RAT SPERM PLASMA-MEMBRANE MANNOSIDASE - LOCALIZATION AND EVIDENCE FORPROTEOLYTIC PROCESSING DURING EPIDIDYMAL MATURATION

Previous studies from this laboratory have identified a novel cu-D-man nosidase on the sperm plasma membranes of several species, including m an, which may have a role in fertilization. The polyclonal antibody ra ised against an isoform of the enzyme purified from rat epididymal flu id was found to cross-react with the alpha-D-mannosidase activity pres ent in the detergent-solubilized spermatozoa and sperm plasma membrane s. In the present study, we have used affinity-purified as well as mon ospecific anti-mannosidase IgG to demonstrate that the sperm mannosida se is an integral plasma membrane component of the rat sperm and is lo calized on the periacrosomal region of the sperm head. In addition, we demonstrate proteolytic processing of the membrane-bound alpha-D-mann osidase during maturation of spermatozoa. The membrane fractions prepa red from testis, and spermatozoa from the caput, corpus, and cauda reg ions of the epididymis, were solubilized in SDS and resolved by SDS-PA GE. The resolved polypeptides, when subjected to Western blot analysis using affinity-purified anti-mannosidase IgG as the primary antibody, revealed the presence of three specific immunoreactive bands (apparen t M(r), 135, 125, and 115 kDa) in the membranes from testis, caput, an d corpus spermatozoa. However, the cauda sperm plasma membranes showed only one immunoreactive band of apparent M(r) 115 kDa. The disappeara nce of the 135-and 125-kDa forms and the appearance of a sharp 118-kDa band on cauda spermatozoa suggests a precursor-product relationship b etween various molecular forms of the enzyme. Trypsin treatment of tes ticular and caput sperm membranes largely converted the precursor form s to the mature (115-kDa) form. The in vitro proteolysis resulted in a n elevated level of the alpha-D-mannosidase activity in the caput (but not cauda) sperm plasma membrane. Inclusion of trypsin inhibitors (be nzamidine and aprotinin) largely prevented the conversion of precursor form to the mature form. These data are consistent with the observed increase in the levels of sperm enzyme activity as spermatozoa move fr om the caput to the cauda region and suggest that the increase is due to the conversion of enzymatically inactive/less active high molecular weight precursor forms (135 and 125 kDa) into enzymatically active ma ture form (115 kDa)during sperm maturation. (C) 1995 Academic Press, I nc.