HYALURONIDASE ACTIVITY OF MACAQUE SPERM ASSESSED BY AN IN-VITRO CUMULUS PENETRATION ASSAY

A model system consisting of cynomolgus macaque sperm and ovulated ham ster ova-cumulus complexes (OCCs) was utilized to study the role of th e sperm protein PH-20 in cumulus penetration. The hyaluronidase activi ty of solubilized macaque sperm PH-20 was evaluated using an ELISA-lik e microplate assay prior to and following the addition of the hyaluron idase inhibitors heparin (0-100 mu g/ml) and apigenin (250 mu M), as w ell as the Ig fraction of a polyclonal antibody raised against purifie d recombinant macaque PH-20 (R10; 10-400 mu g/ml). Sperm motility foll owing exposure to enzyme inhibitors was evaluated using computer-aided sperm motility analysis. Macaque sperm were labeled with the permeant fluorescent nuclear dye, Hoechst 33342, and were coincubated with ovu lated hamster OCCs for 30 min at 37 degrees C. The addition of heparin , apigenin, or R10 antibody to solubilized sperm extracts resulted in a linear dose-dependent decrease in hyaluronidase activity(P < .01). I n the heterologous cumulus penetration assay, fluorescently labeled ma caque sperm that were pretreated with heparin (1-100 mu g/ml), apigeni n (250 mu M), or R10 antibody (Ig fraction, 10-400 mu g/ml) demonstrat ed a dose-dependent decrease in the ability to penetrate hamster OCCs (P < 0.01), in the absence of effects on sperm motility. In the homolo gous assay, experiments using macaque OCCs and fluorescently labeled m acaque sperm confirmed that the same concentrations of heparin and R10 antibody similarly suppressed spermatozoal cumulus penetration (P < . 01). These results suggest that macaque sperm PH-20-derived hyaluronid ase participates in cumulus penetration in this species, and that this model system is useful for further studies into primate gamete intera ction. (C) 1997 Wiley-Liss, Inc.