sperm cytoplasm after sperm-egg membrane fusion. The Ca(2+) increase in the isolated, cell-free cytoplasm indicates that the endoplasmic reticulum is the major Ca(2+) store for the Ca(2+) 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.     

The kinetics of monospermic and polyspermic fertilization in free-spawning marine invertebrates

The equation of Vogel et al. (1982) is widely used in fertilization studies of free-spawning marine invertebrates to predict the percentage of viable eggs that will be fertilized at any specified levels of gamete concentration and contact time. Here, the random collision model that underlies the Vogel et al. equation is extended to distinguish between monospermic and polyspermic fertilization, and separate equations for the percentages of monospermic and polyspermic fertilization are obtained. These equations provide an explanation for empirical observations which have shown a decreased percentage of successful egg development at high sperm concentrations. Comparison is made with an earlier heuristic attempt (Styan, 1998) to predict the extent of polyspermic fertilization, and it is found that this earlier method can underestimate the percentage of polyspermic fertilization by up to 10 percent. Moreover, the approach used here retains the flexibility to model changes in sperm concentration due to dispersal mechanisms, and is able to model different mechanisms for the block to polyspermy.     

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.     

Sperm Incorporation Independent of Fertilization Cone Formation in the Danio Egg

The responses of the egg to insemination in a modified Fish Ringer's solution (FRS) were examined in eggs of the zebrafish ( Brachydanio rerio ) primarily by scanning electron microscopy. FRS is a physiological saline which temporarily inhibits parthenogenetic activation of the egg for 5–8 min. Spermatozoa were collected in a small volume of water and pipetted over eggs in FRS. Eggs inseminated in FRS typically incorporated the fertilizing sperm within 3–4 min. Inseminated cells showed an absence of a fertilization cone and no cortical granule exocytosis. The deep conical depression in the egg surface beneath the micropyle remained unaltered. Control eggs inseminated in tank water developed a large fertilization cone during sperm incorporation. Occasionally, eggs inseminated in water were observed to incorporate the entire sperm head prior to egg activation. Our results corroborate earlier findings showing that in the zebrafish, cortical granule exocytosis, fertilization cone formation and elevation of the sperm entry site are not triggered by the fertilizing sperm in experimental conditions (18, 19). Furthermore, sperm incorporation requires neither egg activation nor formation of a fertilization cone in this fish.     

Electron microscopic observations on sperm entry and pronuclear formation in naked eggs of the rose bitterling in polyspermic fertilization

Unfertilized eggs of the rose bitterling (Rhodeus ocellatus ocellatus) were squeezed out of females that had an elongated ovipositor and were dechorionated mechanically with fine forceps in physiological saline. The dechorionated eggs were transferred into fresh water then inseminated at once by spermatozoa of the same species. A large number of spermatozoa was found on the surface of eggs that had not yet had cortical reaction following insemination. The surface of the naked eggs responded by formation of many small cytoplasmic protrusions (viz., fertilization cones) at sperm attachment sites. The formed fertilization cones were rosettelike structures formed by the aggregation of some bleblike swellings devoid of microvilli and microplicae. About 10 min after insemination, the fertilization cones retracted, but marks of their presence characterized by less microvilli and microplicae remained in the eggs 15 min after insemination. Many spermatozoa penetrated into the cytoplasm of each naked egg. The sperm nuclear envelope disappeared by means of vesiculation resulting from fusion of the inner and outer membranes. The sperm nucleus decondensed and developed into a larger male pronucleus. Smooth-surfaced vesicles surrounded the decondensing sperm nucleus and formed the new male pronuclear envelope. Sperm mitochondria and flagella were found in the egg 15 min after insemination. The response of the egg surface to sperm entry and pronucleus formation are discussed.     

A long-lasting electrically mediated block, due to the egg membrane hyperpolarization at fertilization, ensures physiological monospermy in eggs of the crab Maia squinado

