The spermatozoan plasma and acrosomal membranes are partitioned into domains of distinct molecular and structural composition which perform specific functions at defined steps of fertilization. The long range goals of this proposal are to define the molecular and structural mechanisms which generate the mosaic character of the spermatozoan membranes and to identify the role of cytoskeletal assemblies in sperm development and fertilization. Four specific aims are directed towards these goals. Aim one is to define the role of the spermatid cytoskeleton in acrosome morphogenesis. Quick-freeze, deep-etch freeze-fracture and thin section electron microscopy will be employed to define the spatial organization and membrane interactions of the subacrosomal f-actin network in hamster spermatids. Affinity-isolation protocols will be utilized to purify spermatid actin-binding proteins and ultrastructural immunochemistry will be utilized to define the role of actin-binding proteins in acrosome morphogenesis. Aim two is to define the mechanisms which generate the unique molecular and structural properties of the inner acrosomal membrane. Inner acrosomal membrane-specific monoclonal antibodies and ultrastructural immunocytochemistry will be employed to identify protein trafficking pathways which establish domain-specific protein localizations; specific protein-protein interactions between the inner acrosomal membrane and perinuclear cytoskeleton which function in membrane domain formation will be identified. Aim three is to identify the mechanism of assembly of the membrane skeleton of the outer acrosomal membrane and to define its roles in acrosome function. The protein trafficking and sorting pathways utilized to establish the outer acrosomal membrane domain will be identified. It will be determined if the acrosomal lamina functions both to immobilize integral membrane proteins and to segregate the acrosomal matrix into distinct compartments. Aim four is to identify polypeptides of the inner acrosomal membrane which function during fertilization to bind the zona pellucida and the egg plasma membrane. Purified inner acrosomal membranes will be tested for their ability to bind the zona and the egg plasma membrane and to block sperm-egg interactions in vitro. Completion of these specific aims will provide new data on the mechanisms which establish the different domains of the acrosomal membrane and provide new insights into the function of domain-specific membrane proteins during spermiogenesis and fertilization.