Sv. Prasad et al., EVALUATING ZONA-PELLUCIDA STRUCTURE AND FUNCTION USING ANTIBODIES TO RABBIT 55 KDA ZP PROTEIN EXPRESSED IN BACULOVIRUS EXPRESSION SYSTEM, Molecular reproduction and development, 43(4), 1996, pp. 519-529
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.