The Calcium Transient In Sea Urchin Eggs During Fertilization Requires The Production Of Inositol 1,4,5-Trisphosphate

The production of inositol 1,4,5-trisphosphate (InsP(3)) has been reported to mediate the transient rise in intracellular Ca(2+) activity ([Ca(2+)](i)) in sea urchin eggs during fertilization. However, direct evidence of an absolute requirement for generation of InsP(3) during fertilization is still lacking. We investigated this question by blocking the InsP(3) synthesizing enzyme phospholipase C (PLC) during fertilization with U73122, an aminosteroid. U73122 inhibited the sperm-induced Ca(2+) release in a dose-dependent manner, although above 15 mu M U73122 eggs showed an elevated resting [Ca(2+)](i) and a lower fertilization rate. The inhibition of Ca(2+) transient by U73122 was not due to a failure of fertilization, since incorporated sperm nuclei were evident in eggs used to measure the Ca(2+) response. U73122 also prevented the accompanying rise in intracellular pH (pH(i)), which is mediated by the activation of the Na(+)-H(+) antiporter. The antiporter is regulated through activation of protein kinase C by 1,2-diacylglycerol, which is the other hydrolytic product of phosphatidylinositol 4,5-bisphosphate by PLC. Further evidence of the specificity of U73122 action was inhibition of the increase in InsP(3) mass during the first 2 min of fertilization. In addition, U73122 inhibited the GTP gamma S-induced Ca(2+) release and pH(i) rise in unfertilized eggs. These results suggested that the transient rise in Ca(2+) in sea urchin during fertilization requires the production of InsP(3). (C) 1998 Academic Press.