Sperm surface protein PH-20 is bifunctional: one activity is a hyaluronidase and a second, distinct activity is required in secondary sperm-zona binding.
In previous studies, we have found that the sperm membrane protein PH-20 acts during two different stages of fertilization. On acrosome-intact sperm, PH-20 has a hyaluronidase activity that is required for sperm penetration through the cumulus cell layer that surrounds the oocyte. On acrosome-reacted sperm, PH-20 has a required function in sperm-zona binding (secondary binding). Because hyaluronic acid (HA) has been detected in the zona pellucida, secondary sperm-zona adhesion could depend on repetitive binding and hydrolysis of HA by PH-20 acting as a hyaluronidase. Alternatively, PH-20 may be bifunctional and have a second, different activity required for secondary binding. To distinguish between these two possibilities, in this study we used reagents that inhibit either PH-20's function in sperm-zona binding or its hyaluronidase activity. We found that an anti-PH-20 monoclonal antibody that inhibited sperm-zona binding (approximately 90%) had no effect on hyaluronidase activity. Conversely, apigenin, a hyaluronidase inhibitor, blocked PH-20 hyaluronidase activity 93% without inhibiting sperm-zona binding. Similarly, another anti-PH-20 monoclonal antibody that inhibited hyaluronidase activity 95% only partially inhibited sperm-zona binding (approximately 45%). We also extensively pretreated oocytes with hyaluronidase to remove all accessible HA on or in the zona pellucida and found little or no effect on secondary sperm-zona binding. Our results suggest that PH-20 is bifunctional and has two activities: a hyaluronidase activity and a second, separate activity required for secondary sperm-zona binding.
Hunnicutt
G R
GR
University of Connecticut Health Center, Department of Physiology, Farmington 06030-3505, USA.
Primakoff
P
P
Myles
D G
DG
D000177:Acrosome / Q000502:physiology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002448:Cell Adhesion / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D020843:Chamomile; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005419:Flavonoids / Q000494:pharmacology; D006168:Guinea Pigs; D006821:Hyaluronoglucosaminidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008297:Male; D009822:Oils, Volatile / Q000494:pharmacology; D010946:Plants, Medicinal; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000378:metabolism / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D004791:Enzyme Inhibitors; D005419:Flavonoids; D009822:Oils, Volatile; D006821:Hyaluronoglucosaminidase; C066852:hyaluronidase PH-20