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.     

Molecular characteristics of porcine Fas-associated death domain (FADD) and procaspase-8

To reveal the intracellular signal transduction molecules involved in granulosa cell apoptosis in porcine ovarian follicles, we cloned the porcine Fas-associated death domain (FADD), an adaptor protein for the cell death receptor, and procaspase-8, an initiator caspase. Porcine FADD (pFADD) was 636 bp (211 amino acids: aa) long and showed 74.0 and 65.4% homology with human and murine FADD, respectively. Porcine procaspase-8 (pprocaspase-8) was 1,431 bp (476 aa) long and 70.6 and 63.4% homologous with human and murine procaspase-8, respectively. To confirm the apoptosis-inducing abilities, we constructed pFADD and pprocaspase-8 cDNA expression vectors with enhanced green fluorescence protein (EGFP) and then transfected them into human uterine cervix tumor (HeLa-K), human granulosa cell-derived (KGN), murine granulosa-derived tumor (KK1), and porcine granulosa cell-derived (JC410) cells. When pFADD and pprocaspase-8 were overexpressed, cell death was induced in these transfected cells. However when caspase-inhibitor p35 was cotransfected, cell death was inhibited. The pFADD and pprocaspase-8 genes are well conserved, as are the physiological functions of their products.     

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.     

BMAL1对小鼠睾丸间质细胞凋亡的影响

实验旨在研究 BMAL1基因对睾丸间质细胞凋亡的影响。实验以小鼠睾丸间质细胞系为研究对象,使用小干扰 RNA(siRNA)抑制 BMAL1的表达,使用流式细胞术、实时荧光定量 PCR(qRT-PCR)和蛋白印迹(Western Blot)检测细胞凋亡水平。结果表明:干扰组睾丸间质细胞凋亡水平显著上升(P<0.05),且伴随着促凋亡蛋白 BAX 显著上调和抑凋亡蛋白 BCL-2 表达下调(P<0.05)。结果显示,BMAL1作为核心的生物钟基因,可抑制小鼠睾丸间质细胞的凋亡。     

Expression of Mitochondria‐Associated Genes (PPARGC1A,NRF‐1, BCL‐2 and BAX) in Follicular Development and Atresia of Goat Ovaries

Most follicles undergo atresia during the developmental process. Follicular atresia is predominantly regulated by apoptosis of granulosa cells, but the mechanism underlying apoptosis via the mitochondria‐dependent apoptotic pathway is unclear. We aimed to investigate whether the mitochondria‐associated genes peroxisome proliferator‐activated receptor‐gamma, coactivator1‐alpha (PPARGC1A), nuclear respiratory factor‐1 (NRF‐1), B‐cell CLL/lymphoma 2 (BCL‐2) and BCL2‐associated X protein (BAX) played a role in follicular atresia through this pathway. The four mitochondria‐associated proteins (PGC‐1α, which are encoded by the PPARGC1A gene, NRF‐1, BCL‐2 and BAX) mainly expressed in granulosa cells. The mRNA and protein levels of PPARGC1A/PGC‐1α and NRF‐1 in granulosa cells increased with the follicular development. These results showed that these genes may play a role in the regulation of the follicular development. In addition, compared with healthy follicles, the granulosa cell in atretic follicles had a reduced expression of NRF‐1, increased BAX expression and increased ratio of BAX to BCL‐2 expression. These results suggested that changes of the mitochondria‐associated gene expression patterns in granulosa cells may lead to follicular atresia during goat follicle development.     

miRNA调节卵泡颗粒细胞凋亡及其机制的研究进展

微小核糖核酸(microRNA,miRNA)是一类内源性非编码RNA,具有广泛的基因表达调控作用,可以在转录后水平通过影响靶基因来调控相应蛋白质的表达,进而调节细胞的生命活动。miRNA在哺乳动物卵泡颗粒细胞中表达,并调控颗粒细胞的凋亡。颗粒细胞作为卵巢卵泡中数量最多的细胞群,在卵泡发育过程中起着至关重要的作用,不仅为卵母细胞提供营养物质,还调控其发育和成熟。颗粒细胞凋亡是导致卵泡闭锁的重要原因,影响卵泡的数量和质量从而影响雌性动物的繁殖性能。颗粒细胞凋亡过程受多种因素的调控。文章简述了miRNA对卵巢颗粒细胞凋亡的调控作用及其机制,其中包括miRNA通过调控激素分泌和细胞凋亡相关因子的表达进而调节颗粒细胞的凋亡,miRNA对颗粒细胞凋亡相关信号通路的影响,miRNA调控颗粒细胞凋亡导致的卵巢相关疾病,并总结了对颗粒细胞凋亡有调控作用的miRNA,以及miRNA在疾病诊断和治疗中的潜在作用,以期为后续相关卵巢疾病的发病机制和治疗方案研究,以及提高雌性哺乳动物生殖性能提供指导和参考。     

