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 InsP3 during fertilization is still lacking. We investigated this question by blocking the InsP3 synthesizing 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+](i) and 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 (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 InsP3 mass during the first 2 rain of fertilization. In addition, U73122 inhibited the GTPγS-induced Ca2+ release and pH(i) rise in unfertilized eggs. These results suggested that the transient rise in Ca2+ in sea urchin during fertilization requires the production of InsP3.