Regulation of granulosa cell apoptosis by death ligand–receptor signaling

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.     

Regulation mechanism of selective atresia in porcine follicles: regulation of granulosa cell apoptosis during atresia

More than 99% of follicles undergo a degenerative process known as "atresia", in mammalian ovaries, and only a few follicles ovulate during ovarian follicular development. We have investigated the molecular mechanism of selective follicular atresia in mammalian ovaries, and have reported that follicular selection dominantly depends on granulosa cell apoptosis. However, we have little knowledge of the molecular mechanisms that control apoptotic cell death in granulosa cells during follicle selection. To date, at least five cell death ligand-receptor systems [tumor necrosis factor (TNF)alpha and receptors, Fas (also called APO-1/CD95) ligand and receptors, TNF-related apoptosis-inducing ligand (TRAIL; also called APO-2) and receptors, APO-3 ligand and receptors, and PFG-5 ligand and receptors] have been reported in granulosa cells of porcine ovaries. Some cell death ligand-receptor systems have "decoy" receptors, which act as inhibitors of cell death ligand-induced apoptosis in granulosa cells. Moreover, we showed that the porcine granulosa cell is a type II apoptotic cell, which has the mitochondrion-dependent apoptosis-signaling pathway. Briefly, the cell death receptor-mediated apoptosis signaling pathway in granulosa cells has been suggested to be as follows. (1) A cell death ligand binds to the extracellular domain of a cell death receptor, which contains an intracellular death domain (DD). (2) The intracellular DD of the cell death receptor interacts with the DD of the adaptor protein (Fas-associated death domain: FADD) through a homophilic DD interaction. (3) FADD activates an initiator caspase (procaspase-8; also called FLICE), which is a bipartite molecule, containing an N-terminal death effector domain (DED) and a C-terminal DD. (4) Procaspase-8 begins auto-proteolytic cleavage and activation. (5) The auto-activated caspase-8 cleaves Bid protein. (6) The truncated Bid releases cytochrome c from mitochondrion. (7) Cytochrome c and ATP-dependent oligimerization of apoptotic protease-activating factor-1 (Apaf-1) allows recruitment of procaspase-9 into the apoptosome complex. Activation of procaspase-9 is mediated by means of a conformational change. (8) The activated caspase-9 cleaves downstream effector caspases (caspase-3). (9) Finally, apoptosis is induced. Recently, we found two intracellular inhibitor proteins [cellular FLICE-like inhibitory protein short form (cFLIPS) and long form (cFLIPL)], which were strongly expressed in granulosa cells, and they may act as anti-apoptotic/survival factors. Further in vivo and in vitro studies will elucidate the largely unknown molecular mechanisms, e. g. which cell death ligand-receptor system is the dominant factor controlling the granulosa cell apoptosis of selective follicular atresia in mammalian ovaries. If we could elucidate the molecular mechanism of granulosa cell apoptosis (follicular selection), we could accurately diagnose the healthy ovulating follicles and precisely evaluate the oocyte quality. We hope that the mechanism will be clarified and lead to an integrated understanding of the regulation mechanism.     

Role of Cell Death Ligand and Receptor System on Regulation of Follicular Atresia in Pig Ovaries

Several hundred thousand primordial follicles are present in the mammalian ovary, however, only a limited number develop to the pre-ovulatory stage, and then finally ovulate. The others, more than 99%, will be eliminated through a degenerative process called 'atresia'. The endocrinological regulatory mechanisms involved in follicular development and atresia have been characterized to a large extent, but the precise temporal and molecular mechanisms involved in the regulation of these events have remained unknown. From many recent studies, it is suggested that the apoptosis in ovarian granulosa cells plays a crucial role in follicular atresia. Notably, death ligand–receptor interaction and subsequent intracellular signalling 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 signalling. The roles of death ligands and receptors [Fas ligand (FasL)–Fas, tumour necrosis factor (TNF)α–TNF receptor (TNFR), and TNFα-related apoptosis-inducing ligand (TRAIL)–TRAIL receptor (TRAILR)] and intracellular death-signal mediators [Fas-associated death domain protein (FADD), TNF receptor 1-associated death domain protein (TRADD), caspases, apoptotic protease-activating factor 1 (Apaf1), TNFR-associated factor 2 (TRAF2), and cellular FLICE-like inhibitory protein (cFLIP), etc.] in granulosa cells will be discussed.     

