Porcine zona pellucida ZP3α glycoprotein mediates binding of the biotin‐labeled M_r 55,000 family (ZP3) to boar sperm membrane vesicles

Abstract

The two M(r) 55,000 glycoproteins, ZP3 alpha and ZP3 beta, of porcine zona pellucida copurify as a preparation designated ZP3. Gamete binding assays have implicated ZP3 alpha, but not ZP3 beta, as participating in sperm-zona recognition events. We now report that boar sperm contain membrane-associated binding sites with specificity for ZP3 alpha. Biotin-labeled (b-) preparations of ZP3 bind to intact boar sperm in a saturable manner, with localization on the anterior head region. Membrane vesicles 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 assays using b-ZP3 (0.1 microgram/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 enzyme that trims polylactosamines, enhanced its affinity for membrane receptors. In contrast treatments such as chemical deglycosylation, pronase digestion, or disruption of disulfide bonds abolished the ligand activity of ZP3. Finally, purified ZP3 alpha was an at least 100-fold better antagonist than purified ZP3 beta. The results demonstrate that binding of b-ZP3 to isolated boar sperm membranes is mediated by sperm receptors with specificity for the ZP3 alpha macromolecular component and reveal a complex contribution of both carbohydrate and protein moieties toward the ligand activity of this sperm adhesive zona molecule.

Recombinant Porcine Zona Pellucida Glycoproteins Expressed in Sf9 Cells Bind to Bovine Sperm but Not to Porcine Sperm

“…These assays were performed at least three times, and the data are shown as the means Ϯ S.D. much higher level of sperm binding activity than does rZPB alone is consistent with reports that ZPC promotes the sperm binding activity of ZPB in the native porcine zona (20,23).…”

Hetero-oligomerization-dependent Binding of Pig Oocyte Zona Pellucida Glycoproteins ZPB and ZPC to Boar Sperm Membrane Vesicles

“…pZP(BϩC), pZPB, and pZPC correspond to preparations designated ZP3, ZP3␣, and ZP3␤ in prior publications from this laboratory. Membrane vesicles were isolated from capacitated boar sperm by nitrogen cavitation and differential centrifugation and stored at Ϫ70°C as described previously (22).…”

“…When tested in gamete adhesion assays, pZPB inhibits sperm-zona binding, whereas pZPC is without effect (20,23). Likewise, when tested in solid phase binding assays, biotin-labeled pZPB binds to immobilized boar sperm membranes in a concentration-dependent, saturable manner, whereas biotin-pZPC does not (22). In competition assays, pZPB blocks binding of a biotin-pZP(BϩC) probe to membranes; pZPC fails to compete but, surprisingly, enhances the apparent affinity of pZPB for its cognate zona-binding protein.…”

“…Due to post-translational processing, the three mature glycoproteins from isolated zonae appear on nonreducing SDS gels as two diffuse bands (15); pZPA runs at M r 90,000, whereas pZPB and pZPC co-migrate at M r 55,000. Electrophoretic or chromatographic protocols permit isolation of copurified pZPB and pZPC glycoproteins (15)(16)(17) and such pZP(BϩC) preparations exhibit several important biological activities, including: stimulation of the acrosome reaction in capacitated boar sperm (18), inhibition of sperm-zona attachment (19,20), binding to a sulfated polysaccharide-binding domain on boar sperm proacrosin (21), and binding to isolated boar sperm membrane vesicles (22).…”

Time Course Analyses of Cortical Granule Exocytosis and Zona Pellucida Modifications after Artificial Activation in In-Vitro Matured Porcine Oocytes.

“…Taken together, further research is needed to elucidate whether the different patterns of Ca 2+ oscillation responsible for egg activation have an effect on the time course of CG exocytosis and ZP modification in porcine oocytes. Moreover, in porcine oocytes, the ZP3α glycoprotein family is responsible for the binding of sperm to oocytes, and ZP1 glycoprotein has no effect on sperm-oocyte ZP binding [7,37,38]. Therefore, the exact physiological roles that the reduced amounts of ZP1 and ZP2 glycoproteins play in the block to polyspermy at fertilization in porcine oocytes will be the subject of further studies.…”