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.     

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+.     

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.     

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.     

Voltage Noise Changes during Monospermic and Polyspermic Fertilization of Mature Eggs of the Anuran, Rana temporaria

The electrical response of mature anuran eggs to the fertilizing sperm consists of a rapid depolarization and a decrease in resistance of the plasma membrane (fertilization potential) and serves as a fast block to polyspermy. We report here that the fertilization potential, previously thought to be the earliest electrical response of the egg, is preceded in Rana temporaria by changes in voltage noise. Voltage noise was recorded after insemination and compared in monospermic and NaI-induced polyspermic eggs. Fertilization potential in monospermic eggs arised at 1 min 45 sec to 2 min 15 sec after insemination, and that in NaI-induced polyspermic eggs did at 3 min to 3 min 30 sec after insemination. However, the increase in voltage noise was detected at the similar time (1–2 min 30 sec) after insemination in both the eggs. The duration of voltage noise increase before the fertilization potential was larger in polyspermic eggs (50–105 sec) than in monospermic eggs (10–40 sec). Polyspermic fertilization in Rana temporaria induced by NaI was checked by visualizing multiple sperm entry sites with the scanning microscope. The process of sperm entry and the development of the fertilization body are similar to those occurring with monospermic fertilization; furthermore all supernumerary sperm fuse only with the animal hemisphere of the egg. Although the physiological basis of the changes in voltage noise is unclear, these alterations appear to be the earliest electrical response to sperm yet reported.     

Benzohydroxamic acid induces polyspermic fertilization in the sea urchin Arbacia punctulata

Benzohydroxamic acid (BHA) is a competitive inhibitor of the sea urchin sperm peroxidase. We now report that addition of BHA to fertilization cultures of Arbacia punctulata promotes polyspermy. This effect is dose and sperm density dependent. The cortical reaction (elevation of the fertilization envelope) is not retarded by BHA. BHA must be added to the cultures before the eggs complete the cortical reaction at 60 sec post insemination in order to induce polyspermy. Since sea urchin eggs release H2O2 during the cortical reaction at fertilization, these findings support our hypothesis that the sperm peroxidase has a functional role in helping to prevent polyspermy.     

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.     

SED1 function during mammalian sperm-egg adhesion

A prerequisite for successful fertilization is the species-specific binding of sperm to the extracellular coat of the egg. Gamete binding triggers the release of sperm hydrolytic enzymes that digest a path through the egg coat, thus bringing sperm into proximity with the egg plasma membrane where gamete fusion occurs. Although some components of the sperm membrane and the egg coat that participate in sperm-egg interactions have been identified, results from targeted deletions and gene substitutions indicate that other, as yet unidentified, gamete receptors must contribute to sperm-egg binding. Recent studies implicate the bi-motif protein, SED1, as being required for successful sperm-egg adhesion in mouse. SED1 contains Notch-like EGF repeats as well as discoidin/F5/8 complement domains--motifs that mediate a variety of cell-cell and cell-matrix interactions. SED1's ability to promote gamete adhesion resides within its two discoidin/F5/8C domains, which are able to dock to substrates as diverse as phospholipid membranes and extracellular matrices. SED1 is also expressed in a wide range of tissues and epithelia, where it may function similarly as an adhesive protein facilitating cell-cell and/or cell-matrix interactions.     

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.     

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.     

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.     

Concanavalin A Modifies Allo-specific Sperm-Egg Interactions in the Ascidian, Ciona intestinalis

In the self sterile ascidian, Ciona intestinalis , the spermatozoa rarely bind to the vitelline coat of autologous eggs and never penetrate it. We report here that concanavalin A (ConA), a lectin recognizing mannose or glucose residues of carbohydrates, can modify these self- and nonself-specific sperm-egg interactions. When eggs were pretreated with 0.1–0.5 mg/ml of ConA, about two thousand spermatozoa became attached to the autologous vitelline coat within five minutes of insemination. The effect of ConA was not modified by the addition of D-mannose or pretreatment of spermatozoa with ConA, showing that ConA does not function merely as a ligand bridging the sperm and vitelline coat. In contrast to the marked enhancement of sperm-egg binding, ConA did not facilitate the penetration of spermatozoa through the autologous vitelline coat. Even in non-autologous insemination, it blocked the sperm penetration and, consequently, fertilization did not occur, as shown by Rosati et al. (1978). D-Mannose, when mixed with ConA in advance, completely abolished this inhibitory effect of ConA. Lotus agglutinin, a fucose-binding lectin, was less effective and wheat germ agglutinin and soy bean agglutinin had no effect on sperm entry in the perivitelline space. The results of this study are discussed in relation to the possible involvement of mannosyl and/or glucosyl glycoconjugates in allo-specific sperm-egg interactions.     

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.     

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.     

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.     

