Bid and Bax Are Involved in Granulosa Cell Apoptosis During Follicular Atresia in Porcine Ovaries

Abstract

Abstract. More than 99% of follicles undergo "atresia" during follicular development and growth. Follicular atresia is predominantly regulated by granulosa cell apoptosis. However, the intracellular signaling pathway of apoptosis in granulosa cells has not been revealed. In the present study, we examined changes in the expression of BH3-interacting domain death agonist (Bid) and Bcl-2-associated X protein (Bax), which are considered to promote the cell death ligand/ receptor-mediated process in mitochondrion-dependent type II apoptosis, in porcine granulosa cells during atresia. Levels of mRNA and protein of Bid and Bax were determined by the reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting techniques, respectively. Levels of Bid and Bax mRNA and protein were markedly increased in granulosa cells of early atretic follicles compared with those of healthy follicles. In situ hybridization and immunohistochemical staining revealed that mRNA and protein of Bid and Bax were present in the granulosa cells, though only traces were found in healthy follicles; however, strong staining was noted in atretic follicles. These results indicate that Bid and Bax appear to be signal transduction factors in granulosa cells during follicular atresia and appear to play proapoptotic roles and confirm that the porcine granulosa cell is a mitochondrion-dependent type II apoptotic cell. Key words: Bcl-2-associated X protein (Bax), BH3-interacting domain death agonist (Bid), Follicular atresia, Mitochondrion-dependent type II apoptotic cell, Pig ovary (J. Reprod. Dev. 57: [421][422][423][424][425][426][427] 2011) n mammalian ovaries, more than 99% of follicles degenerate at various stages of follicular growth and development [1]. The degeneration is explained, at least in part, by the apoptosis of granulosa cells [2][3][4][5][6]. In the early stages of follicular atresia in pig ovaries, biochemical and morphological characteristics typical of apoptosis, namely nuclear fragmentation, chromatin condensation and cell shrinkage, are observed in scattered granulosa cells located on the inner surface of the follicular wall, but not in cumulus cells, oocytes or the cells of internal or external thecal layers [7,8]. However, the intracellular signal transduction pathway of apoptosis in granulosa cells of pig ovaries is not well understood.Selective apoptotic cell death is induced by cell death ligand/ receptor systems, including Fas ligand (FasL; also called Apo-1 ligand or CD95 ligand) and Fas (also called Apo-1 or CD95), tumor necrosis factor (TNF)-α and TNF receptors (TNFRs) and TNFrelated apoptosis-inducing ligand (TRAIL) and TRAIL receptors (DR4 and 5) [9][10][11]. The cell death ligand/receptor-mediated apoptotic signaling pathway is suggested to be as follows [12][13][14][15][16][17][18]: When trimerized cell death ligands bind with trimerized death receptors located on the cell membrane, the receptors are activated. An adaptor protein (Fas-associated death domain protein: FADD) binds with an activated rece...

The role of BH3-only proteins in apoptosis within the ovary

“…Though a comprehensive study of the role of BID in cell death within the ovary using knockout mice is yet to be published, several studies have hinted at important roles in follicular atresia and corpora lutea regression (Yadav et al 2005, Goyeneche et al 2006, Sai et al 2011, Paulose et al 2012, Craig et al 2013. More than 99% of the growing follicles in the ovary are destined for atresia, which is usually first evident by apoptosis of the granulosa cells.…”

“…More than 99% of the growing follicles in the ovary are destined for atresia, which is usually first evident by apoptosis of the granulosa cells. Notably, BID expression increases during follicular atresia in the porcine ovary and knockdown of BID expression in human granulosa cell-derived KGN cells can suppress apoptosis (Sai et al 2011(Sai et al , 2012, suggesting a role for BID in regulating the normal process of follicular atresia. In addition to this natural process, BID may also be responsible for triggering follicular atresia following exposure to exogenous environmental toxicants such as di-n-butyl phthalate and the organochlorine pesticide methoxychlor, which are known to increase follicular atresia (Paulose et al 2012, Craig et al 2013.…”

Rescue from dominant follicle atresia by follicle-stimulating hormone in mice

“…It is clear that granulosa cell apoptosis is the major cause of antral and dominant follicular atresia (Manabe et al, 2008;Sai et al, 2011); however, the mechanism of granulosa cell apoptosis remains unclear.…”

Effect of RNA Interference of BID and BAX mRNAs on Apoptosis in Granulosa Cell-derived KGN Cells

“…However, there is insufficient information on the role of intracellular signaling molecules in granulosa cells, especially downstream of the activated caspase-8. Previously, we revealed high levels of BID and BAX mRNA and protein in the granulosa cells of early atretic follicles but low levels in those of healthy follicles, indicating that BID and BAX are involved in granulosa cell apoptosis during atresia in porcine ovaries [8]. In the present study, to confirm the roles of BID and BAX in the apoptosis, we examined the effects of suppressing BID or BAX mRNA expression with the RNAi technique on apoptosis in human granulosa cell-derived KGN cells.…”

“…In porcine ovarian follicles in the early stages of atresia, characteristics typical of apoptosis are observed in scattered granulosa cells, but not in cumulus cells, oocytes or the cells of internal or external thecal layers [7]. As previously reported [8], it is considered that selective granulosa cell apoptosis is induced by death ligand and receptor systems as follows: When trimerized death ligands bind with trimerized death receptors located on the cell membrane, the receptors are activated. An adaptor protein (Fasassociated death domain protein) binds with activated receptors to construct death-inducing signal complex (DISC), which binds procaspase-8.…”