The sperm plasma and acrosomal membranes are partitioned into domains of distinct molecular composition which perform specific functions required for mammalian fertilization. The long range goals of this proposal are to define the molecular processes which assemble discrete membrane domains and to identify the biochemical mechanisms regulating the fusion between the periacrosomal plasma membrane and the outer acrosomal membrane during the acrosome reaction. Four aims are proposed addressing these goals. Aim one is to identify the function of the periacrosomal plasma membrane protein PM52 (sperad) in sperm capacitation and/or the acrosome reaction. Immunoprecipitation and blot overlay analyses will be performed to identify ligands for the cytoplasmic domain of PM52 and permeant peptide mimetics of the proline- rich domain of PM52 will be tested for effects on capacitation and the acrosome reaction. Aim 2 is to determine if docking interactions between the periacrosomal plasma membrane and the outer acrosomal membrane generate domain-specific protein localization. Immunoblotting and blot overlays will identify PM52 binding proteins in the outer acrosomal membrane and immunofluorescence experiments will test if disruption of PM52-outer acrosomal membrane interaction results in protein redistribution. Aim 3 is to determine if germ cell isoforms of syntaxin and synaptophysin function in the membrane fusion events of the acrosome reaction. Immunolocalization and immunoprecipitation analyses will be performed to determine if these proteins assemble into a membrane fusion complex required for acrosomal exocytosis. Aim 4 is to identify protein-protein interactions regulating the assembly of the outer acrosomal membrane-acrosomal matrix complex. Biochemical and ultrastructural experiments will be performed to identify proteins of the outer acrosomal membrane which bind to, and regulate the distribution of, acrosomal matrix assemblies within the acrosome. Completion of these studies will provide new insights into the assembly of the acrosomal segment and of its functions during mammalian fertilization and the findings have potential application to strategies for fertility regulation and/or improvement.