Involvement of Trypsin-Like Activity in Binding of Mouse Spermatozoa to Zonae Pellucidae

Work from a number of laboratories has shown that fertilization is blocked in the presence of protease inhibitors, although the specific site of inhibition has not been identified. The present experiments were designed to discriminate between sperm binding to zonae pellucidae as opposed to sperm penetration through zonae, so as to assess the effect of protease inhibitors on these two distinct events. Exposure of capacitated mouse spermatozoa to a variety of protease inhibitors directed against trypsin blocked sperm binding to zonae in a concentration-dependent manner. A chymotrypsin-directed inhibitor was not capable of blocking sperm binding to zonae. The trypsin inhibitors did not affect sperm penetration through zonae nor gamete membrane fusion if the sperm had established a firm association with the zona surface before addition of the inhibitors. Previous incubation of zona-intact eggs with the inhibitors did not lead to a reduction in sperm binding, indicating that the activity affected by the inhibitors is borne by spermatozoa. Interaction between spermatozoa and the zona surface appeared to be the specific locus of inhibition; sperm binding to zona-free eggs (i.e., binding to the egg plasma membrane) was unaltered by the trypsin inhibitors. These results suggest a reevaluation of the function of proteases in fertilization focusing on their role in initial sperm contact with the zona pellucida.