Purification of native human zona pellucida glycoproteins from eggs: Binding characteristics to human spermatozoa, hyper-activation, acrosomal exocytosis, and sperm-oocyte interaction

Abstract

Fertilization starts with the binding of the spermatozoa to the zona pellucida (ZP) of the oocyte. Such binding is a carbohydrate-mediated event and consists of a series of tightly regulated events. Molecular interactions between spermatozoon and ZP in human are not well characterized due to limited availability of oocytes for research. Our current technology cannot generate recombinant human ZP glycoproteins with native glycosylation. In this study, human ZP glycoproteins, hZP2 (~120 kDa), hZP3 (~58 kDa) and hZP4 (~ 65 kDa) were purified from zonae pellucidae (purity .88%) by immunoaffinity columns. The binding sites of the purified native hZP3 and hZP4 were localized to the acrosome region of the capacitated human spermatozoa, and were lost after acrosome reaction. Purified hZP2 bound to this region only in acrosomereacted spermatozoa. Differential binding of the three glycoproteins to the post-acrosomal region and the mid-piece of the spermatozoa was observed. Both hZP3 and hZP4 induced acrosome reaction and inhibited spermatozoa-ZP binding time- and concentration-dependently to different extents. In addition, hZP3, but not hZP2 and hZP4, induced hyperactivation. These biological activities of the glycoproteins were dependent partly on their glycosylation. Both the N-linked and O-linked glycosylation contributed to the observed activities of the ZP glycoproteins in humans, though the former seemed to have a greater impact. The results describe for the first time, the biological activities of the purified human ZP glycoproteins from the native source.