Characterization of a membrane antigen from rabbit testis and sperm isolated by using monoclonal antibodies and effect of its antiserum on fertility.

An antigen was isolated from deoxycholate-solubilized rabbit testis and sperm by using an immunosorbent column containing IgG from a monoclonal antibody (8C10.5) that inhibits fertility. Elution was by stepwise increases in pH (8.0, 10.0, and 11.4), with the pH 11.4 fraction after recycling through the column showing a single band at 63 kilodaltons in slab NaDodSO4/PAGE with a silver stain. The antigen molecule was composed of two subunits, which on two-dimensional PAGE showed many spots within the same molecular size range (50-70 kilodaltons) but differing in charge. The antigen isolated either from testis or sperm showed mainly the same spots. The antigen is periodic acid/Schiff positive and contained 21% carbohydrate. An asialo-derivative of the antigen did not change its characteristics on NaDodSO4/PAGE. This glycoprotein resolved into two types of polypeptides, those binding and those not binding to a lens culinaris lectin column; some of the polypeptides appeared common to both fractions. Murine antiserum against the antigen neither agglutinated nor immobilized rabbit sperm but in immunofluorescence reacted with the plasma membrane of viable rabbit sperm as well as with murine and human sperm. Fertilization of female rabbits inseminated with treated sperm was not affected, but, by 9 days, fertility was significantly reduced (21% of controls). The postfertilization antifertility effect was not due to parthenogenic activation or to polyspermy. The antiserum reacted with one specific band in the one- and two-dimensional gel electrophoretic transfer blot procedure and was unaffected by absorption with different somatic tissues.