Edward L. Chambers

The activation of sea urchin eggs by the divalent ionophores A23187 and X-537A.

Abstract The divalent ionophores A23187 and X-537A induce parthenogenesis in sea urchin eggs. This results from their ability to mobilize intracellular Ca 2+ , which is implicated in both artificial parthenogenesis as well as the natural fertilization process. A23187 causes expulsion of cortical granules and elevation of the fertilization membrane within 0.5–9 min followed by an initiation of cell cleavage. The broader spectrum ionophore X-537A is less potent, but the production of cytoplasmic aberrations are more apparent. In contrast to the sperm-activated egg, the initial phase of ionophore induced activation is accompanied either by relatively insignificant changes in membrane resistance, or an increase.

Correlative ultrastructural and electrophysiological studies of sperm-egg interactions of the sea urchin, Lytechinus variegatus☆

The sequence of ultrastructural events following the onset of the sperm-induced conductance increase in eggs of the sea urchin, Lytechinus variegatus, was investigated. Eggs voltage clamped at -20 mV were fixed 1 to 20 sec after onset of the conductance increase caused by single sperm. Continuity between the plasma membranes of the sperm and egg was first detected 5 sec after onset of the conductance increase. The earliest stages of formation of the fertilization cone coincided with the establishment of continuity of the gamete plasma membranes. At 6 to 8 sec after the initial conductance increase cortical granule dehiscence was first observed in the immediate vicinity where continuity of the gamete plasma membranes had occurred. These observations are consistent with the conclusion that opening of ion channels at fertilization precedes fusion of the sperm and egg plasma membranes, while exocytosis of cortical granules is initiated following fusion of the sperm and egg plasma membranes.

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 (Em) 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 Em more positive than +18 mV, even though many sperm may attach, the egg remains completely inert (Jaffe, Nature (London) 261, 68-71, 1976). At Em from +17 to -90 mV, all inseminated eggs elevate normal fertilization envelopes, although substantially increased concentrations of sperm are required at Em from +17 to +12 mV. Whether cleavage occurs depends on the clamped Em. When clamped at Em from +17 to -25 mV, 100% of activated eggs cleave. However, when clamped at Em from -26 to -75 mV the percentage of activated eggs which cleave progressively decreases. At clamped Em 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 Em 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.

Voltage clamp studies of fertilization in sea urchin eggs: II. Current patterns in relation to sperm entry, nonentry, and activation

Following attachment of a sperm to the surface of a sea urchin egg clamped at a membrane potential (Vm) more positive than +17 mV, no changes in membrane conductance can be detected, the sperm does not enter egg, and no morphological changes can be detected. At Vm from +17 to -100 mV three characteristically different types of current profiles are observed: Type I are activation currents in eggs penetrated by a sperm. These have three phases, which occur in all eggs clamped at Vm from +17 to -20 mV and in decreasing percentages at clamped Vm more negative than -20 mV (to -75 mV). Complete fertilization envelopes are elevated, relatively large mound-shaped fertilization cones form, and the eggs develop to normal embryos. Type II are sperm transient currents in eggs not penetrated by a sperm, the eggs otherwise remaining in the unfertilized state. These transients are simpler and shorter than type I currents, and are observed only at clamped Vm more negative than -20 mV. Type III are modified activation currents in eggs not penetrated by a sperm. These have three phases, are observed only at clamped Vm more negative than -20 mV, and are the only type of activation current seen at clamped Vm more negative than -75 mV. Complete fertilization envelopes are elevated, the fertilization cones are small and filament-like, and the eggs fail to cleave. We conclude that (a) the sperm transient currents (type II) and phase 1 of the activation currents (type I and III) are similar events generated by a sperm-initiated localized conductance increase, (b) the abrupt decrease of current which terminates the sperm transients and phase 1 of type III currents results from a turnoff of the sperm-induced conductance increase and signals that the sperm will not enter the egg, and (c) the occurrence of phase 2 during an electrophysiological response induced by a sperm indicates that the egg is activating.

