Expression and Localization of Fas Ligand and Fas During Atresia in Porcine Ovarian Follicles

Self Cite

Abstract. In mitochondrion-dependent type II apoptosis, BH3-interacting domain death agonist (BID) and BCL-2-associated X protein (BAX) promote death ligand and receptor-mediated cell death. In porcine ovaries, the levels of BID and BAX increase in follicular granulosa cells during atresia. In the present study, to confirm the pro-apoptotic activity of BID and BAX in granulosa cells, we examined the effect of RNA interference of BID or BAX on apoptosis using a human ovarian granulosa tumor cell line, KGN. By reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, expression of BID and BAX was detected in KGN cells. Then, we suppressed BID and BAX mRNA expression in KGN cells using small interfering RNA (siRNA). When BID or BAX was suppressed, a significant decrease in the apoptotic cell rate was noted. In granulosa-derived cells, BID and BAX showed pro-apoptotic activity. These results suggest that BID and BAX act as signal-transducing factors in mitochondrion-dependent type II apoptosis. Key words: BCL-2-associated X protein (BAX), BH3-interacting domain death agonist (BID), Mitochondrion-dependent type II apoptosis, RNA interference (RNAi) (J. Reprod. Dev. 58: [112][113][114][115][116] 2012) I 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 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. Then, procaspase-8 is truncated and activated in DISC. Two types of cell death ligand and receptor-dependent signaling pathways, type I and II, have been found. Granulosa cells undergo mitochondrion-dependent type II apoptosis. Activated caspase-8 shortens BH3-interacting domain death agonist (BID) protein [9,10], and the truncated-BID (tBID) stimulates BCL-2-associated X protein (BAX). Then, BAX is transferred to the outer membrane of the mitochondrion and releases cytochrome c [11,12]. Cytochrome c binds with apoptotic protease-activating factor 1 and procaspase-9, and this complex is called an apoptosome [13]. The procaspase-9 is activated in the apoptosome and shortens procaspase-3, and the activated caspase-3 truncates caspase-3-activated DNase (CAD) [14,15]. The shortened CAD moves into the nucleus and cuts the genomic DNA leading to apoptosis.A large amount of caspase-8 is activated in type I apoptosis, but a small amo...

Section: Discussionmentioning

confidence: 99%

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

Sai

Matsuda

Goto

et al. 2012

Self Cite

“…Our previous studies demonstrated that TNF superfamily and TNFR superfamily proteins are also expressed in porcine ovarian follicles and that they are involved in regulation of survival and apoptosis of granulosa cells [27,[29][30][31][32]. Not only death receptors, but also decoy receptors were expressed in porcine ovarian follicles.…”

Section: Discussionmentioning

confidence: 99%

“…As previously reported [27], ovaries obtained from gilts at a local slaughterhouse were used. For the isolation of total RNA, tertiary follicles (3-4 mm in diameter) were dissected out using scalpels and classified into the healthy, early atretic and progressed atretic stages based on their morphology under a dissection microscope.…”

Section: Tissue Collectionmentioning

confidence: 99%

“…TNF-related apoptosis-inducing ligand (TRAIL) and its receptors, death receptor-4 (DR4), death receptor-5 (DR5) and decoy receptor-1 (DcR1), were expressed in porcine granulosa cells [29][30][31], and expression of DcR1 is reduced in granulosa cells in atretic follicles [31]. In addition, removal of DcR1 from the cellular surface sensitizes granulosa cells to TRAIL induced-apoptosis in vitro [30].FasL and Fas has also been considered to participate in apoptosis during follicular atresia in the mammalian ovary, based on their expression in ovarian follicles [27,[33][34][35][36][37][38][39][40][41][42] and pro-apoptotic activities of FasL and antibodies to Fas in follicular cells. Increases in Fig.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Changes in the Expression of Decoy Receptor 3 in Granulosa Cells During Follicular Atresia in Porcine Ovaries

