Relevance of Glycosylation of Human Zona Pellucida Glycoproteins for Their Binding to Capacitated Human Spermatozoa and Subsequent Induction of Acrosomal Exocytosis

Abstract

To delineate the functional aspects of zona pellucida (ZP) glycoproteins during fertilization in human, in the present study, fluorochrome-conjugated Escherichia coli (E. coli)- and baculovirus-expressed recombinant human ZP glycoprotein-2 (ZP2), -3 (ZP3), and -4 (ZP4) were employed. In an immunofluorescence assay, capacitated human sperm exhibited binding of the baculovirus-expressed recombinant ZP3 as well as ZP4 to either acrosomal cap or equatorial region whereas acrosome-reacted sperm failed to show any binding to the acrosomal cap. Using double labeling experiments, simultaneous binding of ZP3 and ZP4 to the acrosomal cap was observed suggesting the possibility of different binding sites of these proteins on the sperm surface. No binding of ZP2 was observed to the capacitated sperm. However, acrosome-reacted sperm (20.00 +/- 1.93%) showed binding of ZP2 that was restricted to only equatorial region. Interestingly, E. coli-expressed recombinant human zona proteins also showed very similar binding profiles. Competitive inhibition studies with unlabeled recombinant human zona proteins revealed the specificity of the above binding characteristics. Binding characteristics have been further validated by an indirect immunofluorescence assay using native human heat solubilized isolated zona pellucida. Employing baculovirus-expressed recombinant ZP3 and ZP4 with reduced N-linked glycosylation and respective E. coli-expressed recombinant proteins, it was observed that glycosylation is required for induction of acrosomal exocytosis but its absence may not compromise on their binding ability. These studies have revealed the binding profile of individual human zona protein to spermatozoa and further strengthened the importance of glycosylation of zona proteins for acrosomal exocytosis in spermatozoa.