Two-dimensional polyacrylamide gel electrophoresis characterization of APz, a sperm protein involved in zona binding in the pig and evidence for its binding to specific zona glycoproteins

A boar sperm integral plasma membrane protein (APz) involved in the adhesion of uncapacitated and capacitated sperm to the porcine zona pellucida (ZP) has been characterized by two-dimensional polyacrylamide gel electrophoresis (PAGE) and tested for its ability to bind to various zona glycopeptides. APz shows microheterogeneity and focuses over a wide pH range, with predominant forms focusing above pH 7. The protein, when excised from nonreducing polyacrylamide gels, inhibited sperm-egg binding and bound heat-solubilized zonae preventing these zonae from blocking sperm binding to eggs. In an indirect assay, a polyclonal monovalent antibody, which blocks sperm-egg binding and which is absorbed by APz, was used to determine the ability of zona glycopeptides to prevent the sperm-egg blocking activity of the antibody from being absorbed by intact sperm. When whole heat-solubilized ZP was added to sperm at doses that block sperm-egg binding and the excess ZP was removed, the sperm-egg blocking activity of the antibody was not absorbed by these sperm, and antibody-containing supernatants blocked the binding of untreated sperm to eggs as effectively as antibody that was not mixed with fresh sperm. When alpha ZP3 was used in the same manner, sperm-egg blocking activity again was not absorbed by antibody-treated cells. Beta ZP3, however, failed to block sperm-egg binding and failed to absorb the sperm-egg blocking activity of the antibody. These findings support the argument that the action of APz is physiologically significant and involves specific binding sites on the ZP3 component of the ZP.