The biosynthesis of acrosomal enzymes by isolated spermatogenic cells will be investigated to determine whether these enzymes are processed in a manner similar to that used to deliver acid hydrolases to lysosomes. Guinea pig sperm will be prepared and induced to undergo the acrosome reaction with the ionophore A23187. The soluble acrosomal components released from the sperm will be collected and used as the source for two acrosomal enzymes: proacrosin/acrosin and beta-galactosidase. Monoclonal antibodies will be prepared against these enzymes and used to study their synthesis by purified populations of guinea pig pachytene spermatocytes, round spermatids, and condensing spermatids. Using immunoblotting of cell extracts, immunofluorescence, and immunoprecipitation of extracts from metabolically labeled spermatogenic cells, the monoclonal antibodies will be used to determine what cell types are capable of synthesizing proacrosin and beta-galactosidase. Using pulse- chase labeling followed by immunoprecipitation, the processing of precursor forms of proacrosin and beta-galactosidase will be examined in spermatogenic cell cultures. Experiments will also be performed to determine whether mannose-6-phosphate groups are attached to the newly-synthesized proacrosin and beta- galactosidase. Each spermatogenic cell population will be assayed for mannose-6-phosphate receptor by immunoblotting, immunofluorescence, and as receptor activity assay. Spermatogenic cells will also be tested for their ability to internalize bovine testicular beta-galactosidase (a model lysosomal enzyme), guinea pig proacrosin, and guinea pig sperm beta-galactosidase via mannose-6-phosphate receptor-mediated endocytosis. The experiments performed during this project will provide important new information concerning the synthesis, processing and transport of acrosomal enzymes. Increased information concerning the development of the acrosome will advance our understanding of the regulation of male fertility.
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