Structural Relationship of Sperm Soluble Hyaluronidase to the Sperm Membrane Protein PH-201

Abstract

The sperm plasma membrane protein PH-20 has a hyaluronidase activity that enables acrosome-intact sperm to pass through the cumulus cell layer of the egg. In this study we analyzed the relationship of guinea pig PH-20 and the "classical" soluble hyaluronidase released at the time of the acrosome reaction of guinea pig sperm. PH-20 is a membrane protein, anchored in the plasma and inner acrosomal membranes by a glycosyl phosphatidyl inositol anchor. Several types of experiments indicate a structural relationship of PH-20 and the soluble hyaluronidase released during the acrosome reaction. First, an antiserum raised against purified PH-20 is positive in an immunoblot of the soluble protein fraction released during the acrosome reaction. In the released, soluble protein fraction, the anti-PH-20 antiserum recognizes a protein of approximately 64 kDa, i.e., identical in molecular mass to PH-20 (approximately 64 kDa). Second, the enzymatic activity of the released hyaluronidase is completely inhibited (100%) by the anti-PH-20 antiserum. Third, almost all (97%) of the soluble hyaluronidase is removed from the released protein fraction by a single pass through an affinity column made with an anti-PH-20 monoclonal antibody. These findings suggest that the released, soluble hyaluronidase is a soluble form of PH-20 (sPH-20). During the acrosome reaction, PH-20 undergoes endoproteolytic cleavage into two disulfide-linked fragments whereas the released sPH-20 is not cleaved, suggesting the possible activity of a membrane-bound endoprotease 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 acrosome reaction.

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

“…The molecular size of Hyal5 was identical under nonreducing and reducing conditions, whereas 52-kDa PH-20 in the AI fraction of wild-type mice was separated into two polypeptides with the sizes of 43 and 18 kDa only under reducing conditions. These results demonstrate that Hyal5 is a single-chain molecule structurally distinguishable from PH-20 consisting of two chains covalently linked by one of two preexisting disulfide bridges (8,17,18,30). Indeed, the endoproteolytic cleavage site sequences in guinea pig and mouse PH-20, Arg 346 -Ser 347 , and Arg 347 -Ala 348 , respectively, are replaced by Thr 347 -Met 348 in Hyal5 (Figs.…”

“…Guinea pig PH-20 is known to be a GPI-anchored protein containing four domains (8,17,18): the signal peptide domain at the N terminus, the catalytic domain as hyaluronidase, the ZPbinding domain, and the recognition domain for attachment to GPI at the C terminus (Fig. 1B).…”

“…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).…”

“…Because cumulus cells are embedded in the extracellular matrix abundant in hyaluronan (11), a glycosylphosphatidylinositol (GPI)-anchored sperm hyaluronidase, PH-20, has long been believed to catalyze the hyaluronan degradation, thus enabling acrosome-intact (AI) sperm to penetrate the cumulus mass (9,(12)(13)(14)(15)(16)(17). A possible involvement of PH-20 in the binding of acrosomereacted (AR) sperm to ZP, known as secondary sperm͞ZP binding, has been also suggested (18).…”

Acrosin Accelerates the Dispersal of Sperm Acrosomal Proteins during Acrosome Reaction

“…When anti-PH2O antibody was used, several regions of sperm were stained; the acrosome and sperm head were strongly and weakly stained, respectively. This result may be due to the fact that PH2O is present in the sperm as two forms: a soluble form locating in the acrosome, and a membrane-anchored form on the plasma and inner acrosomal membranes (15,16). Although the sperm tail was also stained by anti-PH2O antibody, the signal may be nonspecific.…”