PH-20 but not acrosin is involved in sperm penetration of the macaque zonapellucida

In this study, we investigated the functions of PH-20 and acrosin during th e interaction of macaque sperm with the zona pellucida. Both of these sperm enzymes have been reported to be present on the inner acrosomal membrane o f acrosome reacted sperm, and have been suggested to play a role during sec ondary sperm-zona binding in other species. Anti-macaque PH-20 IgG, anti-pi g acrosin IgG and soybean trypsin inhibitor (SBTI) were used as probes for immunolocalization of the two proteins at the ultrastructural level, and as reagents for blocking sperm penetration of the macaque zona pellucida in v itro, As a control, we performed similar studies with antibodies to CD-46, which is also located on the inner acrosomal membrane, but has no known fun ction in sperm-zona pellucida interaction, After labeling with anti-acrosin IgG, gold label was not present on the sperm surface before the acrosome r eaction, but was detected over the entire head of sperm that were induced t o acrosome react with calcium ionophore A23187. In contrast, when sperm wer e induced to acrosome react by binding to intact zona pellucida, acrosin wa s present in the acrosomal shroud but not on the inner acrosomal membrane, Similar results were obtained when SBTI was used as a probe for enzyme loca lization. PH-20 and CD-46 were demonstrated on the inner acrosomal membrane of sperm induced to acrosome react by ionophore treatment and by zona bind ing. Neither anti-acrosin IgG nor anti-CD-46 IgG affected sperm penetration of the zona at concentrations up to 300 mu g/ml, but zona penetration was blocked completely when anti-PH-20 IgG (100 mu g/ml) was present during spe rm-oocyte interaction, Ultrastructural observations of oocytes incubated wi th anti-PH-20 IgG showed that acrosomal shrouds were present on the zona su rface but no sperm had begun to penetrate into the zona substance. We concl ude that anti-PH-20 IgG prevented sperm penetration of the macaque zona pel lucida by interference with secondary sperm-zona binding, rather than prima ry sperm-zona binding or the zona-induced acrosome reaction. Acrosin was no t detected on the inner acrosomal membrane of sperm that are induced to acr osome react after zona binding, and acrosin does not appear to be critical for sperm penetration of the macaque zona pellucida, Mol. Reprod. Dev, 53:3 50-362, 1999. (C) 1999 Wiley-Liss, Inc.