miR-495-3p对山羊卵巢颗粒细胞功能的影响

旨在探究miR-495-3p对山羊卵巢颗粒细胞功能的影响及作用机制.本研究选取健康的3～4月龄大足黑山羊母羊,收集卵巢颗粒细胞,利用miR-495-3p模拟物(mimics)和抑制物(inhibitor)构建过表达和抑制模型,通过流式细胞术检测细胞凋亡和周期,ELISA分析颗粒细胞的雌二醇(E2)和孕酮(P4)分泌,采...     

Relationship between apoptosis and proliferation in granulosa and theca cells of cystic follicles in sows

To investigate the causes of the occurrence and persistence of porcine cystic follicles, we evaluated the apoptosis and proliferation of follicular cells in these cysts. Apoptotic frequencies were examined by TUNEL assay and the expression of apoptosis regulators (XIAP, bax, bc1-2 and caspase-3) by immunohistochemistry, Western blotting and real-time quantitative PCR; cell proliferation activity was evaluated by PCNA immunohistochemistry and proliferation of in vitro cultured granulosa and theca cells. The low apoptotic frequency and weak proliferative activity were found in cystic follicles. Low frequency of apoptosis might be associated with decreased amounts of apoptotic-related factors (bax and caspase-3) and increased amounts of anti-apoptotic factors (XIAP and bcl-2) in cystic follicles. Significantly lower proliferation activity was detected in granulosa and theca cells from cystic follicles, and lesser PCNA-positive cells were found in cystic follicles. Our results indicate that the programmed cell death and cell proliferation system were altered in cystic follicles. The disorder between apoptosis and proliferation was responsible for maintaining a static condition without degeneration, which leads to the long-term persistence of follicles. These findings provide important novel insights into the pathogenesis of follicular cysts in sows.     

Vitamin E regulates bovine granulosa cell apoptosis via NRF2-mediated defence mechanism by activating PI3K/AKT and ERK1/2 signalling pathways

High-yield dairy cows are usually subject to high-intensive cell metabolism and produce excessive reactive oxygen species (ROS). Once ROS is beyond the threshold of scavenging ability, it can induce oxidative stress, imperilling the reproductive performance of cows. The study was to investigate the effects of vitamin E (VE) on H₂O₂-induced proliferation and apoptosis of bovine granulosa cells and the underlying molecular mechanism. Granulosa cells were pretreated with VE for 24 hr and then treated with H₂O₂ for 6 hr. The results showed that VE treatment decreased the intracellular ROS levels, increased the MDA content, and improved the antioxidant enzyme activity in a dose-dependent manner. Furthermore, VE treatment promoted the proliferation and inhibited apoptosis in granulosa cells by up-regulation of CCND1 and BCL2 levels and down-regulation of P21, BAX, and CASP3 levels. The cytoprotective effects of VE were attributed to the activation of the NRF2 signalling pathway. Knockdown of the NRF2 impaired the cytoprotective effects of VE on granulosa cells. Besides, the PI3K/AKT and ERK1/2, but not the p38 signalling pathway is involved in the regulation of VE-mediated cell proliferation and apoptosis. The PI3K/AKT inhibitor LY294002 and ERK1/2 inhibitor SCH772984 inhibited the VE-induced granulosa cell proliferation and promoted apoptosis, whereas the p38 inhibitor SB203580 had the opposite effects. These results were confirmed by proliferation and apoptosis-related gene expression at mRNA and protein levels. The results also showed that the PI3K/AKT inhibitor LY294002 and ERK1/2 inhibitor SCH772984 inhibited VE-induced NRF2, GCLC, GCLM, and HO-1 expression, whereas the p38 inhibitor SB203580 not. Overall, the results demonstrated that VE-regulated granulosa cell proliferation and apoptosis via NRF2-mediated defence system by activating the PI3K/AKT and ERK1/2 signalling pathway.     

Does Apoptosis Occur during Follicular Atresia in the Follicular Walls of the Porcine Ovary?

