Apoptosis in Porcine Granulosa Cells During Follicular Atresia Is Regulated by Anti-Apoptotic cFLIP.

More than 99% of follicles are selectively eliminated via a degenerative process called gatresiah during follicular development and growth. Follicular atresia is indicated to begin with granulosa cell apoptosis. To reveal the molecular mechanisms of follicle selection, we examined changes in the expression levels of cellular FLICE-like inhibitory protein (cFLIP), which is a homologue of an intracellular apoptosis inducer (procaspase-8/FLICE), has two alternative splicing isoforms (cFLIP short and long form: cFLIPS and cFLIPL, respectively) and inhibits apoptosis initiated by death receptors, in porcine granulosa cells during atresia. In granulosa cells, we detected mRNA and protein of cFLIPL but no cFLIPS. mRNA and protein of cFLIPL were highly expressed in granulosa cells of healthy follicles and rapidly decreased during atresia. We examined changes in the localization of cFLIPL mRNA and protein in pig ovaries using in situ hybridization and immunohistochemistry, respectively, and found that they were abundant in granulosa cell layer of healthy follicles in comparison with that in those of atretic follicles. Moreover, when cFLIPL was stably expressed in granulosa cell-derived KGN cells following transfection of an expression vector, the cell death ligand/receptor-mediated apoptosis was inhibited. Suppression of cFLIPL by small interfering RNA (siRNA) spontaneously induced cell death in cultured KGN cells. We suggested that cFLIPL plays an anti-apoptotic role in the granulosa cells of healthy follicles of pig ovaries, and that cFLIPL is an essential pro-survival factor for granulosa cells and acts as a key determinant of follicular growth and/or atresia by regulating granulosa cell apoptosis.