The PH-20 Protein in Cynomolgus Macaque Spermatozoa: Identification of Two Different Forms Exhibiting Hyaluronidase Activity

Abstract

In these experiments, we have characterized the bifunctional sperm protein PH-20 in macaque sperm and studied its hyaluronidase activity. Intact sperm were evaluated before the acrosome reaction (AR), and a soluble form of PH-20 released during acrosomal exocytosis was also investigated. Western blots of SDS-PAGE of acrosome-intact sperm extracts revealed a 64-kDa form of PH-20 was recognized by a polyclonal antibody (R-10) raised in rabbits against purified, recombinant cynomolgus macaque 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 > or = pH 7. The majority of enzyme activity in acrosome-intact sperm extracts occurred at neutral pH, while the soluble hyaluronidase activity released at the AR was predominantly acid-active. Hyaluronidase activity of PH-20 at different pH optima was investigated using hyaluronic acid substrate gel electrophoresis, and results indicated that the 64-kDa polypeptide had a broad range, with the majority of activity at neutral pH (pH 7). The 53-kDa polypeptide in sperm extracts only exhibited activity at acid pH (pH 4). The hyaluronidase activities of both enzymes could be inhibited by apigenin. The soluble PH-20 hyaluronidase 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 membranes, 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 soluble acid-active form at the time of the AR. The generation of the soluble form of PH-20 may result from the action of acrosomal enzymes, which could include proteases, glycosidases, and phospholipases.

Identification of a hyaluronidase, Hyal5, involved in penetration of mouse sperm through cumulus mass

“…These data may weaken the possibility that the ZP-binding domains of Hyal5 and PH-20 function in the sperm-egg interaction. It has been reported that two forms of hyaluronidase are present in sperm of several mammalian species: PH-20 GPI-anchored on the plasma and inner acrosomal membranes and a soluble enzyme (sPH-20) released during acrosome reaction (17,34,35). In cynomolgus macaque, the GPI-anchored form exhibits the maximum activity at neutral pH, whereas sPH-20 is active only at acid pH (34,35).…”

Sperm penetration through cumulus mass and zona pellucida

“…Following the acrosome reaction PH-20 is present on the inner acrosomal membrane and on the plasma membrane overlying the equatorial segment, indicating that PH-20 may migrate from the plasma membrane of the posterior sperm head to the inner acrosomal membrane after acrosomal exocytosis. A soluble form of monkey PH-20 is also released during the acrosome reaction (Cherr et al, 1996). It is thus conceivable that the localization of mouse Ph-20 and Hyal5 is essentially similar to that of guinea pig and monkey PH-20 on the acrosome-intact and acrosome-reacted sperm, although we need to ascertain the PH-20 migration during the acrosome reaction.…”

Hyaluronidase reduces human breast cancer xenografts in SCID mice

“…The 2 bands may represent the 2 forms of the enzyme, related to the soluble and membrane-bound forms of PH-20. 16,17 Separation of the 2 forms of the enzyme, the 75 and 58 kDa bands, was achieved by tandem gel filtration on Superose 12. Single bands were obtained on SPAGE, indicating that there was not equilibrium between the 2 forms of the enzyme (Fig.…”