PARENT SESSION

(T623) cFLIP REGULATES GRANULOSA CELL APOPTOSIS DURING FOLLICULAR ATRESIA IN PORCINE OVARIES.

Matsuda-Minehata, Fuko¹, Goto, Yasufumi², Inoue, Naoko³, Manabe, Noboru², ¹ University of Tokyo, Tokyo, Japan² University of Tokyo, Ibaraki-Iwama, Japan³ Nagoya University, Nagoya, Japan

ABSTRACT- More than 99% of follicles are selectively degenerated (atresia) during follicular development and growth. Follicular atresia is indicated to begin with granulosa cell apoptosis. Recently, we detected the expression of anti-apoptotic factor, cellular Flice-like inhibitory protein (cFLIP), in porcine granulosa cells. cFLIP is a homologue of an intracellular apoptosis inducer (procaspase-8/Flice), and has two alternative splicing isoforms: cFLIP short form (cFLIP_S) and long form (cFLIP_L). The expression levels of cFLIP_S and cFLIP_L mRNA and protein in porcine granulosa cells were determined by RT-PCR and Western blotting, respectively. cFLIP_L mRNA and protein were highly expressed in granulosa cells of healthy follicles and decreased during atresia. cFLIP_S mRNA levels in granulosa cells did not show any change among stages of follicular development, and its protein level was extremely low. We examined changes in the localization of cFLIP mRNAs in pig ovaries using in situ hybridization and found that cFLIP_L is abundant in granulosa cells of healthy follicles in comparison with that in those of atretic follicles. Immunohistochemical analyses demonstrated that the cFLIP protein is highly expressed in the granulosa cell layer of healthy follicles but weakly expressed in that of atretic follicles. Next, we performed over expression or inhibition analyses of cFLIP and anti-apoptotic effect of cFLIP were demonstrated in cultured granulosa cell lines. These results suggested that cFLIP, especially cFLIP_L, plays an anti-apoptotic role in the granulosa cells of healthy follicles of pig ovaries, and that cFLIP could be a major survival factor that determines whether growth or atresia occurs in porcine follicles.

KEY WORDS: porcine cFLIP, apoptosis, granulosa cell, follicular atresia