The PH-20 protein in cynomolgus macaque spermatozoa: Identification of two different forms exhibiting hyaluronidase activity

Gary N. Cherr, Stuart A Meyers, Ashley I. Yudin, Catherine A. VandeVoort, Diana G. Myles, Paul Primakoff, James W. Overstreet

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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 ≥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.

Original languageEnglish (US)
Pages (from-to)142-153
Number of pages12
JournalDevelopmental Biology
Volume175
Issue number1
DOIs
StatePublished - Apr 10 1996

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Hyaluronoglucosaminidase
Macaca
Spermatozoa
Acrosome Reaction
Acrosome
Proteins
Acids
Enzymes
Apigenin
Peptides
Immunoglobulin Fab Fragments
Membranes
Phospholipases
Antibodies
Glycoside Hydrolases
Exocytosis
Hyaluronic Acid
Electrophoresis
Polyacrylamide Gel Electrophoresis
Peptide Hydrolases

ASJC Scopus subject areas

  • Developmental Biology

Cite this

The PH-20 protein in cynomolgus macaque spermatozoa : Identification of two different forms exhibiting hyaluronidase activity. / Cherr, Gary N.; Meyers, Stuart A; Yudin, Ashley I.; VandeVoort, Catherine A.; Myles, Diana G.; Primakoff, Paul; Overstreet, James W.

In: Developmental Biology, Vol. 175, No. 1, 10.04.1996, p. 142-153.

Research output: Contribution to journalArticle

Cherr, Gary N. ; Meyers, Stuart A ; Yudin, Ashley I. ; VandeVoort, Catherine A. ; Myles, Diana G. ; Primakoff, Paul ; Overstreet, James W. / The PH-20 protein in cynomolgus macaque spermatozoa : Identification of two different forms exhibiting hyaluronidase activity. In: Developmental Biology. 1996 ; Vol. 175, No. 1. pp. 142-153.
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