cFLIP regulates death receptor-mediated apoptosis in an ovarian granulosa cell line by inhibiting procaspase-8 cleavage

More than 99% of follicles in mammalian ovaries undergo atresia, but the mechanisms regulating the strict selection process are still unclear. Granulosa cell apoptosis is considered the trigger of follicular atresia, which occurs in advance of the death of an oocyte. Cellular FLICE-like inhibitory protein (cFLIP), a homologue of procaspase-8 (also called FLICE), is an intracellular anti-apoptotic protein. It is expressed in granulosa cells of porcine ovaries, where its levels decreases during follicular atresia. We hypothesized that cFLIP regulates granulosa cell apoptosis by acting as a pro-survival factor. In the present study, to further reveal the function of cFLIP in granulosa cells, we examined the anti-apoptotic mechanism of cFLIP using KGN, a human granulosa tumor cell line. Fas-mediated apoptosis was induced by co-treatment with anti-Fas antibody (CH-11), which acts as an agonist of Fas-ligand, and cycloheximide (CHX). When cFLIP was stably expressed in KGN cells following transfection of an expression vector, the Fas-mediated apoptosis was inhibited. Suppression of cFLIP by small interfering RNA (siRNA) spontaneously induced cell death. Silencing of cFLIP promoted cleavage of procaspase-8, and the cell death caused by cFLIP siRNA was completely blocked by a caspase-8 inhibitor (Z-IETD-FMK), indicating that cFLIP regulates apoptosis in KGN cells by inhibiting cleavage of procaspase-8. In conclusion, cFLIP is an essential pro-survival factor for granulosa cells, and it prevents granulosa cell apoptosis by inhibiting procaspase-8 activation.     

Gene expression of resistin and TNF-α in adipose tissue of Japanese Black steers and Holstein steers

The aim of the present study was to compare the expression of adipose tissue mRNA related to glucose metabolism between Japanese Black steers (n = 5) and Holstein steers (n = 5). We examined the expression of the resistin, tumor necrosis factor‐α (TNF‐α), glucose transporter 1 (GLUT1) and growth hormone receptor (GHR) genes using real‐time polymerase chain reaction of cDNA in adipose tissue. The cDNA sequence identified by 5′/3′‐rapid amplification of cDNA and the deduced amino acid sequence were highly conserved in human, porcine and murine resistin. Expression of resistin mRNA was significantly greater in Holstein steers than in Japanese Black steers. In contrast, expression of TNF‐α mRNA was slightly greater in Japanese Black steers. Expression of GHR mRNA was significantly greater in Japanese Black steers compared with the Holstein steers, although there was no significant difference in the expression of GLUT1 mRNA. However, the plasma non‐esterified fatty acid (NEFA), glucose, insulin and growth hormone concentrations did not differ between Japanese Black and Holstein steers. The present results show that there is a difference in the expression level of mRNA related to glucose metabolism between Japanese Black steers and Holstein steers.     

Expression of ski in the granulosa cells of atretic follicles in the rat ovary

The aim of the present study was to locate Ski protein, a product of cellular protooncogene c-ski, in rat ovaries in order to predict the possible involvement of Ski in follicular development and atresia. First, expression of c-ski mRNA in the ovaries of adult female rats was confirmed by RT-PCR. Then, ovaries obtained on the day of estrus were subjected to immunohistochemical analysis for Ski and proliferating cell nuclear antigen (PCNA) in combination with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). Ski was expressed in granulosa cells that were positive for TUNEL, but negative for PCNA, regardless of the size of follicles. Expression of Ski in TUNEL-positive granulosa cells, but not in PCNA-positive granulosa cells, was also verified in immature hypophysectomized rats having a single generation of developing and atretic follicles by treatment with equine chorionic gonadotropin. These results indicate that Ski is profoundly expressed in the granulosa cells of atretic follicles, but not in growing follicles, and suggests that Ski plays a role in apoptosis of granulosa cells during follicular atresia.     

