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(799) CHANGES IN mRNA EXPRESSION OF TNF AND RECEPTOR FAMILY IN GRANULOSA CELLS DURING FOLLICULAR ATRESIA IN PIG OVARIES. Inoue, Naoko1, Nakayama, Mizuho1, Matsuda, Fuko1, Maeda, Akihisa1, CHENG, Yuan1, Manabe, Noboru1, 1 Unit of Anatomy and Cell Biology, Kyoto, Japan ABSTRACT- In mammalian ovaries, more than 99% of the follicles undergo a degenerative process, atresia, during follicle development. Recently, our findings have suggested that the degeneration of atretic follicles can be explained, at least in part, by the apoptotic cell death of granulosa cells. Tumor necrosis factor alpha (TNF) has two contrary roles, cell death and cell proliferation by binding to TNF receptor (TNFR) 1 or 2. When TNF-binding TNFR2 interacts with TNF receptor associated factor 2 (TRAF2; an indicator of a survival factor), a survival signal is induced. The expression of Fas ligand (FasL) and Fas has been demonstrated in porcine granulosa cells, however, the detailed changes in the expression and localization of their mRNAs have not yet been determined. We demonstrated the changes in expression and localization of signaling molecule (TNF, TRAF2, FasL and Fas) mRNAs by in situ hybridization during follicular atresia in pig ovaries, and then we quantified their mRNAs by real-time polymerase chain reaction (PCR). For in situ hybridization, formalin-fixed paraffin-embedded tissue sections of ovaries were prepared. For PCR analysis, mRNA was prepared from isolated granulosa cells of healthy, early atretic and progressed atretic follicles. TNF and TRAF2 were demonstrated in the outer zone of the granulosa layer (proliferating cell-rich area) of healthy follicles, but trace levels of both were seen in the granulosa layer of atretic follicles, suggesting that TNF may act as a survival factor in porcine follicles. FasL mRNA was highly expressed in the granulosa cells of early and progressed atretic follicles, and a positive reaction for Fas mRNA was detected in those of follicles of all stages, indicating that the FasL and Fas system may act as an inducer of granulosa cell apoptosis. KEY WORDS: TNF family, Pig ovary , Apoptosis, Follicular atresia |
