PARENT SESSION

(W615) GRANULOSA CELL IS A TYPE II APOPTOTIC CELL THAT REGULATES FOLLICULAR ATRESIA IN PIG OVARIES.

Sai, Takafumi¹, Matsui, Toshikatsu¹, Goto, Yasufumi¹, Matsuda-Minehata, Fuko¹, Inoue, Naoko², Maeda, Akihisa¹, Cheng, Yuan¹, Li, Junyou¹, Manabe, Noboru¹, ¹ University of Tokyo, Ibaraki-Iwama, Japan² Nagoya University, Nagoya, Japan

ABSTRACT- Apoptosis in granulosa cells plays crucial roles in ovarian follicular atresia, but the intracellular regulating mechanism, especially the mitochondrion-dependent apoptosis signaling pathway, remains largely unknown. We examined whether the mitochondrial pathway is associated with granulosa cell apoptosis during atresia in porcine ovaries. The mRNAs of both caspase-9 and apoptotic protease-activating factor-1 (Apaf1), which are major signal-transducing components in the mitochondrion-dependent pathway, were detected in granulosa cells prepared from healthy, early atretic and progressed atretic follicles by real-time reverse transcription-polymerase chain reaction. No changes in the expression level of Apaf1 mRNA were seen during follicular atresia, but that of caspase-9 mRNA increased during atresia. Apaf1 protein was steadily detected in granulosa cells prepared from healthy, early atretic and progressed atretic follicles by Western blotting, but high expression of procaspase-9 (inactive precursor of caspase-9) protein was detected only in granulosa cells of healthy follicles. Decreased procaspase-9 protein was demonstrated during follicular atresia. In relation to the diminution of procaspase-9 protein, the proteolytic activity of caspase-9 increased during atresia. Intensive expression of caspase-9 mRNA was demonstrated in the granulosa cells of early atretic and progressed atretic follicles but not in those of healthy follicles. Moreover, in vitro studies using primary cultured granulosa cells showed that cytochrome C release from mitochondria is essential for apoptotic cell death. These results indicate that the mitochondrion-dependent signaling pathway, which is mediated by Apaf1 and caspase-9 proteins and cytochrome C, plays crucial roles in determining the fate of granulosa cells during atresia in porcine ovaries. We conclude that the porcine granulosa cell is a type II apoptotic cell with a mitochondrion-dependent apoptosis signaling pathway.

KEY WORDS: GRANULOSA CELL, TYPE II APOPTOTIC CELL, ATRESIA, OVARY