Sugimoto

Kagawa

Morita

et al. 2010

Self Cite

Abstract. During follicular development in mammalian ovaries, the majority of follicles undergo atresia. One of the characteristics of this process is apoptotic cell death in granulosa cells. Several death ligands and receptors, including Fas ligand (FasL) and Fas, have been detected in ovarian follicles and also demonstrated to be capable of inducing apoptosis in follicular cells. Decoy receptor 3 (DcR3) competes with Fas to bind FasL but lacks intracellular death domains, thus inhibiting the induction of apoptosis by the FasL/Fas system. In the present study, we examined the expression of putative porcine DcR3 (pDcR3) mRNA in porcine ovarian follicles. Total RNA was extracted from granulosa cells and thecal layer cells of tertiary follicles at healthy, early atretic and progressed atretic stages, and the expression of pDcR3 mRNA was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR). The nucleic acid sequence in the coding region had 80% homology to that of human DcR3, and the deduced amino acid sequence was 73% identical to that of human DcR3. In an in situ hybridization experiment, pDcR3 mRNA expression was confirmed in granulosa and thecal layers, in both healthy and atretic follicles. Quantitative real time RT-PCR analysis showed that the expression of pDcR3 mRNA was weaker in granulosa cells of atretic follicles than those of healthy follicles. No notable changes were seen in the thecal layer cells. These results suggest that DcR3 plays a significant role in the regulation of apoptosis in granulosa cells, but not in thecal layer cells, during atresia. Key words: Apoptosis, Decoy receptor-3 (DcR3), Follicular atresia, Granulosa cell, Porcine ovary (J. Reprod. Dev. 56: [467][468][469][470][471][472][473][474] 2010) n mammalian ovaries, the majority of follicles undergo a degenerative process known as atresia during follicular growth and maturation. Atretic follicles can be identified by their morphology, typically karyopyknosis in granulosa cells [1,2]. Karyopyknosis is, however, also a typical morphological characteristic of apoptotic cells [3,4]. Earlier studies [5,6] have shown that degeneration of granulosa cells in atretic follicles is also associated with oligonucleosomal fragmentation of DNA, a biochemical hallmark of apoptosis, in agreement with the morphological observations. Our previous studies [7][8][9][10][11] demonstrated that porcine granulosa cells undergo apoptosis during follicular atresia through the histochemical and biochemical detection of DNA fragmentation and by electron microscopy. In antral follicles, apoptotic cells occurred at the inner surface of the granulosa layer at an early stage of follicular atresia and then diffused in the granulosa layer as atresia advanced. In thecal layer of atretic follicles, apoptotic cells were observed but at a low frequency compared with that in the granulosa layer.Although it has been demonstrated that apoptotic cell death plays a significant role in ovarian follicular atresia, the mechanisms regulating apoptosis in granu...

“…Dev. 56: [230][231][232][233][234][235] 2010) poptosis is a crucial mechanism in ovarian function through its significant contribution to cell deletion during follicular atresia [1][2][3][4][5] and luteal regression [6]. It is generally accepted that apoptosis is the main type of cell death during structural luteolysis in many species [7].…”

mentioning

confidence: 99%

Expression and Localization of cFLIP, an Anti-apoptotic Factor, in the Bovine Corpus Luteum

Hojo

Al-zi’abi

Komiyama

et al. 2010

Self Cite

Abstract. The objective of the present study was to investigate the potential mechanisms regulating cellular FLICE-like inhibitory protein (cFLIP), an anti-apoptotic factor, in the bovine corpus luteum (CL). Expression of cFLIP mRNA was highest at the developing stage and then decreased significantly during the mid, late and regressed stages (P<0.05). Western blot analysis revealed that expression of the long isoform of cFLIP (cFLIPL) protein was high during the early and developing luteal stages, remained steady during the mid and late luteal stages and then decreased significantly (P<0.05) by the regressed stage. However, the expression levels of the short isoform of cFLIP (cFLIPS) remained low during the early, developing and mid luteal stages. Immunostaining of cFLIP was strongest in the cytoplasm of luteal and non-luteal cells, including endothelial and immune cells, remained high during the early, developing and mid luteal stages and then decreased significantly (P<0.05) in the late and regressed luteal stages. Immunostaining of cFLIP was observed only in macrophage-like cells in the regressing CL. However, cultured mid luteal cells had a higher percentage of cFLIP-positive cells and a lower percentage of TUNEL-positive cells than luteal cells treated with tumor necrosis factor α (TNF)/interferon γ (IFNG; P<0.01). These results indicate downregulation of cFLIP during structural luteal regression, suggesting that cFLIP plays a survival role in the bovine CL. Key words: Anti-apoptotic factor, Apoptosis, Bovine, Cellular FLICE-like inhibitory protein (cFLIP), Corpus luteum (J. Reprod. Dev. 56: [230][231][232][233][234][235] 2010) poptosis is a crucial mechanism in ovarian function through its significant contribution to cell deletion during follicular atresia [1][2][3][4][5] and luteal regression [6]. It is generally accepted that apoptosis is the main type of cell death during structural luteolysis in many species [7]. Apoptosis is a highly regulated process, and various factors are known to be involved in apoptosis in the corpus luteum (CL), such as hormones (progesterone), cytokines [tumor necrosis factor (TNF), interferons, interleukins] and others [BCL2 family, FAS and Fas ligand (FASLG) system, caspase family members, reactive oxygen species (nitrogen oxide, etc.)] [8]. A better understanding of pro-and anti-apoptotic factors may improve manipulation techniques of luteolysis, which is a key element in estrous cycle control in clinical bovine reproduction. Although many proteins (e.g., BCL2 family proteins, FASLG and caspases) have been demonstrated to be involved in the apoptotic cascade process during luteolysis [8], little information about one anti-apoptotic factor, cellular FLICE-like inhibitory protein, cFLIP, is available in the bovine CL.cFLIP is an important regulator of death receptor-mediated apoptosis [2,[9][10][11]. At the mRNA level, cFLIP exists as multiple splice variants, but at the protein level, it occurs in two endogenous forms, cFLIP long (cFLIPL) and cFLIP short (cFLIPS) [12]. ...