In humans, zona pellucida glycoprotein-1 binds to spermatozoa and induces acrosomal exocytosis.
BACKGROUND
It has been suggested that the zona pellucida (ZP) may mediate species-specific fertilization. In human the ZP is composed of four glycoproteins: ZP1, ZP2, ZP3 and ZP4. In the present study, the expression profile of ZP1 in human oocytes and ovaries, and its role during fertilization, is presented.


METHODS
Human ZP1 (amino acid residues 26-551) was cloned and expressed in both non-glycosylated and glycosylated forms and its ability to bind to the capacitated human spermatozoa and to induce acrosomal exocytosis was studied. Monoclonal antibodies (MAbs), specific for human ZP1 and devoid of reactivity with ZP2, ZP3 and ZP4 were generated and used to localize native ZP1 in oocytes and ovarian tissues.


RESULTS
The MAbs generated against ZP1 recognized specifically the zona matrix of secondary and antral follicles, ovulated oocytes, atretic follicles and degenerating intravascular oocytes, but failed to react with the Fallopian tube, endometrium, ectocervix and kidney. Escherichia coli and baculovirus-expressed recombinant human ZP1 revealed bands of approximately 75 and approximately 85 kDa, respectively, in western blot. Lectin binding studies revealed the presence of both N- and O-linked glycosylation in baculovirus-expressed ZP1. Fluorescein isothiocyanate-labelled E. coli- and baculovirus-expressed recombinant ZP1 bound to the anterior head of capacitated spermatozoa, however, only baculovirus-expressed ZP1 induced acrosomal exocytosis in capacitated sperm suggesting the importance of glycosylation in mediating the acrosome reaction. The human ZP1-mediated acrosome reaction involved the activation of both T- and L-type voltage-operated calcium channels, but does not activate the G(i)-coupled receptor pathway. Inhibition of protein kinase A and C significantly also reduced the ZP1-mediated induction of the acrosome reaction.


CONCLUSIONS
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.
Ganguly
Anasua
A
Reproductive Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
Bukovsky
Antonin
A
Sharma
Raj K
RK
Bansal
Pankaj
P
Bhandari
Beena
B
Gupta
Satish K
SK
D000177:Acrosome / Q000502:physiology*; D020101:Acrosome Reaction; D000911:Antibodies, Monoclonal / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D004527:Egg Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005089:Exocytosis; D005260:Female; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D006080:Ovarian Follicle / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis; D013094:Spermatozoa / Q000378:metabolism*; D000072216:Zona Pellucida Glycoproteins
D000911:Antibodies, Monoclonal; D004527:Egg Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C000606514:ZP1 protein, human; C000606515:ZP2 protein, human; C000606517:ZP3 protein, human; D000072216:Zona Pellucida Glycoproteins