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(468) ANTI-APOPTOTIC PROTEIN, FLIP, IS EXPRESSED IN GRANULOSA CELLS OF PORCINE OVARIAN FOLLICLES. Matsuda, Fuko1, Goto, Yasufumi1, Matsui, Toshikatsu1, Inoue, Naoko1, Imakawa, Kazuhiko2, Manabe, Noboru1, 1 Unit of Anatomy and Cell Biology, Kyoto, Japan2 Laboratpry of Animal Breeding, Tokyo, Japan ABSTRACT- In mammalian ovaries, less than 1% of follicles reach ovulation, while the remainder undergoes atresia. Granulosa cell apoptosis plays a crucial role in the atretic process, the mechanisms of which are still largely unclear. Flice (procaspase-8) inhibitory protein (FLIP) is an intracellular anti-apoptotic factor. Its structure is similar to that of procaspase-8, but it has no proteolytic activity. FLIP interacts with the adapter protein, FADD, and precursor protease, procaspase-8, and inhibits cell-death receptor-dependent apoptosis induction. We identified porcine FLIP genes, the FLIP-long and -short forms, which show high homology with those of humans and mice, and examined the contribution of FLIPs on granulosa cell apoptosis during follicular atresia. FLIP mRNA was detected in granulosa cells by RT-PCR, and FLIP protein expression was confirmed by Western blotting. Protein expression levels were higher in the granulosa cells of healthy follicles than those of atretic follicles. Immunohistochemistry for FLIP also revealed that FLIP was localized in granulosa cells. These data indicate that FLIP may contribute to the survival of healthy follicles. Then, to evaluate the anti-apoptotic activity of FLIP, we performed an in vitro experiment. Porcine FLIP (pFLIP) expression vector was constructed and transfected into HeLa cells, and then apoptosis was induced by adding anti-Fas antibody and cycloheximide to the transfected cells. The apoptosis induction rate in pFLIP-transfected HeLa cells was lower than that in empty vector-transfected cells, indicating that pFLIP acts as a functional anti-apoptotic factor, the as human or murine FLIP. In conclusion, FLIP acts as a survival factor and plays important roles in follicle selection in porcine ovaries. KEY WORDS: Apoptosis, Follicular atresia, Porcine FLIP, Granulosa cell |
