EVALUATING ZONA-PELLUCIDA STRUCTURE AND FUNCTION USING ANTIBODIES TO RABBIT 55 KDA ZP PROTEIN EXPRESSED IN BACULOVIRUS EXPRESSION SYSTEM

A cDNA encoding the rabbit 55 kDa ZP protein was expressed using a bac ulovirus expression system and was evaluated for its ability to elicit antibodies which may interfere with sperm-ZP interaction. The express ed glycosylated protein, BV55, was purified by wheat germ agglutinin l ectin affinity chromatography. Antisera made in guinea pigs immunized with BV55 (GP-alpha-BV55) is specific for the 55 kDa rabbit ZP protein . Indirect immunofluorescence studies indicate that GP-alpha-BV55 loca lizes to a filamentous meshwork on the surface of the ZP of isolated r abbit eggs. Immunohistochemical analysis of rabbit ovaries demonstrate d that this antigen is localized within the ZP of primary and more adv anced stage ovarian follicles but is not detected in primordial follic les. In addition, the 55 kDa antigen was detected in the granulosa cel ls of secondary stage follicles but not in the oocyte. GP-alpha-BV55 e ffectively blocked the binding of rabbit sperm to rabbit eggs in vitro . However, Fab fragments generated from GP-alpha-BV55 failed to block sperm binding, suggesting that the inhibitory effect of GP-alpha-BV55 was due to stearic hindrance rather than specific blocking of a sperm receptor site. Although the Fab fragment did not inhibit sperm binding , additional studies demonstrated that biotinylated BV55 protein bound to rabbit sperm in the acrosomal region in a manner consistent with l igand activity in the sperm-ZP interaction, and that BV55 bound to rab bit sperm in a dose-dependent manner. These studies therefore demonstr ate that antibodies against recombinant ZP proteins recognize the nati ve intact ZP and inhibit sperm-ZP interaction. They also provide evide nce that the rabbit 55 kDa ZP protein, which is the homolog of the pig ZP3 alpha sperm receptor protein, has sperm receptor activity. (C) 19 96 Wiley-Liss, Inc.