Disulfide formation in bovine zona pellucida glycoproteins during fertilization: evidence for the involvement of cystine cross-linkages in hardening of the zona pellucida

The time for solubilization of the bovine zona pellucida in a hypotonic buf fer containing 5% (v/v) beta-mercaptoethanol and 7 mol urea l(-1) increased by 10% after fertilization. Coupling with a specific fluorescent thiol pro be, monobromobimane (mBBr), was markedly greater in the zona pellucida of o varian eggs compared with fertilized eggs, indicating that the cysteine res idues in the zona pellucida of unfertilized eggs are oxidized to cystines d uring fertilization. After endo-P-galactosidase digestion to remove N-acety llactosamine repeats of the carbohydrate chains, three zona pellucida glyco proteins (ZPA, ZPB and ZPC) coupled with the fluorescent bimane groups were fractionated efficiently by reverse-phase HPLC. Estimation of bimane group s in the three components and SDS-PAGE revealed that intramolecular disulfi de bonds in ZPA and intra- and intermolecular disulfide bonds in ZPB were f ormed during fertilization, but oxidation of cysteine residues in ZPC was l ow. Specific proteolysis of ZPA during fertilization was also observed. The se results indicate that the formation of disulfide linkages together with specific proteolysis result in the construction of a rigid zona pellucida s tructure, which is responsible for hardening of the zona pellucida.