MONOCLONAL-ANTIBODIES AGAINST PIG OVARIAN FOLLICULAR GRANULOSA-CELLS INDUCE APOPTOTIC CELL-DEATH IN CULTURED GRANULOSA-CELLS

Two monoclonal antibodies capable of inducing, granulosa cell apoptosi s were produced against granulosa cells prepared from antral follicles of pig ovaries. The healthy follicles, 4-5 mm in diameter, were disse cted 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 immunofluores cent staining using frozen ovarian sections. The isolated spleen cells prepared from immunized mice producing antibodies against the granulo sa cells were fused with Sp2/O-Agl4 mouse myeloma cells by standard hy bridization techniques. Two hybridoma clones, PFG-1 and PFG-2, which p roduced specific IgM antibodies against granulosa cells were selected. Western blotting analysis revealed that PFG-1 and PFG-2 antibodies sp ecifically 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 ce lls of healthy follicles. When the isolated granulosa cells prepared f rom healthy follicles were cultured in medium containing 0.1 or 10 mu g/ml PFG-1 or PFG-2 antibodies, respectively, the cells underwent apop tosis as determined by nuclear morphology, DNA electrophoresis and flo w cytometric analysis. In conclusion, these two monoclonal antibodies against granulosa cells have cell-killing activity in cultured granulo sa cells.