Formation of the rabbit zona pellucida and its relationship to ovarian follicular development

The zona pellucida is the unique extracellular glycoprotein matrix which is assembled during growth of the mammalian oocyte. The present studies were carried out to examine the formation of this structure in relation to the differentiation of ovarian cell types during follicular development. Specific antibodies were developed against total rabbit ZP proteins as well as against ZP proteins electrophoretically purified by high-resolution two-dimensional polyacrylamide electrophoresis gels (2D-PAGE). Antibodies were characterized by (a) immunoelectrophoresis, (b) a Staphylococcus aureus protein A binding assay, and (c) immunoblotting following 2D-PAGE separation of ZP proteins. Immunoperoxidase localization with these antibodies was used to determine the stage of ovarian follicular development at which ZP antigens first appear as well as to evaluate the cellular and extracellular distribution of these proteins throughout folliculogenesis. The ZP proteins were first observed in the cytoplasm and at the periphery of the oocytes surrounded by a thin squamous follicular cell layer. No staining was observed in the cytoplasm of follicle cells during early folliculogenesis. As the ZP matrix was assembled extracellularly, the intensity of staining of the outer and inner regions could be distinguished. This differentiation of the matrix coincided with the differentiation of the follicular cells into a multilayer cell complex. At this stage, specific ZP proteins are localized within the cytoplasm of the inner layers of these follicular cells. The staining is then diminished in cells of preantral follicles. These studies demonstrate that the formation of the ZP is an excellent model system to study the early stages of follicular development and cell differentiation.