Monoclonal Antibodies against Pig Ovarian Follicular Granulosa Cells Induce Apoptotic Cell Death in Cultured Granulosa Cells

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Abstract
Two monoclonal antibodies capable of inducing granulosa cell apoptosis were produced against granulosa cells prepared from antral follicles of pig ovaries. The healthy follicles, 4-5 mm in diameter, were dissected from the ovaries of gilts, and then granulosa cells were isolated. BALB/c female mice were immunized with the isolated granulosa cells. Antibodies against the granulosa cells were detected by immunofluorescent staining using frozen ovarian sections. The isolated spleen cells prepared from immunized mice producing antibodies against the granulosa cells were fused with Sp2/O-Ag14 mouse myeloma cells by standard hybridization techniques. Two hybridoma clones, PFG-1 and PFG-2, which produced specific IgM antibodies against granulosa cells were selected. Western blotting analysis revealed that PFG-1 and PFG-2 antibodies specifically recognized cell-membrane proteins with molecular weights of 55 and 70 kD and isoelectric points of 5.9 and 5.4, respectively. The monoclonal antibodies immunohistochemically reacted with granulosa cells of healthy follicles. When the isolated granulosa cells prepared from healthy follicles were cultured in medium containing 0.1 or 10 μg/m l PFG-1 or PFG-2 antibodies, respectively, the cells underwent apoptosis as determined by nuclear morphology, DNA electrophoresis and flow cytometric analysis. In conclusion, these two monoclonal antibodies against granulosa cells have cell-killing activity in cultured granulosa cells.