This proposal for renewal extends previous work which has identified a protein complex involved in the attachment of boar sperm to the porcine zona pellucida (ZP). Experiments are described to determine the origin of the zona binding protein (ZBP), its chemical and immunological properties and the structural basis of its interaction with ZP. Chemical and structural analyses include determining the amino acid and carbohydrate composition of ZBP, its relation to the lipid bilayer, and its association with other plasma membrane proteins which may function as receptors for ZBP or cooperate in its action. Glycoproteins of the zona that react specifically with 125I-labelled ZBP will be determined using immunoprecipitation techniques and radioautographic analysis of 2-D PAGE separated transblots of ZP treated with labelled ZBP. Proteolytic digestion of ZBP and ZP will be carried out to identify, isolate, and determine the chemical properties of interacting sites on these adhesion molecules. Specific polyclonal antiserum to ZBP will be used to determine its tissue and species specificity, its localization on sperm during maturation and capacitation and to determine if ZBP originates from the epididymal secretion. Procedures to accomplish these objectives also include preparative 2-D PAGE and affinity chromatography to isolate ZBP in quantity, western blotting, radioautography and ELISA to identify and quantitate ZBP. Indirect fluorescence microscopy will be used to locate ZBP on sperm and to determine whether ZBP is secreted by the epididymis. It is anticipated that these experiments will provide new insights into how mammalian sperm develop fertilizing capacity and how the epididymis functions in sperm maturation. It is also anticipated that new knowledge on the molecular mechanisms of cell-cell adhesion will be obtained.
