The long term objective of this project is to understand the creation and maintenance of sperm surface topography and the molecular basis of fertilization. Previous evidence indicates that the PH-20 surface protein functions is sperm-zona binding.
Aim I asks how the PH-20 membrane protein is targeted to two different membranes during spermiogenesis. Experiments using techniques of gene cloning and sequencing will test the hypothesis that either two genes or two mRNAs encoded two forms of the PH-20 protein that are differentially targeted.
In Aim II the change in localization of PH-20 during epididymal passage will be studied, using techniques of fluorescence intensity measurements and fluorescence redistribution after photobleaching (FRAP). Another alteration that occurs during epididymal passage is the appearance of the integral membrane protein, PH-30 which our evidence indicates has a role in sperm-egg plasma membrane fusion. The mechanism of its appearance will be investigated. Because of the previous finding that two membrane proteins, PT-1 and PH-20, are freely diffusing on the sperm surface, it will be tested if these proteins are anchored in the membrane by covalent attachment to a lipid, phosphatidyl inositol (PI). During the last grant period, it was discovered that acrosome-intact and acrosome-reacted guinea pig sperm can both initiate sperm-zona binding. We will identify sperm and zona components involved in the binding of each type of sperm. In addition, sperm interactions with the egg plasma membrane will be analyzed. These studies on sperm surface topography and sperm-egg interactions are designed to advance the knowledge of sperm surface organization and regulation of sperm function during fertilization. Understanding of these phenomena are important in both the areas of infertility and contraception.
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