Regulation of ovarian folliculogenesis by IGF and BMP system in domestic animals

Involvement of insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in ovarian folliculogenesis has been extensively studied during the last decade. In all mammalian species, IGF-I stimulates granulosa cell proliferation and steroidogenesis. The concentrations of IGF-I and -II do not vary during terminal follicular growth and atresia. In contrast, the levels of IGFBP-2 and -4, as well as IGFBP-5 in ruminants, dramatically decrease and increase during terminal follicular growth and atresia, respectively. These changes are responsible for an increase and a decrease in IGF bioavailability during follicular growth and atresia, respectively. They are partly explained by changes in ovarian expression. In particular, expression of IGFBP-2 mRNA decreases during follicular growth in ovine, bovine and porcine ovaries, and expression of IGFBP-5 mRNA dramatically increases in granulosa cells of bovine and ovine atretic follicles. Changes in IGFBP-2 and -4 levels are also due to changes in intrafollicular levels of specific proteases. Recently, we have shown that the pregnancy-associated plasma protein-A (PAPP-A) is responsible for the degradation of IGFBP-4 in preovulatory follicles of domestic animals. Expression of PAPP-A mRNA is restricted to the granulosa cell compartment, and is positively correlated to expression of aromatase and LH receptor. From recent evidence, the bone morphogenetic protein (BMP) family would also play a key role in ovarian physiology of domestic animals. In particular, we and others have recently shown that a non-conservative substitution (Q249R) in the bone morphogenetic protein-receptor type IB (BMPR-IB) coding sequence is fully associated with the hyperprolific phenotype of FecB(B)/FecB(B) Booroola ewes. BMP-4 and GDF-5, natural ligands of BMPR-IB, strongly inhibit secretion of progesterone by ovine granulosa cells in vitro, but granulosa cells from FecB(B)/FecB(B) ewes are less responsive than those from FecB(+)/FecB(+) to the action of these peptides. It is suggested that in FecB(B)/FecB(B) ewes, Q249R substitution would impair the function of BMPR-IB, leading to a precocious differentiation of granulosa cells and of follicular maturation. Interestingly, recent findings have described mutations in BMP-15 gene associated with hyperprolific phenotypes in Inverdale and Hanna ewes, suggesting that the BMP pathway plays a crucial role in the control of ovulation rate.     

Does 2-hydroxyflutamide Inhibit Apoptosis in Porcine Granulosa Cells? - An In Vitro Study

In mammalian ovaries, the majority of follicles are lost before ovulation by atresia. This degenerative process is initiated or caused by granulosa cell apoptosis. To reveal the androgen-dependent mechanism of selective follicular atresia, the culture model system for agonism and antagonism of the androgen receptor has been established. We examined the influence of an androgen receptor antagonist, 2-hydroxyflutamide (2-Hf), on the incidence of apoptosis in cultured porcine granulosa cells. They were incubated (6 and 12-h) in the presence of testosterone (T, 10(-7)M), 2-Hf (1.7×10(-4) M) or both T and 2-Hf (T+2-Hf), and then analyzed by flow cytometry with fluorescein labelled annexin V. To better imitate in vivo conditions, the intact porcine follicles (6-8 mm in diameter) have been incubated in an organ culture system with the addition of the same factors. Sections obtained from follicles fixed after culture were stained with hematoxylin and eosin, and the presence of apoptosis-related DNA strand breaks was evaluated by the TUNEL method. Estradiol and progesterone concentrations in the culture media were measured by radioimmunoassays. The addition of T or 2-Hf to the culture media caused an increase in the number of apoptotic granulosa cells, while treatment with T+2-Hf decreased it in both in vitro and organotypic models. Follicles cultured with the addition of T or 2-Hf exhibited morphological changes indicating follicular atresia. Granulosal estradiol secretion was considerably stimulated by T+2-Hf. The highest increase in follicular estradiol secretion was observed after the anti-androgen addition. In both granulosal and follicular cultures, the production of progesterone declined in the presence of T or 2-Hf but increased after their simultaneous addition. In conclusion, androgen receptor antagonist 2-Hf attenuates induction of granulosa cell apoptosis in the presence of a high T level. The nature of this protective mechanism as yet is unknown and requires further research.     

猪卵巢细胞中凋亡抑制因子cFLIP在卵巢卵泡和黄体中的表达

采用免疫组织化学法检测猪卵巢中细胞凋亡调控蛋白即细胞内FLICE样抑制蛋白（Cellular FLICE-like inhibitory protein,cFLIP）在猪卵巢卵泡和黄体中的表达水平。结果显示,在健康卵泡和功能性黄体中均能够观察到cFLIP的高度表达,而闭锁卵泡和退化黄体中cFLIP的表达显著减弱。结果表明,cFLIP作为一种细胞凋亡抑制因子,参与猪卵巢中的卵巢闭锁和黄体退化。