Baculovirus-expressed recombinant human zona pellucida glycoprotein-B induces acrosomal exocytosis in capacitated spermatozoa in addition to zona pellucida glycoprotein-C.
To facilitate our understanding of the role of zona pellucida glycoproteins during fertilization in humans, recombinant human zona pellucida glycoprotein-A (hZPA), -B (hZPB) and -C (hZPC) were obtained by using Escherichia coli and baculovirus expression systems. Analysis by SDS-PAGE and Western blot of the Ni-NTA affinity purified recombinant proteins revealed that the baculovirus-expressed hZPA, hZPB and hZPC have an apparent molecular weight of approximately 110, approximately 70-75 and approximately 65 kDa, respectively, as compared to approximately 80, approximately 65 and approximately 50 kDa of the respective E. coli-expressed proteins. Lectin binding studies revealed that the baculovirus-expressed recombinant zona proteins were glycosylated. Major oligosaccharides were represented by strong reactivity with Concanavalin A (mannose alpha 1-3 or mannose alpha 1-6 residues) and Jacalin (alpha-O glycosides of Gal or GalNAc moieties). A significant increase in acrosomal exocytosis was observed when capacitated human sperm were incubated in vitro with baculovirus-expressed hZPB (P=0.0005) and hZPC (P=0.0005) The E. coli-expressed hZPB, hZPC and baculovirus-expressed hZPA failed to induce any significant increase (P>0.05) in acrosome reaction. In contrast to hZPC, the acrosome reaction induced by recombinant hZPB was not inhibited by pertussis toxin. These studies, for the first time, have demonstrated that in humans, ZPB also induces acrosomal exocytosis through a Gi independent pathway.
Chakravarty
Sanchita
S
Gamete Antigen Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110 067, India.
Suraj
K
K
Gupta
Satish Kumar
SK
D020101:Acrosome Reaction; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D004527:Egg Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D005089:Exocytosis; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D037342:Pertussis Toxin / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D013075:Sperm Capacitation / Q000187:drug effects*; D013094:Spermatozoa / Q000187:drug effects*; D000072216:Zona Pellucida Glycoproteins
D004527:Egg Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C000606517:ZP3 protein, human; C082833:ZP4 protein, human; D000072216:Zona Pellucida Glycoproteins; D037342:Pertussis Toxin