THE PH-20 PROTEIN IN CYNOMOLGUS MACAQUE SPERMATOZOA - IDENTIFICATION OF 2 DIFFERENT FORMS EXHIBITING HYALURONIDASE ACTIVITY

In these experiments, we have characterized the bifunctional sperm pro tein PH-20 in macaque sperm and studied its hyaluronidase activity. In tact sperm were evaluated before the acrosome reaction (AR), and a sol uble form of PH-20 released during acrosomal exocytosis was also inves tigated. Western blots of SDS-PAGE of acrosome-intact sperm extracts r evealed a 64-kDa form of PH-20 was recognized by a polyclonal antibody (R-10) raised in rabbits against purified, recombinant cynomolgus mac aque sperm PH-20. The soluble components released during the AR which were recognized by the R-10 antibody included both the 64-kDa form and a 53-kDa form of PH-20. An ELISA-like procedure for determining PH-20 hyaluronidase activity indicated that acrosome-intact sperm exhibited two peaks of hyaluronidase activity near pH 4 and greater than or equ al to pH 7. The majority of enzyme activity in acrosome-intact sperm e xtracts occurred at neutral pH, while the soluble hyaluronidase activi ty released at the AR was predominantly acid-active. Hyaluronidase act ivity of PH-20 at different pH optima was investigated using hyaluroni c acid substrate gel electrophoresis, and results indicated that the 6 4-kDa polypeptide had a broad range, with the majority of activity at neutral pH (pH 7). The 53-kDa polypeptide in sperm extracts only exhib ited activity at acid pH (pH 4), The hyaluronidase activities of both enzymes could be inhibited by apigenin. The soluble PH-20 hyaluronidas e activity released during the AR was primarily of the acid-active 53- kDa form. fine structural localization of PH-20 using Fab fragments of R-10 IgG demonstrated that PH-20 was associated not only with sperm m embranes, but also with the dispersing acrosomal contents. These data suggest that the more neutral-active form of PH-20 (64 kDa) is present on the plasma and inner acrosomal membranes and gives rise to the sol uble acid-active form at the time of the AR. The generation of the sol uble form of PH-20 may result from the action of acrosomal enzymes, wh ich could include proteases, glycosidases, and phospholipases. (C) 199 6 Academic Press, Inc.