In response to fertilization, the membrane potential (Em) of the crab egg hyperpolarizes from about -50 mV to about -80 mV in 400 msec. To establish whether this fast hyperpolarization is correlated with physiological polyspermy or conversely mediates an electrical block to polyspermy, we examined the morphological and electrophysiological characteristics of eggs from the crab Maia squinado. Fertilized naturally spawned eggs were found to be physiologically monospermic and their average Em was constant at -77 +/- 0.5 mV. To examine a possible electrical block ensuring this monospermy, unfertilized eggs were voltage clamped at various Em values ranging from +20 to -90 mV, inseminated, and examined morphologically. All eggs clamped at +20 to -65 mV responded by developing a fertilization current, If. It consisted of an outwardly directed K+ current in one or several steps, each caused by a single spermatozoon interacting with the egg membrane. The percentage of eggs clamped at values more negative than -65 mV, which responded at insemination by developing an If, decreased and dropped to 0 at -80 mV. This indicated that the membrane processes occurring during the contact between gametes and eliciting an electrical response by the egg membrane are voltage dependent. Further, the spermatozoon never penetrated into eggs voltage clamped at a Em between +20 and -60 mV and at voltages more negative than -75 mV. Em values between -65 and -75 mV were required for spermatozoon incorporation into the egg, indicating that sperm entry is also voltage dependent. It is proposed that the hyperpolarization of the egg membrane in response to fertilization constitutes a long-lasting electrical block to polyspermy in crab eggs.     

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.     

Fertilization in Discoglossus pictus (Anura). I. Sperm-egg interactions in distinct regions of the dimple and occurrence of a late stage of sperm penetration

The heterogeneity of the egg surface with respect to receptivity to sperm was investigated in Discoglossus pictus; in this species fertilization occurs only in an indentation called the dimple, at the center of the animal hemisphere. Following insemination sperm are seen in the outermost jelly layers and in the lens-shaped jelly plug, converging to the dimple center, D1. A fertilization potential (FP) is recorded 30 sec following insemination. About 30 min after fertilization, when fertilization cones can be detected easily, immotile sperm are found at the center of the cone, where 10 min later they accomplish penetration. After 15 min the cone regresses and the second polar body is extruded. In eggs where the plug was experimentally displaced with respect to the dimple, spermatozoa contacted the sides of the dimple and simple protrusions formed but not cones. Spermatozoa do not elicit a normal FP in these regions but small step depolarizations which may be followed by a gradual rise to a positive plateau potential. Such eggs do not develop. In the protrusions, sperm may be only partially incorporated and the unpenetrated portion appears to degenerate. We conclude that at least two regions exist in the dimple: D1, where the FP is triggered, cones are formed, sperm penetration is fully accomplished and development is initiated; and D2 + D3 where the electrical response is not a normal FP, cones do not form, total sperm penetration does not occur, and development is not initiated.     

Voltage clamp studies of fertilization in sea urchin eggs: I. Effect of clamped membrane potential on sperm entry,activation, and development

To analyze the role of the activation potential (a positive shift of the membrane potential which occurs following sperm attachment) in fertilization and development of the sea urchin egg, unfertilized Lytechinus variegatus eggs were voltage clamped at membrane potentials (E_m) from +20 to ?90 mV, and then inseminated. Either a fast two electrode voltage clamp, or a single electrode switched voltage clamp was used. The clamp was maintained for 3 to 15 min after initiation of a conductance increase. At E_m more positive than +18 mV, even though many sperm may attach, the egg remains completely inert (Jaffe, Nature (London)261, 68–71, 1976). At E_m from +17 to ?90 mV, all inseminated eggs elevate normal fertilization envelopes, although substantially increased concentrations of sperm are required at E_m from +17 to +12 mV. Whether cleavage occurs depends on the clamped E_m. When clamped at E_m from +17 to ?25 mV, 100% of activated eggs cleave. However, when clamped at E_m from ?26 to ?75 mV the percentage of activated eggs which cleave progressively decreases. At clamped E_m between ?76 and ?90 mV, none of the activated eggs cleave. All monospermic voltage clamped eggs that cleave develop to normal swimming blastulae. In all eggs that fail to cleave (clamped at E_m more negative than ?30 mV), sperm penetration is blocked, the sperm is lifted off the egg surface as the fertilization envelope rises, and a sperm aster never forms. Preventing formation of the fertilization envelope by prior disruption of the vitelline layer with dithiothreitol does not promote entry of the sperm. In conclusion, preventing the depolarization normally associated with fertilization suppresses sperm entry in the sea urchin egg, yet activation proceeds. Present evidence suggests an effect of the electrical field across the plasma membrane in suppressing sperm entry.     

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.     

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.     

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.     

