These studies revealed for the first time that in humans ZP1, in addition to ZP3 and ZP4, binds to capacitated spermatozoa and induces acrosomal exocytosis.

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Recent studies document that the zona pellucida matrix of human oocytes is composed of four glycoproteins designated as ZP1, ZP2, ZP3, and ZP4 instead of three as proposed in mouse model. In the present study, investigations were carried out to find the presence of the fourth ZP glycoprotein in the ovaries of non-human primates namely bonnet monkey (Macaca radiata). Employing total RNA isolated from bonnet monkey ovaries, the complementary deoxyribonucleic acid (cDNA) encoding bonnet monkey ZP1 (up to furin cleavage site) was successfully amplified by reverse transcribed polymerase chain reaction (RT-PCR). The deduced amino acid (aa) sequence of bonnet monkey ZP1 revealed 96.0% identity with human ZP1. The 21 cysteine residues present in bonnet monkey ZP1 were conserved in human, mouse, rat, quail, and chicken. Simultaneously, polyclonal antibodies were generated in mice against synthetic peptides corresponding to human ZP1 (P1, 137-150 aa; P2, 223-235 aa; P3, 237-251 aa; P4, 413-432 aa; and P5, 433-451 aa). Employing anti-peptide antibodies that were devoid of cross-reactivity as determined by ELISA with human/bonnet monkey recombinant ZP2, ZP3, and ZP4, the presence of ZP1 in the ovaries of bonnet monkey and human oocytes was demonstrated by indirect immunofluorescence. The antibodies against peptides P3 and P4 reacted only with the ZP of bonnet monkey ovaries and not other ovarian associated cells. The data presented in this manuscript provide evidence, for the first time, that the bonnet monkey ZP matrix is composed of four glycoproteins.

BackgroundThe human egg coat, zona pellucida (ZP), is composed of four glycoproteins designated as zona pellucida glycoprotein-1 (ZP1), -2 (ZP2), -3 (ZP3) and -4 (ZP4) respectively. The zona proteins possess the archetypal 'ZP domain', a signature domain comprised of approximately 260 amino acid (aa) residues. In the present manuscript, attempts have been made to delineate the functional significance of the 'ZP domain' module of human ZP1, corresponding to 273-551 aa fragment of human ZP1.MethodsBaculovirus-expressed, nickel-nitrilotriacetic acid affinity chromatography purified 'ZP domain' of human ZP1 was employed to assess its capability to bind and subsequently induce acrosomal exocytosis in capacitated human spermatozoa using tetramethyl rhodamine isothiocyanate conjugated Pisum sativum Agglutinin in absence or presence of various pharmacological inhibitors. Binding characteristics of ZP1 'ZP domain' were assessed employing fluorescein isothiocyanate (FITC) labelled recombinant protein.ResultsSDS-PAGE and immunoblot characterization of the purified recombinant protein (both from cell lysate as well as culture supernatant) revealed a doublet ranging from ~35-40 kDa. FITC- labelled 'ZP domain' of ZP1 binds primarily to the acrosomal cap of the capacitated human spermatozoa. A dose dependent increase in acrosomal exocytosis was observed when capacitated sperm were incubated with recombinant 'ZP domain' of human ZP1. The acrosome reaction mediated by recombinant protein was independent of Gi protein-coupled receptor pathway, required extra cellular calcium and involved both T- and L-type voltage operated calcium channels.ConclusionsResults described in the present study suggest that the 'ZP domain' module of human ZP1 has functional activity and may have a role during fertilization in humans.

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