CALCIUM MOBILIZATION AND INFLUX DURING SPERM EXOCYTOSIS

We have previously shown that two intracellular events which occur dur ing capacitation of bovine sperm are the formation of actin filaments on the plasma and outer acrosomal membranes and the attachment of a PI P2-specific phospholipase C (PLC) to this membrane bound F-actin. This PLC plays an essential role in sperm exocytosis (acrosome reaction). In the present report, we further elucidated the role of this PLC usin g a PIP2-specific PLC of bacterial origin. This PLC is different from the endogenous sperm PLC in that it is calcium independent and not inh ibited by neomycin. Here we report using bovine sperm that this bacter ial PLC can restore actin release from extracted membranes as well as membrane fusion in a cell-free assay when the endogenous PLC is inhibi ted by neomycin. The sperm PLC requires 2 mu M calcium for half maxima l activation, while half maximal actin release from extracted plasma m embranes occurs at 80 mu M. Extracted sperm membranes were examined fo r calcium pumps and channels. Sperm plasma membranes were found to pos sess a thapsigargin insensitive calcium pump and calcium channels whic h are opened by phosphorylation by protein kinase C. The acrosomal mem brane possesses a calcium pump which is inhibited by thapsigargin and calcium channels which are opened by cAMP. These observations are disc ussed in terms of a model of acrosomal exocytosis which involves a cal cium rise that occurs in two stages resulting from calcium mobilizatio n from internal stores followed by influx of extracellular calcium.