The proposed research program will utilize monoclonal antibodies to investigate the role of protein determinants in a) the assembly and structural organization of the sperm mucleus; and b) the extragenomic contribution of sperm to embryonic development. The required monospecific antibodies are to be isolated using hybridomatechnology following syngeneic and xenogeneic immunization with nuclei and perinuclear theca of mouse spermatozoa. Nuclear antigens are to be identified and characterized by polyacrylamide gel electrophoresis and Western blot techniques. Localization of selected antigens to discrete nuclear domains and/or specific chromatin structures will be defined by light and electron microscopy. The latter ogjective is to be accompliched using selectd monoclonal antibody probes in conjunction with second antibody or protein A conjugated to fluorescein isothiocyanate, rhodamine, ferritin or colloidal gold. These antibody probes are also to be utilized in immunohistochemical studies to determine the fate of the nuclear antigens after fertilization and their role during formation of the male pronucleus and subsequent development of gynogenones and normal embryos. Finally, the extragenomic contribution of sperm to embryogenesis is to be determined by microinjecting nuclear components into parthenogenones.
