STRUCTURAL RELATIONSHIP OF SPERM SOLUBLE HYALURONIDASE TO THE SPERM MEMBRANE-PROTEIN PH-20

The sperm plasma membrane protein PH-20 has a hyaluronidase activity t hat enables acrosome-intact sperm to pass through the cumulus cell lay er of the egg. In this study we analyzed the relationship of guinea pi g PH-20 and the ''classical'' soluble hyaluronidase released at the ti me of the acrosome reaction of guinea pig sperm. PH-20 is a membrane p rotein, anchored in the plasma and inner acrosomal membranes by a glyc osyl phosphatidyl inositol anchor. Several types of experiments indica te a structural relationship of PH-20 and the soluble hyaluronidase re leased during the acrosome reaction. First, an antiserum raised agains t purified PH-20 is positive in an immunoblot of the soluble protein f raction released during the acrosome reaction. In the released, solubl e protein fraction, the anti-PH-20 antiserum recognizes a protein of s imilar to 64 kDa, i.e., identical in molecular mass to PH-20 (similar to 64 kDa). Second, the enzymatic activity of the released hyaluronida se is completely inhibited (100%) by the anti-PH-20 antiserum. Third, almost all (97%) of the soluble hyaluronidase is removed from the rele ased protein fraction by a single pass through an affinity column made with an anti-PH-20 monoclonal antibody. These findings suggest that t he released, soluble hyaluronidase is a soluble form of PH-20 (sPH-20) . During the acrosome reaction, PH-20 undergoes endoproteolytic cleava ge into two disulfide-linked fragments whereas the released sPH-20 is not cleaved, suggesting the possible activity of a membrane-bound endo protease on PH-20. We searched for a cDNA encoding sPH-20 but none was found. This result suggests that sPH-20 may arise from the enzymatic release of PH-20 from its membrane anchor, possibly at the time of acr osome reaction.