ISOLATION AND CHARACTERIZATION OF A PROTEIN WITH HOMOLOGY TO ANGIOTENSIN-CONVERTING ENZYME FROM THE PERIACROSOMAL PLASMA-MEMBRANE OF EQUINESPERMATOZOA

The periacrosomal plasma membrane of spermatozoa is involved in sperm binding to oviductal epithelial cells and to the zona pellucida. A pro tein of 68-70 kD molecular mass was purified biochemically from the is olated periacrosomal plasma membrane of equine spermatozoa as a possib le receptor for adhesion of spermatozoa to oviductal epithelial cells. A polyclonal antibody raised in rabbits against the purified equine s perm membrane protein recognized the 70 kD and an antigenically relate d 32 kD protein in preparations of isolated periacrosomal sperm plasma membrane and in detergent extracted ejaculated and epididymal spermat ozoa. A larger protein (similar to 110 kD) was detected in equine test is. Two antigenically related proteins (64 and 45 kD) were recognized on the plasma membrane of cynomolgus macaque spermatozoa, In vitro spe rm-binding assays were performed in the presence of antigen-binding fr agments or IgG purified from the polyclonal antiserum to investigate a possible function of the isolated protein in binding of equine sperma tozoa to homologous oviductal epithelial cells or zona pellucida. Incu bation with antigen-binding fragments or IgG purified from the antiser um did not inhibit binding of equine spermatozoa either to oviductal e pithelial cells or to the zona pellucida. On ultrastructural examinati on, the antibody bound exclusively to the cytoplasmic side of the peri acrosomal plasma membrane of equine and macaque spermatozoa. Microsequ ence analysis of 13 residues of sequence showed strong homology with a number of angiotensin converting enzymes: An 84% identity was identif ied with testis specific and somatic forms of human and mouse angioten sin-converting enzyme. Immunocytochemistry and immunoblot analysis est ablished that the protein is specific for the periacrosomal membrane o f ejaculated, epididymal, and testicular stallion spermatozoa. (C) 199 7 Wiley-Liss, Inc.