The formation of flagella and the synthesis of proacrosin will be investigated in primary cultures of mouse round spermatids. Initial experiments are designed to determine whether spermatids that do not make flagella in culture lack the centrioles necessary for axonemal nucleation. The requirements of protein, RNA, and tubulin biosynthesis for flagella formation will also be examined. The length of time for cell culture will be increased to ascertain the extent of tail maturation that can be obtained in vitro. Antibodies to the major fibrous sheath protein will be used to determine whether this component appears in the newly-formed flagella. The synthesis of proacrosin will also be examined to determine whether proacrosin contains N-linked oligosaccharides and whether these carbohydrate side chains are processed to the """"""""complex"""""""" type. The carbohydrate side chains will be analyzed for mannose-6-phosphate groups. If these residues are found, it suggests that proacrosin is directed to the acrosome by the same phosphomannosyl receptor system that transports lysosomal hydrolases to the lysosome. The experiments of this proposal will provide the bases for long-term biochemical studies of mammalian spermiogenesis.
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