This proposal for renewal expands earlier works and describes new experiments designed to isolate in large quantities the proteins of the plasma membrane of boar spermatozoa which are involved in specific binding to the porcine zona pellucida. A secondary objective is to identify proteins that are involved in other events in fertilization, particularly sperm-egg fusion. Monovalent antibodies prepared from rabbits immunized with purified plasma membrane polypeptides will be used to determine whether several polypeptides, shown to interact strongly to zonae particles in vitro (or other polypeptides) prevent the binding of sperm to the zonae of eggs and fertilization in vivo and in vitro. In vivo fertilization will involve the addition of washed sperm (with and without monovalent antibody to PM and PM polypeptides) directly into the surgically exposed uterus and oviducts of superovulated gilts. Fusion experiments will be carried out in vitro using the zona-free hamster assay. Antibodies will be prepared from polypeptides separated by preparative SDS-PAGE and by chromatography in weakly denaturing non-ionic detergents. Proteins shown to block in vivo fertilization will be isolated in quantity and tested for their ability to block directly sperm-egg interactions. Their carbohydrate and amino acid composition will also be determined. Location of these polypeptides on the sperm surface will be determined by immunocytochemical methods that include fluorescence microscopy and transmission electron microscopy. It is anticipated that results from these experiments will provide a focus for determining how the sperm plasma membrane (and its complementary receptor on the zona) organize to initiate events involved in sperm activation and fertilization.
