Abstract
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.
Highlights
The 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
Characteristics of baculovirus-expressed recombinant human ZP1273-551aa The nucleotide sequencing of the cDNA encoding ZP1273-551aa, PCR amplified from human ovarian cDNA library, revealed three changes at 858, 867 and 1590 nt positions as compared to human ZP1 sequence already published in Genbank (NM_207341)
Recombinant human ZP1273-551aa expressed in Sf21 insect cells was present both in the cell lysate as well as the 10× concentrated culture supernatant (Figure 1, panel b and c respectively)
Summary
The 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 ZP matrix plays a crucial role by serving as a substrate for sperm binding, as well as an agonist for regulated exocytosis of the spermatozoon’s acrosomal vesicle and facilitates avoidance of polyspermy [1]. Human ZP matrix is composed of 4 glycoproteins designated as zona pellucida glycoprotein-1 [ZP1; 638 amino binding to the human sperm and induction of acrosome reaction [16], whereas in murine model, ZP1 has been postulated to cross-link the filaments formed by ZP2ZP3 heterodimers [17] and may not have any direct role in induction of acrosome reaction [18]. The role of ZP-C sub-domain is not as yet clearly defined
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