FERTILIZATION IN VOLTAGE-CLAMPED SEA URCHIN EGGS

Following insemination of the voltage-clamped sea urchin egg a characteristic component of the activation current is the initial shoulder with abrupt onset. This is the counterpart of the shoulder of the activation potential, and has a duration of ~12s, equal to that of the latent period. After attaining a maximum, the shoulder of the activation current is followed by a large increase in the inward current culminating in the major peak at ~3Is. One of the most interesting findings in voltage clamp studies of fertilization is that the shoulder phase can be fully, or partially, dissociated from the subsequent phases of the activation current by holding the egg’s membrane potential (Vm) in the neighborhood of the resting value (−70 mV). When the dissociation occurs, either complete or partial, the attached sperm fails to enter the egg. When the dissociation is complete, the isolated shoulder (duration ~11s, now termed a sperm transient current) terminates abruptly, subsequent phases of the activation current do not occur, and the egg otherwise remains in the unfertilized state. When the dissociation is partial, the same isolated shoulder (a step-like current profile, abrupt turn-on and turn-off, duration of ~ I2s) is observed, but from 5 to 25s after return of the current to the holding level, the delayed second or major current phase of a modified activation current occurs, accompanied by delayed elevation of the fertilization envelope. Cleavage fails to occur. Dissociation of the shoulder component from the subsequent phases of the activation current together with suppression of sperm entry is also observed in oocytes (germinal vesicle stage) when single sperm attach. Oocytes have a Vm of –70 mV, and, because of the 15– to 20–fold higher membrane conductance compared to that of eggs, single sperm can depolarize the oocytes’ Vm by only 7 to 8 mV.

Sperm-induced currents at fertilization in sea urchin eggs injected with EGTA and neomycin

Membrane currents were measured in single voltage-clamped sea urchin eggs (Lytechinus pictus and Lytechinus variegatus) that were injected with either EGTA or neomycin and inseminated. Although egg activation and the fertilization calcium wave were prevented by injection of either of these compounds, sperm attached and still elicited inward currents. Sperm-induced currents in EGTA-injected eggs had an abrupt onset, quickly reached a maximum, and then slowly declined in amplitude. Sperm incorporation occurred readily in EGTA-injected eggs. Similar results were obtained with another calcium chelator, BAPTA. In neomycin-injected eggs, sperm-induced currents generally had an abrupt onset and, in contrast to EGTA-injected eggs, the currents usually cut off rapidly. Sperm failed to enter the neomycin-injected eggs and the duration of sperm-induced currents in neomycin-injected eggs was markedly dependent upon the voltage-clamp holding potential, with shorter duration currents occurring at -70 than at -20 mV. The lability of the initial interaction between sperm and egg at negative holding potentials may explain why activation often fails when the egg membrane is voltage clamped at these potentials (Lynn et al., Dev. Biol. 128, 305-323, 1988).

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

Depolarization of the sea urchin eggs 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 oocytes 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.

Nicotinic acetylcholine receptors of the neuronal type occur in the plasma membrane of sea urchin eggs

The addition of acetylcholine (ACh, 100 microliters of 10 microM) to the bath in the vicinity of unfertilised sea urchin eggs (Lytechinus variegatus) suspended in sea water (SW) abruptly depolarises the membrane potential (Vm) of the eggs from the resting value of approximately -70 mV. This results in the firing of the eggs action potential, followed by partial repolarisation. Similar addition of ACh to eggs voltage clamped at -70 mV induces an inward current of abrupt onset with peak amplitude of -1.26 +/- 0.20 nA (SE, n = 81). When the eggs are clamped at a Vm more positive than -70 mV, the peak amplitude of the ACh-induced inward current decreases, becoming 0 at a clamped Vm of approximately -20 mV. Further positive shift of the Vm fails to cause reversal of the current. Oocytes clamped at -70 mV exhibit similar inward current responses following application of ACh. Since ACh stimulates both nicotinic and muscarinic receptors (nACh-R and mACh-R, respectively), the effects of exposing eggs to the agonists and antagonists for each type of receptor were examined. For unfertilised eggs clamped at -70 mV the application of 100 microM (-)-nicotine hydrogen tartrate, an agonist of the nACh-R, induces an inward current response similar to that elicited by 10 microM ACh, but of smaller peak amplitude. In contrast, the application of (+)-muscarine chloride, an agonist of the mACh-R, fails to induce any response. Antagonists of the nACh-R inhibit either the neuronal type of nACh-R or the skeletal muscle type of nACh-R. The effect of the antagonists on the amplitudes of the ACh-induced inward current response was determined by superfusing individual eggs clamped at -70 mV with the desired antagonist dissolved in SW, followed by the addition of 100 microliters of 10 microM ACh in the vicinity of the egg. Mecamylamine chloride, an antagonist of the neuronal nACh-R at a concentration of 1 microM, markedly decreases the response to ACh, while at a concentration of 10 microM the response to ACh is abolished. Hexamethonium chloride, another inhibitor of the nACh-R of the neuronal type, also diminishes the ACh-induced response, but at a concentration of 10 microM the response is not completely abolished. Exposure of eggs to alpha-bungarotoxin, an antagonist of the skeletal muscle nACh-R at concentrations up to 250 nM for periods of 30 min, has no effect on the ACh-induced response. The effects of two antagonists of the mACh-R, atropine sulphate and QNB (R-(-)-3-quinuclidinyl benzilate) were also examined. Exposure of eggs to 1 microM atropine does not affect the ACh-induced response, but at concentrations of 10 microM atropine the amplitude of the ACh-induced inward current is significantly reduced. The exposure of eggs to QNB, a highly specific antagonist of the mACh-R, at concentrations up to 50 nM, has no effect on the ACh-induced response. Consequently, the likely explanation for the inhibitory effect of atropine is that at high concentrations atropine cross-reacts with the nACh-R. These findings reveal the presence in unfertilised sea urchin eggs of an ACh-R resembling the neuronal nACh-R. No evidence could be obtained that these receptors have a role in sperm entry, activation of the egg, or early development.

