Abstract
The prevention of polyspermy is essential for the successful progression of normal embryonic development in most sexually reproducing species. In external fertilizers, the process of fertilization induces a depolarization of the egg's membrane within seconds, which inhibits supernumerary sperm from entering an already-fertilized egg. This fast block requires an increase of intracellular Ca(2+) in the African clawed frog, Xenopus laevis, which in turn activates an efflux of Cl(-) that depolarizes the cell. Here we seek to identify the source of this intracellular Ca(2+) Using electrophysiology, pharmacology, bioinformatics, and developmental biology, we explore the requirement for both Ca(2+) entry into the egg from the extracellular milieu and Ca(2+) release from an internal store, to mediate fertilization-induced depolarization. We report that although eggs express Ca(2+)-permeant ion channels, blockade of these channels does not alter the fast block. In contrast, insemination of eggs in the presence of Xestospongin C-a potent inhibitor of inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release from the endoplasmic reticulum (ER)-completely inhibits fertilization-evoked depolarization and increases the incidence of polyspermy. Inhibition of the IP(3)-generating enzyme phospholipase C (PLC) with U73122 similarly prevents fertilization-induced depolarization and increases polyspermy. Together, these results demonstrate that fast polyspermy block after fertilization in X. laevis eggs is mediated by activation of PLC, which increases IP(3) and evokes Ca(2+) release from the ER. This ER-derived Ca(2+) then activates a Cl(-) channel to induce the fast polyspermy block. The PLC-induced cascade of events represents one of the earliest known signaling pathways initiated by fertilization.