SPERM EXOCYTOSIS RECONSTRUCTED IN A CELL-FREE SYSTEM - EVIDENCE FOR THE INVOLVEMENT OF PHOSPHOLIPASE-C AND ACTIN-FILAMENTS IN MEMBRANE-FUSION

We used a cell-free system to study membrane fusion during sperm exocy tosis (acrosome reaction), Extracted bovine sperm plasma and outer acr osomal membranes were labeled with chlorophyll a or DCY, respectively, The occurrence of membrane fusion is indicated by the ability of the probes to diffuse from one membrane species to another which is reveal ed by resonance energy transfer between the two probes, We have previo usly shown using this system that the requirement of capacitation for sperm exocytosis is retained in cell-free membrane fusion, and that th e pH and calcium dependence of the cell-free fusion mimics those of ex ocytosis in intact cells, In the present report we further characteriz e the fusion of sperm membranes which we observe in our assay, Phospho proteins and phospholipases were found to be involved in the membrane fusion step of sperm exocytosis. Protein kinases, phosphatases, and G( i)-like proteins, while involved in exocytosis in intact cells, are no t involved specifically in the membrane fusion step of exocytosis. The role of membrane bound F-actin in regulating membrane fusion was also studied using fluorescently labeled phalloidin, The results show that cortical F-actin has two roles in regulating sperm exocytosis. One is to form a scaffolding to hold phospholipase C at the membrane, It als o functions as a physical barrier to membrane fusion which is removed by the increases in intracellular calcium and pH which precede fusion.