Effects of native human zona pellucida glycoproteins 3 and 4 on acrosome reaction and zona pellucida binding of human spermatozoa

Acrosome reaction is crucial to the penetration of spermatozoa through the zona pellucida (ZP). Glycosylation of ZP glycoproteins is important in spermatozoa-ZP interaction. Human ZP glycoprotein-3 (ZP3) is believed to initiate acrosome reaction. Recently, human ZP4 was also implicated in inducing acrosome reaction. These studies were based on recombinant human ZP proteins with glycosylation different from their native counterparts. In the present study, the effects of native human ZP3 and ZP4 on acrosome reaction and spermatozoa-ZP binding were investigated. Native human ZP3 and ZP4 were immunoaffinity-purified. They induced acrosome reaction and inhibited spermatozoa-ZP binding time- and dose-dependently to different extents. These biological activities of human ZP3 and ZP4 depended partly on their glycosylation, with N-linked glycosylation contributing much more significantly than O-linked glycosylation. Studies with inhibitors showed that both human ZP3- and ZP4-induced acrosome reactions were protein kinase-C, protein tyrosine kinase, T-type Ca²⁺ channels, and extracellular Ca²⁺ dependent. G-protein also participated in human ZP3- but not in ZP4-induced acrosome reaction. On the other hand, protein kinase-A and L-type Ca ²⁺ channels took part only in human ZP4-induced acrosome reaction. This manuscript describes for the first time the actions of purified native human ZP3 and ZP4 on acrosome reaction and spermatozoa-ZP binding.