The Calcium Transient in Sea Urchin Eggs during Fertilization Requires the Production of Inositol 1,4,5-Trisphosphate

Abstract

The production of inositol 1,4,5-trisphosphate (InsP3) has been reported to mediate the transient rise in intracellular Ca2+activity ([Ca2+]i) in sea urchin eggs during fertilization. However, direct evidence of an absolute requirement for generation of InsP3during fertilization is still lacking. We investigated this question by blocking the InsP3synthesizing enzyme phospholipase C (PLC) during fertilization with U73122, an aminosteroid. U73122 inhibited the sperm-induced Ca2+release in a dose-dependent manner, although above 15 μM U73122 eggs showed an elevated resting [Ca2+]iand a lower fertilization rate. The inhibition of Ca2+transient by U73122 was not due to a failure of fertilization, since incorporated sperm nuclei were evident in eggs used to measure the Ca2+response. U73122 also prevented the accompanying rise in intracellular pH (pHi), 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 InsP3mass during the first 2 min of fertilization. In addition, U73122 inhibited the GTPγS-induced Ca2+release and pHirise in unfertilized eggs. These results suggested that the transient rise in Ca2+in sea urchin during fertilization requires the production of InsP3.