| Abstrakt: |
ZP3α, 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 (α8–18) corresponding to an amino-terminal sequence of ZP3α (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 ZP3α and ZP3β core proteins demonstrated reactivity of antipeptide sera with homologous protein. Preincubation of antisera with cognate peptide blocked binding to ZP3α thus confirming site-specific binding of antibodies to the targeted sequence in the intact protein. Anti-KLH-α8–18 sera reacted poorly with fully glycosylated ZP3α, but ZP3α immunoreactivity was markedly enhanced by digestion of polylactosamines with endo-β-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 KLH-α8–18 KLH-α8–18 sera significantly diminished attachment of boar spermatozoa to zonae. These studies demonstrate that the amino-terminal domain of pig oocyte zona protein, ZP3α, 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. |