Stages leading to and following fusion of sperm and egg plasma membranes

The site of gamete interaction of electrophysiologically recorded Lytechinus variegatus eggs, fixed with osmium tetroxide (OsO4) and/or glutaraldehyde (GTA) at varying intervals after the onset of the increase in membrane conductance induced by an attached sperm, has been examined by high-voltage and conventional transmission electron microscopy. Although GTA and a GTA-OsO4 mixture induced different electrical responses, specimens prepared with the two fixatives were ultrastructurally similar. In specimens observed within 5 s of the change in conductance, the acrosomal process projected through the vitelline layer and abutted the egg plasma membrane. A conspicuous layer of bindin surrounded the acrosomal process and connected the sperm to the eggs vitelline layer. In a fortuitous specimen fixed within 4 s following the change in conductance, the area of contact between the gamete plasma membranes possessed a trilaminar structure that separated the eggs and sperms cytoplasms. The morphology of this area of contact was consistent with previously proposed intermediates of membrane fusion. Five to six seconds after the change in conductance, the sperm was connected to the egg via a narrow cytoplasmic bridge that consisted of the former acrosomal process and a projection of the egg cortex. The region of the bridge midway between the fused gametes was encircled by dense material that marked the site of sperm-egg fusion. Gamete interactions in which the activation potential was recorded (unclamped egg) were comparable in time and ultrastructure to events taking place in voltage-clamped eggs except for one major difference. Intact cortical granules (one to three) were observed beneath the tip of the incorporating sperm in unclamped eggs fixed following the onset of the activation potential, whereas all cortical granules dehisced in clamped eggs.

Gamete Interactions and the Initiation of Egg Activation in Sea Urchins

The earliest perceivable response of echinoid eggs to the fertilizing sperm is a transient depolarization, the activation or fertilization potential (Steinhardt et al., 1971; Jaffe, 1976; Chambers and de Armendi, 1979). This change in electrical activity involves the appearance of sperm associated ion channels, which depolarize the plasma membrane, as well as the opening of voltage-dependent calcium channels (Chambers and de Armendi, 1979). These changes constitute Phase 1 (Lynn et al., 1988; Chambers, 1989). In voltage clamped eggs, sperm which induce Phase 1 either enter or fail to enter the egg. If sperm penetration occurs, the inward current of Phase 1, initiating Phase 2, continues to increase; if sperm penetration fails to occur the inward current is abruptly severed. During Phase 2 a large, rapid and transient increase in intracellular free calcium (Steinhardt and Epel, 1974; Steinhardt et al., 1977; Whitaker and Steinhardt, 1982; Jaffe, 1983) propagates in the form of a wave from the point of gamete interaction to the opposite pole of the egg (Eisen et al., 1984; Swan and Whitaker, 1986; Yoshimoto et al., 1987). This wave of increased intracellular calcium is initiated following a latent period (Phase 1) of approximately 12 sec and stimulates the egg from its quiescent state to proliferation and embryogenesis (Chambers, 1989).