Voltage response to fertilization and polyspermy in sea urchin eggs and oocytes

Successful collision rates of sperm with eggs and oocytes of the sea urchins Psammechinus microtuberculatus and Paracentrotus lividus have been studied using an electrophysiological method. A monospermic response in eggs consists of a 1- to 2-mV step depolarization of the egg plasma membrane accompanied by an increase in voltage noise. The step precedes the main positive-going depolarization by approximately 13 sec at room temperature. If other successful collisions occur during this 13-sec period (indicated by additional steps), the egg is polyspermic. It is shown by direct observation that each step depolarization signifies the entry of a single sperm. No evidence for an electrically mediated fast block was found. The average rate of successful sperm-egg encounters increases with sperm density, although individual steps appear to occur randomly. Step depolarizations also occur in oocytes, however, they usually decay after several seconds and are not followed by a large, positive-going depolarization. The rate of occurrence of such steps increases with sperm density over the range 10⁵ to 10⁹ sperm/ml. The original evidence of Rothschild and Swann for a fast partial block is compared with a model of polyspermy suggested by our experiments. Reasonable agreement between our method of counting successful collisions (in oocytes and eggs) and the method used by Rothschild and Swann (for eggs) was obtained for sperm densities below 10⁶/ml. The results diverge for higher sperm densities, our method giving higher values. A test for the hypothesis of a fast partial block to polyspermy is suggested, using our method of counting successful sperm-egg collisions.     

Electrical characteristics of ascidian egg fragments

Fragments of ascidian eggs, but at random in any plane and ranging in size from 10 to 90% of the total egg volume, displayed the electrical characteristics of the intact egg, having a resting potential of -86 mV and giving rise to an action potential upon stimulation by electrical current injection. Following insemination, the fragments generated fertilization potentials, comparable to those of intact eggs, although the repolarization phase was shorter. Our data show that there are sufficient ion channels throughout the egg surface to generate action potentials and fertilization potentials in excised egg fragments, irrespective of their global origin. Furthermore, the fertilizing spermatozoon is capable of activating fertilization channels in areas of the egg plasma membrane not destined for sperm entry.     

Effects of electrical stimulation before or after in vitro fertilization on sperm penetration and pronuclear formation of pig oocytes

The effects of exposure of pig oocytes to an electrical pulse on sperm penetration and pronuclear formation were determined before or after in vitro fertilization (IVF). After in vitro maturation (IVM) or after collection from oviducts of unmated gilts, pig oocytes either were not exposed or were exposed to an electrical pulse (a 10 sec pulse at 4.0 V mm^?1 AC followed by a 30 μsec pulse at 120 V mm^?1 DC), followed 30 min later by IVF. The incidence of male pronuclear formation of both IVM and in vivo-matured oocytes at 12 hr after insemination was decreased from 59% and 100%, respectively, to 2% and 36%, respectively, by the electrical pulse, but the penetration rates (88–100%) and polyspermic rates (79–100%) were not affected by exposure to an electrical pulse. Similarly, when pig IVM oocytes were exposed to an electrical pulse at 6 hr after insemination, electrical activation did not decrease penetration rates (93% vs. 90%), polyspermic rates (83% vs. 91%), or number of spermatozoa in penetrated oocytes (4.0 ± 0.5 vs. 4.6 ± 0.5) but did decrease the rate of male pronuclear formation from 58% to 18%. When oocytes were examined at 6 hr after insemination, 75% of them had been penetrated and resumed meiotic progression, but all sperm heads in penetrated oocytes were fully condensed or only partially decondensed. The percentage of penetrated eggs with multiple female pronuclei was increased when oocytes were exposed to an electrical pulse in all experimental series. In summary, electrical activation of pig oocytes before or just after IVF does not prevent sperm penetration but does inhibit male pronuclear formation and increases the formation of multiple female pronuclei. © 1993 Wiley-Liss, Inc.     

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.     

Ca2+-induced change of dorsal-ventral polarity in frog (Rana temporaria) eggs

Abstract. The effect of local Ca²⁺ administration 10–20 min after fertilization and during artificial activation was examined in Rana temporaria eggs. Ca²⁺ was injected into the pigmented region near the boundary between pigmented and unpigmented domains. The locations of egg gray crescent (GC) and dorsal lip of blastopore (DLB), as predictors of the dorsal region in embryos, as well as the measurements of angles between GC middle and sperm entry site were observed. In more than 70% of the cases, microinjection of Ca^{2 +} into subcortical cytoplasm and egg pricking in high-Ca^{2 +} solutions induced GC and DLB formation near the injection site. The formation of Ca²⁺-induced GC occurred mostly as in control eggs. In addition premature displacement of the egg surface was observed near the prick site in high-Ca^{2 +} solutions. GC formation occurred by displacement of the pigmented surface in the same direction as earlier wound translocations. These results show that Ca²⁺ injection determines the direction of the surface movement.