Effects of motility inhibitors during sea urchin fertilization : Microfilament inhibitors prevent sperm incorporation and restructuring of fertilized egg cortex, whereas microtubule inhibitors prevent pronuclear migrations

The sensitivity of specific stages of fertilization to microfilament inhibitors (cytochalasins B (CB), D (CD), and E (CE) and phalloidin) and to inhibitors of microtubule assembly (colcemid (CMD), colchicine (CLC), griseofulvin (GSF), maytansine (MAY), nocodazole (NCD), podophyllotoxin (PDP), and vinblastine (VB)) was investigated using differential interference contrast, time-lapse video microscopy of the sea urchin Lytechinus variegatus. Cytochalasins (CDCE>CB) will prevent sperm incorporation if added prior to or simultaneous with insemination. Sperm-egg fusion and the cortical reaction appear normal, but then the subsequent elevation of the fertilization coat lifts and eventually detaches the ‘fertilizing’ sperm from the egg plasma membrane. When the cytochalasins are added after fusion, the forming fertilization cone is rapidly resorbed, and the lateral displacement of the sperm along the egg cortex is terminated; the pronuclear migrations and mitoses occur normally though cytokinesis is never observed. Cytochalasin treatment before or within 2 min of insemination results in the development of aberrant egg cortices, whereas cytochalasin treatments after 2 min post-fusion have little effect. Phalloidin results in large and long-lasting fertilization cones and a retardation of the rate of sperm incorporation. Eggs exposed to any of the microtubule inhibitors 15 min prior to insemination will incorporate the spermatozoon, though the formation of the sperm aster and the accompanying pronuclear migrations are prevented. Interestingly, the final stage of sperm incorporation involving a lateral displacement of the sperm along the egg cortex is greater (27.1 vs 12.4 μm in controls) and faster (5.4 vs 3.5 μm/min in controls) in microtubule-inhibited eggs. GSF and VB, which readily permeate fertilized eggs, will prevent the formation of the sperm aster if added 3 min after sperm-egg fusion, they will prevent the migration of the female pronucleus if added 5 or 7 min after sperm-egg fusion, pronuclear centration if added 10 min post-fusion, and syngamy if added 12 min post-fusion. CLC- or CMD- treated eggs will develop normally if these drugs are photochemically inactivated with 366 nm light within 4 min post-fusion, arguing that sperm incorporation is completely independent of assembling microtubules. These results indicate that microfilament inhibitors will prevent sperm incorporation and the restructuring of the fertilized egg cortex, and that microtubule inhibitors will prevent the formation and functioning of the sperm aster during the pronuclear migrations; an interplay between cortical microfilaments and cytoplasmic microtubules appears required for the successful completion of fertilization.     

The risk of polyspermy in three congeneric sea urchins and its implications for gametic incompatibility and reproductive isolation

Developmental failure caused by excess sperm (polyspermy) is thought to be an important mechanism driving the evolution of gamete-recognition proteins, reproductive isolation, and speciation in marine organisms. However, these theories assume that there is heritable variation in the susceptibility to polyspermy and that this variation is related to the overall affinity between sperm and eggs. These assumptions have not been critically examined. We investigated the relationship between ease of fertilization and susceptibility to polyspermy within and among three congeneric sea urchins. The results from laboratory studies indicate that, both within and among species, individuals and species that produce eggs capable of fertilization at relatively low sperm concentrations are more susceptible to polyspermy, whereas individuals and species producing eggs that require higher concentrations of sperm to be fertilized are more resistant to polyspermy. This relationship sets the stage for selection on gamete traits that depend on sperm availability and for sexual conflict that can influence the evolution of gamete-recognition proteins and eventually lead to reproductive isolation.     

CaMKII can participate in but is not sufficient for the establishment of the membrane block to polyspermy in mouse eggs

Fertilization triggers initiation of development and establishment of blocks on the egg coat and plasma membrane to prevent fertilization by multiple sperm (polyspermy). The mechanism(s) by which mammalian eggs establish the membrane block to polyspermy is largely unknown. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) appears to be the key regulator of several egg activation events (completion of meiosis, progression to embryonic interphase, recruitment of maternal mRNAs). Since sperm-induced increases in cytosolic Ca(2+) play a role in establishment of the membrane block to polyspermy in mouse eggs, we hypothesized that CaMKII was a Ca(2+)-dependent effector leading to this change in egg membrane function. To test this hypothesis, we modulated CaMKII activity in two ways: activating eggs parthenogenetically by introducing constitutively active CaMKIIalpha (CA-CaMKII) into unfertilized eggs, and inhibiting endogenous CaMKII in fertilized eggs with myristoylated autocamtide 2-related inhibitory peptide (myrAIP). We find that eggs treated with myrAIP establish a less effective membrane block to polyspermy than do control eggs, but that CA-CaMKII is not sufficient for membrane block establishment, despite the fact that CA-CaMKII-activated eggs undergo other egg activation events. This suggests that: (1) CaMKII activity contributes to the membrane block, but this not faithfully mimicked by CA-CaMKII and furthermore, other pathways, in addition to those activated by Ca(2+) and CaMKII, also participate in membrane block establishment; (2) CA-CaMKII has a range of effects as a parthenogenetic trigger of egg activation (high levels of cell cycle resumption, modest levels of cortical granule exocytosis, and no membrane block establishment).     

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.