Fertilization in Voltage-Clamped Sea Urchin Eggs

Abstract

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 (V_m) 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 V_m 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’ V_m by only 7 to 8 mV.

These data are consistent with the conclusion that unless depolarization to the neighborhood of 0 mV occurs, the shoulder component is dissociated from subsequent phases of the activation current and sperm entry will not occur. One possibility is that dissociation of the shoulder component accompanied by failure of sperm entry results from a suppressive effect of negative V_m on fusion of the sperm and egg plasma membranes. However, capacitance measurements carried out on eggs using a patch clamp method indicate that coincidentally with the abrupt turn-on of the shoulder current, cytoplasmic continuity between sperm and egg is attained. Nonetheless, the incipient stages of sperm incorporation were shortly terminated since simultaneously with the cutoff of current (which ends the dissociated shoulder component), the fusion event was reversed. Patch clamp measurements have established that the conductance increase which generates the inward shoulder current is not global, but localized to the immediate site of sperm attachment. A consequence of the restriction of the conductance increase to a localized site is that the density (current per unit surface area) could be large. This could result in alteration of the concentration of ions at the site of the entering sperm, and suppress its penetration. This possibility is currently under investigation in ion substitution experiments.