Generation and characterization of site-directed antisera against an amino-terminal segment of a 55 kDa sperm adhesive glycoprotein from zona pellucida of pig oocytes.
ZP3 alpha, a 55 kDa zona pellucida glycoprotein, mediates attachment of boar spermatozoa to zona-encased oocytes. In the study reported here, site-directed antipeptide antibodies were used to probe the topography and biological activity of the amino-terminal domain of this sperm adhesive zona molecule. A synthetic peptide (alpha 8-18) corresponding to an amino-terminal sequence of ZP3 alpha (residues 8-18) was coupled to keyhole limpet haemocyanin (KLH) using either m-maleimidobenzoic acid N-hydroxysuccinimide ester or glutaraldehyde. Male rabbits were immunized with peptide-KLH conjugates using Freund's adjuvant, and antibody production was monitored by ELISA. High-titred antipeptide sera were obtained and further characterized. Western blotting experiments using deglycosylated ZP3 alpha and ZP3 beta core proteins demonstrated reactivity of antipeptide sera with homologous protein. Preincubation of antisera with cognate peptide blocked binding to ZP3 alpha thus confirming site-specific binding of antibodies to the targeted sequence in the intact protein. Anti-KLH-alpha 8-18 sera reacted poorly with fully glycosylated ZP3 alpha, but ZP3 alpha immunoreactivity was markedly enhanced by digestion of polylactosamines with endo-beta-galactosidase, chemical deglycosylation or protein unfolding. Finally, zona-encased pig oocytes were preincubated with the antipeptide sera, washed and exposed to boar spermatozoa. Pretreatment with anti-KLH-alpha 8-18 sera significantly diminished attachment of boar spermatozoa to zonae. These studies demonstrate that the amino-terminal domain of pig oocyte zona protein, ZP3 alpha, is partially occluded, in part by carbohydrate, and that site-directed antipeptide sera targeted to this portion of the molecule can interfere with sperm-zona attachment in vitro.
Yurewicz
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Department of Obstetrics and Gynecology, Wayne State University School of Medicine, C.S. Mott Center for Human Growth and Development, Detroit, MI 48201.
Zhang
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Sacco
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D000818:Animals; D002478:Cells, Cultured; D004527:Egg Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D007106:Immune Sera / Q000302:isolation & purification*; D015151:Immunoblotting; D008297:Male; D008562:Membrane Glycoproteins / Q000276:immunology*; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000276:immunology*; D013084:Sperm-Ovum Interactions / Q000276:immunology; D013094:Spermatozoa / Q000378:metabolism*; D013552:Swine; D013997:Time Factors; D015044:Zona Pellucida / Q000276:immunology*; D000072216:Zona Pellucida Glycoproteins
D004527:Egg Proteins; D007106:Immune Sera; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C000606517:ZP3 protein, human; D000072216:Zona Pellucida Glycoproteins