The PH-20 protein in cynomolgus macaque spermatozoa: identification of two different forms exhibiting hyaluronidase activity.
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.
Cherr
G N
GN
Department of Environmental Toxicology, University of California, Davis, 95616 USA.
Meyers
S A
SA
Yudin
A I
AI
VandeVoort
C A
CA
Myles
D G
DG
Primakoff
P
P
Overstreet
J W
JW
D000177:Acrosome / Q000737:chemistry / Q000201:enzymology; D000818:Animals; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000302:isolation & purification*; D002451:Cell Compartmentation; D020843:Chamomile; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005419:Flavonoids / Q000494:pharmacology; D006821:Hyaluronoglucosaminidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification; D008252:Macaca fascicularis; D008297:Male; D016253:Microscopy, Immunoelectron; D009822:Oils, Volatile / Q000494:pharmacology; D010946:Plants, Medicinal; D013075:Sperm Capacitation / Q000502:physiology*; D013094:Spermatozoa / Q000737:chemistry* / Q000201:enzymology*
D015815:Cell Adhesion Molecules; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007527:Isoenzymes; D009822:Oils, Volatile; D006821:Hyaluronoglucosaminidase; C066852:hyaluronidase PH-20