SPERM SURFACE PROTEIN PH-20 IS BIFUNCTIONAL - ONE ACTIVITY IS A HYALURONIDASE AND A 2ND, 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-inta ct sperm, PH-20 has a hyaluronidase activity that is required for sper m penetration through the cumulus cell layer that surrounds the oocyte . On acrosome-reacted sperm, PH-20 has a required function in sperm-zo na binding (secondary binding). Because hyaluronic acid (HA) has been detected in the zona pellucida, secondary sperm-zona adhesion could de pend on repetitive binding and hydrolysis of HA by PH-20 acting as a h yaluronidase. Alternatively, PH-20 may be bifunctional and have a seco nd, different activity required for secondary binding. To distinguish between these two possibilities, in this study we used reagents that i nhibit either PH-20's function in sperm-zona binding or its hyaluronid ase activity. We found that an anti-PH-20 monoclonal antibody that inh ibited sperm-zone binding (similar to 90%) had no effect on hyaluronid ase activity. Conversely, apigenin, a hyaluronidase inhibitor, blocked PH-20 hyaluronidase activity 93% without inhibiting sperm-zona bindin g. Similarly, another anti-PH-20 monoclonal antibody that inhibited hy aluronidase activity 95% only partially inhibited sperm-zone binding ( similar to 45%). We also extensively pretreated oocytes with hyaluroni dase to remove all accessible HA on or in the zona pellucida and found little or no effect on secondary sperm-zone binding. Our results sugg est that PH-20 is bifunctional and has two activities: a hyaluronidase activity and a second, separate activity required for secondary sperm -zone binding.