Molecular Characteristics of Porcine Fas-associated Death Domain (FADD) and Procaspase-8

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Abstract. Follicular selection predominantly depends on granulosa cell apoptosis in porcine ovaries, but the molecular mechanisms regulating the induction of apoptosis in granulosa cells during follicular selection remain incompletely understood. To determine the role of X-linked inhibitor of apoptosis protein (XIAP), which suppresses caspase-3, -7 and -9 activities and acts as an endogenous inhibitor of apoptotic cell death, in the regulation of granulosa cell apoptosis during follicular atresia, we examined the changes in the expression level and localization of XIAP mRNA and protein in granulosa cells during follicular atresia using reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, Western blotting and immunohistochemistry, respectively. High levels of XIAP mRNA and protein were noted in the granulosa cells of healthy follicles, and decreased levels were noted during follicular atresia. Ιn situ hybridization and immunohistochemistry demonstrated that XIAP mRNA and protein were strongly expressed in the granulosa cells of healthy follicles, but negative/trace stainings were noted in those of atretic follicles. The present findings strongly indicate that XIAP is a candidate molecule which acts as an anti-apoptotic/pro-survival factor by inhibiting intracellular apoptosis signaling and is involved in the regulation of apoptosis in porcine granulosa cells. Key words: Apoptosis, Caspase, Granulosa cell, Pig ovary, X-linked inhibitor of apoptotsis protein (XIAP) (J. Reprod. Dev. 54: [454][455][456][457][458][459] 2008) n mammalian ovaries, more than 99% of follicles disappear during follicular growth and development, and this appears to be primarily due to apoptosis of follicular granulosa cells, as the biochemical and morphological characteristics of apoptosis have been observed in the granulosa cells of atretic follicles [1][2][3]. Apoptotic stimuli and intracellular apoptosis-signal transduction pathways in granulosa cells have not yet been determined, and many researchers have attempted to confirm the primary trigger of apoptosis and how the intracellular apoptotic signal is transmitted in granulosa cells [4][5][6][7]. Many apoptosis-related factors are implicated in follicular atresia, including cell death ligands and receptors, intracellular pro-and anti-apoptotic molecules, cytokines and growth factors. In particular, cell death ligand-receptor signaling has been revealed to be the dominant regulatory system for apoptosis in granulosa cells [4][5][6][7]. To date, two major intracellular-signal pathways in cell-death ligand and receptor-dependent apoptosis (type I and type II: mitochondrion-independent anddependent pathways, respectively) have been found [8][9][10][11][12]. In type I and II apoptotic cells, apoptotic signaling is initially triggered by members of the cell death ligand and receptor superfamily [Fas ligand (FasL) and Fas (also called Apo-1, CD95 or TNFRsf6), tumor necrosis factor (TNF) α and its receptors (TNFRs), TNFrelated apoptosis-inducing ligand (TRAIL; ...

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confidence: 99%

“…TRADD is not detected in the granulosa cells of healthy follicles, but it is highly expressed in atretic follicles [18]. Another adaptor protein, FADD, is also present in pig granulosa cells [17]. Moreover, the caspase-9 precursor (procaspase-9) decreases, while caspase-9 activity increases as follicular atresia progresses [19].…”

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confidence: 99%

Changes in Expression and Localization of X-linked Inhibitor of Apoptosis Protein (XIAP) in Follicular Granulosa Cells During Atresia in Porcine Ovaries

Cheng

Maeda

Goto

et al. 2008

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“…The TNFα-mediated apoptotic signaling pathway (TNFR1-dependent apoptosis) has been suggested to be as follows [1][2][3][4][5][6][7][8] In mammalian ovaries, more than 99.9% of follicles undergo a degenerative process known as "atresia", and only a few follicles selectively ovulate during ovarian follicular development and growth [15][16][17][18][19][20][21]. Our recent findings [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40] have suggested that apoptosis in follicular granulosa cells plays a crucial role in the regulation of ovarian follicular atresia; however, the intracellular signal transduction pathway for apoptois in granulosa cells is still largely unknown [20,21]. Moreover, we have previously suggested that apoptosis and proliferation in granulosa cells are dominantly regulated by a cell death ligand and receptor system (TNF and TNFR superfamily members) [18][19][20][21].…”

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confidence: 99%

Molecular Cloning of Porcine (Sus scrofa) Tumor Necrosis Factor Receptor 2

Cheng

Maeda

Goto

et al. 2007

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Abstract. Tumor necrosis factor (TNF) α can induce both cell death and proliferation by binding to either TNF receptor (TNFR) 1 or 2. In the granulosa cells of porcine ovaries, TNFα is considered to act as an anti-apoptotic/survival factor during follicular atresia. As a first step toward elucidating the function of TNFR2 in regulating follicular development/growth and atresia in porcine ovaries, we isolated the porcine (Sus scrofa) cDNA encoding TNFR2, which was identified from a cDNA library prepared from the follicular granulosa cells of pig ovaries. Porcine TNFR2 (1,125 bp, 375 amino acid residues), which contains specific amino acid region of transmembrane, indicated high identities with human and murine TNFR2 (78 and 69% at mRNA level, respectively; 73 and 61% at protein level, respectively), suggesting that the function of porcine TNFR2 is similar to that of human and murine homologues. Understanding the expression patterns of porcine TNFR2 mRNA in various organs, which we confirmed by reverse transcription polymerase chain reaction analysis, would help to elucidate the physiological role of TNFR2 in the regulation of apoptosis in porcine organs.

“…Bars=100 μm. [50][51][52][53][54][55][56][57][58]. Their participation in the apoptosis of granulosa cells and follicular atresia has been suggested, although their significance in the regulation of follicular atresia in vivo remains to be clarified.…”

Section: Discussionmentioning

confidence: 99%

“…These findings suggest that follicular cells have certain inhibitors of Fas-mediated apoptosis. There are several reports on anti-apoptotic factors in ovarian follicles, including Fas-associated phosphatase (FAP-1) [45], members of the inhibitor of apoptosis protein (IAP) family [46][47][48][49] and cellular FLICE-like inhibitory protein (c-FLIP) [50][51][52][53][54][55][56][57][58]. Their participation in the apoptosis of granulosa cells and follicular atresia has been suggested, although their significance in the regulation of follicular atresia in vivo remains to be clarified.…”

Section: Discussionmentioning

confidence: 99%

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

Sugimoto

Kagawa

Morita

et al. 2010

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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...