Contents In this experiment, the possibility that the follicular-wall cells' death during ovarian follicular atresia occurs as a result of apoptosis was examined. Programmed cell death or apoptosis is a process whereby cells die in a controlled fashion, triggered by changes in levels of specific physiological stimuli. Morphological transformations of the cells are preceded by endo- nuclease-mediated genomic-DNA cleavage. The analysis of DNA from the theca and granulosa layers of follicles indicated that internucleosomal fragmentation of DNA occurred in atretic granulosa cells but not in atretic theca cells. The healthy granulosa and theca cells in all classes of follicles showed no apoptosis. This paper demonstrates that the death of porcine ovarian-follicle walls can be caused by different processes and, contrary to granulosa cells' apoptosis, either does not or only partly concerns the internal theca layer.     

Sensitivity of skeletal muscle to pro-apoptotic factors

In mononuclear cells, apoptosis leads to DNA fragmentation and cell destruction, regardless of the activated pathway. As regards multinuclear cells, e.g. skeletal muscle fibers, apoptosis rarely induces the death of the entire cell, and it generally affects single nuclei. This process, referred to as nuclear apoptosis, has a negative effect on the expression of genes in the myonuclear domain. Apoptosis may be initiated in muscle cells by external stimuli which activate cell membrane death receptors as well as by internal stimuli which stimulate the mitochondrial release of pro-apoptotic proteins. Reactive oxygen species also play an important role in the initiation of apoptosis. In muscle cells, ROS are produced in response to extracellular reactions or by cell mitochondria. It is, therefore, believed that mitochondria play a central role in apoptosis within skeletal muscle. Skeletal muscles have a well-developed system that protects them against oxidative damage. Myogenic stem cells are an integral part of multinucleated myofibers, and they are critically important for the maintenance of normal muscle mass, muscle growth, regeneration and hypertrophy. The latest research results indicate that myogenic cells are more sensitive to oxidative stress and pro-apoptotic factors than well-differentiated cells, such as myotubes. The complex structure and activity of skeletal muscle prompted research into the role of apoptosis and its intensity under various physiological and pathological conditions. This review summarizes the results of research investigating control mechanisms and the apoptosis process in skeletal muscle fibers, and indicates unresearched areas where further work is required.     

TRAIL-decoy receptor 1 plays inhibitory role in apoptosis of granulosa cells from pig ovarian follicles

Previously, we histochemically examined the localization of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its receptors in porcine ovarian follicles, and demonstrated a marked reduction in the expression of TRAIL-decoy receptor-1 (DcRI) in granulosa cells of atretic follicles. In the present study, to confirm the inhibitory activity of DcR1 in granulosa cells, granulosa cells prepared from healthy follicles were treated with phosphatidylinositol-specific phospholipase C (PI-PLC) to cleave glycophospholipid anchor of DcR1 and to remove DcR1 from the cell surface, and then incubated with TRAIL. PI-PLC treatment increased the number of apoptotic cells induced by TRAIL. The present finding indicated the possibility that TRAIL and its receptors were involved in induction of apoptosis in granulosa cells during atresia, and that DcR1 plays an inhibitory role in granulosa cell apoptosis.     

湖羊PGR基因对卵泡颗粒细胞体外增殖与凋亡的影响

旨在探究孕酮受体(progesterone receptor,PGR)基因对湖羊卵泡颗粒细胞体外增殖与凋亡的影响。利用RT-PCR技术扩增和克隆获得PGR基因编码序列(CDS),通过生物信息学软件对其氨基酸序列及同源性进行比对;用所获得序列构建过表达载体和干扰siRNA,分别转染湖羊颗粒细胞,并用CCK8技术检测颗粒细胞的细胞活力;采用RT-qPCR和Western blot技术,检测细胞周期和凋亡的相关基因或蛋白表达水平。结果显示,羊PGR基因的CDS区全长2736 bp,编码911个氨基酸;与其他物种氨基酸序列的同源性为39.66%~95.44%。干扰PGR基因通过下调CDK4、Bcl-2和上调Caspas3、Caspase8、BAX基因mRNA的表达(P<0.05),进而抑制颗粒细胞的增殖,但对CyclinD1未产生影响(P>0.05);过表达PGR基因通过上调CDK4、CyclinD1、Bcl-2和下调Caspase3、Caspase8、BAX基因mRNA的表达(P<0.05),进而促进颗粒细胞的增殖;BAX蛋白表达变化与对应mRNA的表达趋势一致(P<0.05);过表达PGR基因显著上调PCNA基因表达,而干扰PGR基因则下调PCNA蛋白表达(P<0.05)。研究表明,PGR基因通过调控细胞周期和凋亡关键基因的表达,影响湖羊颗粒细胞的增殖与凋亡,进而调节湖羊卵泡发育。