BIOCHEMICAL-CHARACTERIZATION OF THE PH-20 PROTEIN ON THE PLASMA-MEMBRANE AND INNER ACROSOMAL MEMBRANE OF CYNOMOLGUS MACAQUE SPERMATOZOA

Preparations of sperm membranes (plasma membranes and outer acrosomal membranes) and denuded sperm heads were isolated from macaque sperm, a nd the PH-20 proteins present were characterized by Western blotting, hyaluronic acid substrate gel analysis, and a microplate assay for hya luronidase activity. Because we have shown previously that PH-20 is lo cated on the plasma membrane and not on the outer acrosomal membrane, the PH-20 in the membrane preparations was presumed to be plasma membr ane PH-20 (PM-PH-20). PM-PH-20 had an apparent molecular weight of 64 kDa and the optimum pH for its hyaluranidase activity was 6.5. The PH- 20 associated with denuded sperm heads was localized by immunogold lab el to the persistent inner acrosomal membrane (IAM) and was presumed t o be IAM-PH-20, which included a major 64 kDa form and a minor 53 kDa form. The 53 kDa form was not detected in extracts of denuded sperm he ads from acrosome intact sperm that were boiled in nonreducing sample buffer, but was present in extracts of sperm heads from acrosome react ed sperm and in the soluble material released during the acrosome reac tion, whether or not the samples were boiled. Substrate gel analysis s howed that the hyaluronidase activity of the 53 kDa form of PH-20 was greatest at acid pH, and this activity was probably responsible for th e broader and lower optimum pH of lAM hyaluronidase activity. When hyp otonic treatment was used to disrupt the sperm acrosome and release th e acrosomal contents, less than 0.05% of the total hyaluronidase activ ity was released. The PH-20 protein released by hypotonic treatment wa s the 64 kDa form and not the 53 kDa form, suggesting that its source might be the disrupted plasma membranes. Our experiments suggest that the soluble form of hyaluronidase, which is released at the time of th e acrosome reaction, is derived from the IAM. This soluble hyaluronida se is composed of both the 64 kDa form and 53 kDa form of PH-20. The 5 3 kDa form appears to be processed from the 64 kDa form at the time of the acrosome reaction. (C) 1997 Wiley-Liss, Inc.