Expression and Function of Apoptosis Initiator FOXO3 in Granulosa Cells During Follicular Atresia in Pig Ovaries

Abstract

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...

Follicular Growth and Atresia in Mammalian Ovaries: Regulation by Survival and Death of Granulosa Cells

“…In isolated monkey granulosa cells, gonadotropin depletion stimulated both BAX and CASP3 expressions and induced apoptosis [96]. In human granulosa tumor cells, BCL2L11 was upregulated by FOXO3 overexpression [55].…”

“…Addition of anti-apoptotic molecules rescues granulosa cells from a spontaneous onset of apoptosis [39,40,[42][43][44]. Another FOXO transcription factor, FOXO3, is also indicated to act on follicular atresia in pig ovaries; the expression of FOXO3 in granulosa cells increased during follicular atresia, and an overexpression of FOXO3 induced granulosa cell apoptosis that is likely to be caused by the upregulation of proapoptotic factors such as FAS ligand (FASLG) and BCL2-like 11 (BCL2L11) [55]. The addition of FSH significantly decreased FASLG mRNA levels and attenuated apoptosis in porcine granulosa cells [56].…”

Simultaneous Melatonin Administration Effectively Deprograms the Negative Influence of Neonatal Hypothyroidism on Immature Follicles but not on Mature Follicles and Body and Ovarian Weights

“…A possible avenue of future investigations could be the understanding of the possible role of neonatal hypermelatonemia on permanent resetting of local genetic programmes resulting in the activation of survival factors and/or inhibition of apoptotic factors. In the light of reported importance of expression of oocyte GDF 9 for pre-antral follicular survival and transition to antral follicles [34], and the reported expression of FOXO 3, an apoptotic gene, and its hypothesized up regulation by neonatal programming by hypothyroidism [35], studies on the expression of these candidate genes would be worthwhile. The effect of HPOT + MT on the endocrine axes is marked by reduced titres of both sex hormones but increased thyroid hormone titres.…”

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

“…As previously reported [21][22][23], each protein fraction (10 µg/lane) prepared from a cell sample was separated by 10-20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Atto, Tokyo, Japan) and then transferred onto nitrocellulose membranes (Hybond-C; GE Healthcare). The membranes were stained with 0.2% (w/v) Ponceau S solution (Serva Electrophoresis, Heidelberg, Germany) and immersed in a blocking solution [0.1 M Tris HCl, (pH 7.6), 5% (w/v) skim milk, 0.05 M NaCl and 0.1% (v/v) Tween 20; Sigma] for 1 h at 25 C. Each membrane was incubated with the appropriate primary antibody for 1 h at room temperature (22-25 C): for BID protein, a rabbit polyclonal anti-human BID antibody (diluted 1:1,000 with the blocking solution; Santa Cruz Biotechnology, Santa Cruz, CA, USA); for BAX protein, a rabbit polyclonal anti-human BAX antibody (diluted 1:500 with the blocking solution; Santa Cruz Biotechnology); and for GAPDH protein, a goat polyclonal anti-human GAPDH antibody (diluted 1:200 with the blocking solution; Santa Cruz Biotechnology).…”