Hyaluronidase activity of macaque sperm assessed by an in vitro cumulus penetration assay

A model system consisting of cynomolgus macaque sperm and ovulated hamster ova-cumulus complexes (OCCs) was utilized to study the role of the sperm protein PH-20 in cumulus penetration. The hyaluronidase activity of solubilized macaque sperm PH-20 was evaluated using an ELISA-like microplate assay prior to and following the addition of the hyaluronidase inhibitors heparin (0-100 microg/ml) and apigenin (250 microM), as well as the Ig fraction of a polyclonal antibody raised against purified recombinant macaque PH-20 (R10; 10-400 microg/ml). Sperm motility following 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 ovulated 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). In the heterologous cumulus penetration assay, fluorescently labeled macaque sperm that were pretreated with heparin (1-100 microg/ml), apigenin (250 microM), or R10 antibody (Ig fraction, 10-400 microg/ml) demonstrated a dose-dependent decrease in the ability to penetrate hamster OCCs (P < 0.01), in the absence of effects on sperm motility. In the homologous assay, experiments using macaque OCCs and fluorescently labeled macaque 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 hyaluronidase participates in cumulus penetration in this species, and that this model system is useful for further studies into primate gamete interaction.