The principal aims of proposal fall into four major categories: 1) The isolation and characterization of sperm acrosomal membranes. 2. The purification, characterization and localization of acrosomal enzymes. 3. The determination of the role of these enzymes in the gamete interaction, ie. the acrosome reaction, the proenzyme activation and the penetration of the ovum. 4. The investigation on the immunogenecity of the isolated membranes and acrosomal enzymes as a potential contraceptive tool. The procedures for the isolation of acrosomal membranes and related enzymes have been developed in our laboratory. In short, the acrosomal components are sequentially extracted using methods such as osmotic shock, nitrogen pressure, salt and detergent extractions, and the density gradient centrifugation. The purification of enzymes is done by the conventional chromatography procedures as well as the specific substrate and inhibitor affinity chromatography procedures. For the penetration of the ovum, sperm must undergo the acrosome re-action in order to activate the enzymes and ensure their release during the penetration. We will determine the role of acrosomal phospholipase C in the induction of the acrosome reaction and the role of Cathepsin D and other enzymes in activating proenzyme(s). The effect of purified enzymes will be tested on the rabbit and hamster ova. The enzymes will be used alone and in combination to determine their cooperativity during the sperm penetration of the ovum. The synthetic and natural inhibitors of these enzymes will be screened and their contraceptive potential assessed. The antibodies to the isolated acrosomal components will be raised both in male and female rabbits. The antisera will be used to localize the antigens. The immunized rabbits will also be evaluated for the duration of the circulating antibodies and its effect on their fertility. These studies are expected our knowledge of the events leading to fertilization at the molecular level.
