Generation and Immunohistological Characterization of Monoclonal Antibodies against Rat Ovarian Antigens

Hybridoma cell lines were obtained from mouse splenocytes sensitized to granulosa cells and oocytes collected from rat ovaries at the time of gonadotropin stimulation. Each of the Mabs was characterized by its cell type-specific binding reaction using immunofluorescence, avidin-biotin-peroxidase complex, immunogold-silver staining, and some of the Mabs were characterized by electron microscopic immunocytochemistry. Mabs were divided into four classes. On the basis of the immunohistochemical results and a secondary assay, ELISA with conditioned medium containing granulosa cell-secreted proteins (GSPs) we have concluded: (a) The Mabs (Class 1) reacted with GSPs and recognized antigens located on the plasma membrane of granulosa cells. The reaction depends both on the degree of follicular development and on the position of the cells within the follicles. (b) The general pattern of binding of Mab 2E9 (Class 2) was restricted only to the germinal vesicles of oocytes in antral and Graafian follicles. This result indicated a nuclear modification during the progressive phases of oocyte growth. (c) Same antigens recognized by Mabs from Class 3 and 4 were distributed in the cytoplasm of the oocytes and are a constant feature of all growing oocytes displaying a redistribution during meiotic maturation.