Abstract. In mammalian ovaries, most follicles are lost by atresia before ovulation. It has become apparent that the apoptosis of granulosa cells induces follicular atresia. Forkhead box O3 (FOXO3), also called FKHRL1 (forkhead in rhabdomyosarcoma-like 1), is a proapoptotic molecule that belongs to the FOXO subfamily of forkhead transcription factors. Foxo3-deficient female mice were reported to be infertile because of abnormal ovarian follicular development, but the precise influences of FOXO3 on follicular atresia of mature ovary have not been determined. Therefore, we examined the expression and function of FOXO3 in porcine ovarian follicles and granulosa-derived cells. FOXO3 mRNA levels in granulosa cells of porcine ovaries increased during atresia, while FOXO3 protein was abundant in granulosa cells of early atretic follicles. By immunohistochemistry, the inner surface area of the granulosa layer in early atretic follicles was strongly stained with anti-FOXO3 antibody. The granulosa cells expressing FOXO3 coincided with apoptotic cells, indicating a role of FOXO3 as a proapoptotic factor in granulosa cells of porcine ovaries. In porcine (JC-410) and human (KGN) granulosa-derived cells, cell death was induced by transfection of FOXO3 expression vectors. Expression of the proapoptotic factors Fas ligand (FASLG) and BCL2-like 11 (BCL2L11) was upregulated by FOXO3 in KGN cells. In conclusion, FOXO3 is expressed in porcine ovarian follicles and induces apoptosis in granulosa cells, suggesting that it is a candidate for the initiator of follicular atresia. Key words: Apoptosis, Forkhead box O3 (FOXO3), Granulosa cell, Porcine ovary (J. Reprod. Dev. 57: [151][152][153][154][155][156][157][158] 2011) n mammalian ovaries, most follicles degenerate prior to ovulation by a process called atresia [1,2]. The endocrine regulatory mechanisms involved in follicular development and atresia have been characterized to a large extent [3][4][5], but the precise temporal and molecular events involved have remained unknown. Studies have suggested that the apoptosis of ovarian granulosa cells plays a major role in follicular atresia [3,[6][7][8].The forkhead box O (FOXO) subfamily of forkhead transcription factors, consisting of FOXO1 (FKHR, forkhead in rhabdomyosarcoma), FOXO3 (FOXO3a/FKHRL1, FKHR-like 1), FOXO4 (AFX, acute-lymphocytic-leukaemia-1 fused gene from chromosome X) and FOXO6, participates in diverse processes including cell proliferation, apoptosis, stress resistance, differentiation and metabolism [9,10]. The transcriptional activity of FOXO is controlled by phosphorylation by a phosphatidylinositol 3-kinase (PI3K)-Akt (PKB, protein kinase B) pathway [10][11][12]. Three phosphorylation sites have been found in FOXO3: Thr 32 , Ser 253 and Ser 315 . When phosphorylated, FOXO3 binds to 14-3-3 proteins and is exported out of the nucleus, preventing transcription. Conversely, in the absence of phosphorylation, FOXO3 can translocate to the nucleus and increase the expression of genes.FOXO3 is known to enhance the transc...

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...

Several hundred thousand primordial follicles are present in the mammalian ovary, however, only 1% develop to the preovulatory stage and finally ovulate. The remainder will be eliminated via a degenerative process called ‘atresia’. The endocrinological regulatory mechanisms involved in follicular development and atresia have largely been characterized but the precise temporal and molecular mechanisms involved in the regulation of these events remain unknown. Many recent studies suggest that apoptosis in ovarian granulosa cells plays a crucial role in follicular atresia. Notably, death ligand‐receptor interaction and subsequent intracellular signaling have been demonstrated to be the key mechanisms regulating granulosa cell apoptosis. In this review we provide an overview of granulosa cell apoptosis regulated by death ligand‐receptor signaling. The roles of death ligands and receptors [Fas ligand (FasL)]‐Fas, tumor necrosis factor α (TNFα)‐TNF receptor and TNFα‐related apoptosis‐inducing ligand (TRAIL)‐TRAIL receptor (TRAILR)] and intracellular death‐signal mediating molecules (Fas‐associated death domain protein), TNF receptor 1‐associated death domain protein, caspases, apoptotic protease‐activating factor 1, TNFR‐associated factor 2 and cellular FLICE‐like inhibitory protein in granulosa cells are discussed.

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The results of clinical and experimental studies suggest that type I interferons (IFNs) may have direct antifibrotic activity in addition to their antiviral properties. However, the mechanisms are still unclear; in particular, little is known about the antifibrotic activity of IFN-b and how its activity is distinct from that of IFN-a. Using DNA microarrays, we demonstrated that gene expression in TWNT-4 cells, an activated human hepatic stellate cell line, was remarkably altered by IFN-b more than by IFN-a. Integrated pathway enrichment analyses revealed that a variety of IFN-b-mediated signaling pathways are uniquely regulated in TWNT-4 cells, including those related to cell cycle and Toll-like receptor 4 (TLR4) signaling. To investigate the antifibrotic activity of IFN-b and the involvement of TLR4 signaling in vivo, we used mice fed a choline-deficient l-amino acid-defined diet as a model of nonalcoholic steatohepatitis-related hepatic fibrosis. In this model, the administration of IFN-b significantly attenuated augmentation of the area of liver fibrosis, with accompanying transcriptional downregulation of the TLR4 adaptor molecule MyD88. Our results provide important clues for understanding the mechanisms of the preferential antifibrotic activity of IFN-b and suggest that IFN-b itself, as well as being a modulator of its unique signaling pathway, may be a potential treatment for patients with hepatic fibrosis in a pathogenesis-independent manner.