Localization of species conserved zona pellucida antigens in mammalian ovaries

The mammalian zona pellucida (ZP) consists of three glycoproteins (ZP1, ZP2 and ZP3), which are variably conserved among species at the genomic and amino acid levels. In order to evaluate the expression of ZP during ovarian development, a population of antibodies was selected that recognize species conserved antigenic domains of the three ZP proteins. Domain specific antibodies were selected from sera of rabbits immunized with all three native pig ZP proteins by elution of antibodies bound to each of the three human ZP recombinant proteins expressed from cDNAs, using the baculovirus expression system in insect cells. Immunoblot analysis was used to characterize the specificity of the antibodies and immunohistochemistry was used to evaluate the stage specific expression of ZP proteins during ovarian follicular development of the mouse, baboon and human. This study demonstrates that the conserved domains of all three ZP proteins are localized in the oocyte extracellular ZP matrix as well as in a subset of granulosa cells. However, this expression does vary among species with respect to the stage and cell type during early stages of ovarian follicular development. These antibodies should serve as excellent markers for evaluating early stages of human ovarian follicular development and in the development of contraceptive agents.