Bull sperm plasma and outer acrosomal membranes were analyzed by SDS-PAGE. Analysis of the plasma membrane proteins revealed the presence of a 70 kDa band the prominence of which is enhanced after capacitation. This protein was found to bind to zona pellucida intact oocytes. PAGE analysis of outer acrosomal membrane proteins also reveals the presence of a 70 kDa band, but its prominence decreases after capacitation. This protein also binds to zona pellucida intact oocytes. Futhermore, the 70 kDa outer acrosomal membrane protein is recognized in Western blot analysis by antibodies to plasma membrane proteins and vice versa. The results indicate that the 70 kDa acrosomal and plasma membrane proteins are the same. This 70 kDa protein would thus be a zona pellucida binding protein which is initially stored in the outer acrosomal membrane and transferred to the plasma membrane during capacitation, enabling it to function in egg-sperm binding.

We used a cell-free system to study membrane fusion during sperm exocytosis (acrosome reaction). Extracted bovine sperm plasma and outer acrosomal 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 revealed by resonance energy transfer between the two probes. We have previously shown using this system that the requirement of capacitation for sperm exocytosis is retained in cell-free membrane fusion, and that the pH and calcium dependence of the cell-free fusion mimics those of exocytosis in intact cells. In the present report we further characterize the fusion of sperm membranes which we observe in our assay. Phosphoproteins and phospholipases were found to be involved in the membrane fusion step of sperm exocytosis. Protein kinases, phosphatases, and Gi-like proteins, while involved in exocytosis in intact cells, are not 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 also functions as a physical barrier to membrane fusion which is removed by the increases in intracellular calcium and pH which precede fusion.

No abstract