PORCINE ZONA-PELLUCIDA ZP3-ALPHA GLYCOPROTEIN MEDIATES BINDING OF THEBIOTIN-LABELED M(R)-55,000 FAMILY (ZP3) TO BOAR SPERM MEMBRANE-VESICLES

The two M(r) 55,000 glycoproteins, ZP3alpha and ZP3beta, of porcine zo na pellucida copurify as a preparation designated ZP3. Gamete binding assays have implicated ZP3alpha, but not ZP3beta, as participating in spermzona recognition events. We now report that boar sperm contain me mbrane-associated binding sites with specificity for ZP3alpha. Biotin- labeled (b-) preparations of ZP3 bind to intact boar sperm in a satura ble manner, with localization on the anterior head region. Membrane ve sicles obtained from capacitated sperm by nitrogen cavitation retain b -ZP3 binding sites as determined by an enzyme-linked method employing alkaline phosphatase-conjugated strepavidin. In competitive binding as says using b-ZP3 (0.1 mug/ml) as probe, heat-solubilized zonae and ZP3 were effective competitors, whereas the nonzona molecules fetuin and fucoidin were not. Digestion of ZP3 with endo-beta-galactosidase, an e nzyme that trims polylactosamines, enhanced its affinity for membrane receptors. In contrast treatments such as chemical deglycosylation, pr onase digestion, or disruption of disulfide bonds abolished the ligand activity of ZP3. Finally, purified ZP3alpha was an at least 100-fold better antagonist than purified ZP3beta. The results demonstrate that binding of b-ZP3 to isolated boar sperm membranes is mediated by sperm receptors with specificity for the ZP3alpha macromolecular component and reveal a complex contribution of both carbohydrate and protein moi eties toward the ligand activity of this sperm adhesive zona molecule. (C) 1993 Wiley-Liss, Inc.