HUMAN ZONA-PELLUCIDA GLYCOPROTEINS - CHARACTERIZATION USING ANTIBODIES AGAINST RECOMBINANT NONHUMAN PRIMATE ZP1, ZP2 AND ZP3

Characterization and classification of human zona pellucida glycoprote ins is essential to understand the functions of these components durin g fertilization. To achieve this, antibodies were raised in rabbits ag ainst recombinant non-human primate [Bonnet Monkey (Macaca radiata)] z ona pellucida proteins, bmZP1, bmZP2 and bmZP3 expressed in Escherichi a coli. Antibodies against the three recombinant zona proteins reacted with human zonae as revealed by indirect immunofluorescence. Such ant ibodies were used as specific probes to further characterize human zon a pellucida glycoproteins in Western blot of heat solubilized human zo nae pellucidae (hSIZP) resolved by one dimensional sodium dodecyl sulp hate-polyacrylamide gel electrophoresis (SDS-PAGE). Under non-reduced conditions human (h) hZP1, hZP2 and hZP3 resolved as 60, 100 and 53 kD a bands respectively. Under reduced conditions, dominant reactivity of hZP1, hZP2 and hZP3 was localized to 63, 65 and 58 kDa and faint reac tivity to 53, 96 and 138 kDa bands respectively. In two-dimensional SD S-PAGE, hZP1 was shown to comprise two chains at 63-58 and 55-45 kDa, each consisting of multiple isomers. hZP2 was less acidic when compare d with hZP1 and hZP3 and comprised a major component of 65 kDa and a m inor component of similar to 96 kDa. The 65 kDa component displayed a higher degree of charged isomers in comparison with the 96 kDa compone nt, hZP3 comprised a broad band in the range 68-58 kDa. These studies show conclusively that the hZP1 heavy train overlaps with hZP3 and tha t in previous studies, hZP2 was likely to have been misinterpreted as being hZP1. Our studies failed to distinguish two distinct species of hZP3, unlike previous reports. These studies will further help in our understanding of the nature of human zona pellucida glycoproteins.