Avian TVB (DR5-like) death receptor expression in hen ovarian follicles

TVB is an avian death domain-containing receptor belonging to the TNF receptor family and is proposed to be the ortholog to mammalian DR5. Although TVB receptor activation has been demonstrated to mediate apoptosis in chick embryo fibroblasts, there is essentially no information regarding TVB expression or regulation in the mature hen ovary, and in particular within the follicle granulosa layer where apoptosis is known to promote atresia. Significantly, the TVB receptor represents the fourth death domain-containing receptor (also including Fas, TNF-R1, and DR6) found to be expressed within hen granulosa cells. Levels of TVB expression are higher in prehierarchal follicles actively undergoing atresia compared to healthy follicles. However, increased TVB expression does not precede follicle death induced in vitro. Furthermore, TVB expression within granulosa cells is highest during the final stages of follicle development when follicles are not normally susceptible to undergoing atresia. These results provide evidence that TVB receptor signaling in the ovary may function in a capacity other than solely to mediate granulosa cell death and follicle atresia.     

Intracellular mechanisms regulating cell survival in ovarian follicles

The vertebrate ovary represents a uniquely dynamic organ system charged with the responsibility to initially provide, and subsequently foster, optimal numbers of maturing, viable gametes that will insure the propagation of the species. Seemingly in spite of this charge, >99% of germ cells within the ovaries of mammalian and avian species present at the time of birth or hatch are lost via atresia at some point during the lifespan of the female. The consequence of this ongoing germ cell and ovarian follicle attrition in some species eventually leads to the natural termination of reproductive function (e.g. menopause in humans), while in all species an excessive loss of germ cells frequently results in diminished reproductive potential due to subclinical or clinical infertility. Apoptosis represents the primary pathway by which defective or excessive numbers of follicles are rapidly and effectively eliminated, and this process is actively opposed or entirely suppressed by a variety of cell survival signaling pathways and cellular anti-apoptotic proteins expressed within follicles destined for ovulation. Significantly, such survival mechanisms are regulated by many of the same endocrine-paracrine-autocrine factors that control follicle differentiation. This review will begin by briefly discussing the process of apoptosis, then focus on the varied and often redundant mechanisms that prevent apoptotic cell death in granulosa cells specifically during the late preantral (comparable to the prehierarchal stage of follicle development in avian species) and preovulatory stages of follicle development.     

卵泡颗粒细胞凋亡相关蛋白的研究进展

哺乳动物的卵巢中存在大量卵泡。大多数卵泡在发育过程中发生闭锁而消失,只有少数可以发育到成熟而排卵。卵泡是由卵母细胞与其周围的颗粒细胞构成的。卵泡颗粒细胞的凋亡是卵泡闭锁的主要原因。颗粒细胞凋亡相关蛋白通过参与凋亡通路及凋亡信号转导调节凋亡。本文就哺乳动物卵泡颗粒细胞凋亡相关蛋白的研究进展进行综述。     

Conservation of death receptor-6 in avian and piscine vertebrates.

One of the most recently identified members of the tumor necrosis factor receptor family, death receptor-6 (DR6), has been shown to mediate apoptosis following overexpression in HeLa cells. The avian and piscine orthologs of DR6 have now been identified, and the deduced amino acid sequence for each demonstrates a high level of conservation compared to the mammalian sequence. Expression of dr6 mRNA occurs widely across tissues of both the mature chicken and brook trout. It is now well-established that ovarian follicular atresia occurs via apoptosis originating within the granulosa cell layer. Accordingly, DR6 expression within the ovary was examined to assess the relationship between stage of follicle development and relative levels of this death receptor. Of particular interest was the finding that elevated levels of dr6 mRNA, as well as the translated protein, are expressed in atretic compared to healthy follicles of the hen ovary, thus providing the first association between DR6 expression and apoptosis, in vivo. We conclude that DR6 is a highly conserved and widely expressed death-domain-containing receptor and may be implicated in regulating follicle atresia within the vertebrate ovary.     

Expression and Preliminary Functional Profiling of the let-7 Family during Porcine Ovary Follicle Atresia

Most follicles in the mammalian ovary undergo atresia. Granulosa cell apoptosis is a hallmark of follicle atresia. Our previous study using a microRNA (miRNA) microarray showed that the let-7 microRNA family was differentially expressed during follicular atresia. However, whether the let-7 miRNA family members are related to porcine (Sus scrofa) ovary follicular apoptosis is unclear. In the current study, real-time quantitative polymerase chain reaction showed that the expression levels of let-7 family members in follicles and granulosa cells were similar to our microarray data, in which miRNAs let-7a, let-7b, let-7c, and let-7i were significantly decreased in early atretic and progressively atretic porcine ovary follicles compared with healthy follicles, while let-7g was highly expressed during follicle atresia. Furthermore, flow cytometric analysis and Hoechst33342 staining demonstrated that let-7g increased the apoptotic rate of cultured granulosa cells. In addition, let-7 target genes were predicted and annotated by TargetScan, PicTar, gene ontology and Kyoto encyclopedia of genes and genomes pathways. Our data provide new insight into the association between the let-7 miRNA family in granulosa cell programmed death.     

Growth differentiation factor 9 is antiapoptotic during follicular development from preantral to early antral stage

Ovarian follicular atresia represents a selection process that ensures the release of only healthy and viable oocytes during ovulation. The transition from preantral to early antral stage is the penultimate stage of development in terms of gonadotropin dependence and follicle destiny (survival/growth vs. atresia). We have examined whether and how oocyte-derived growth differentiation factor 9 (GDF-9) and FSH regulate follicular development and atresia during the preantral to early antral transition, by a novel combination of in vitro gene manipulation (i.e. intraoocyte injection of GDF-9 antisense oligos) and preantral follicle culture. Injection of GDF-9 antisense suppressed basal and FSH-induced preantral follicle growth in vitro, whereas addition of GDF-9 enhanced basal and FSH-induced follicular development. GDF-9 antisense activated caspase-3 and induced apoptosis in cultured preantral follicles, a response attenuated by exogenous GDF-9. GDF-9 increased phospho-Akt content in granulosa cells of early antral follicles. Although granulosa cell apoptosis induced by ceramide was attenuated by the presence of GDF-9, this protective effect of GDF-9 was prevented by the phosphatidylinositol 3-kinase inhibitor LY294002 and a dominant negative form of Akt. Injection of GDF-9 antisense decreased FSH receptor mRNA levels in cultured follicles, a response preventable by the presence of exogenous GDF-9. The data suggest that GDF-9 is antiapoptotic in preantral follicles and protects granulosa cells from undergoing apoptosis via activation of the phosphatidylinositol 3-kinase/Akt pathway. An adequate level of GDF-9 is required for follicular FSH receptor mRNA expression. GDF-9 promotes follicular survival and growth during the preantral to early antral transition by suppressing granulosa cell apoptosis and follicular atresia.     

Gap junctional communication and connexin43 expression in relation to apoptotic cell death and survival of granulosa cells.

Ovarian follicular atresia in all vertebrates is mediated via apoptosis that is initiated in the granulosa cell layer. Here we investigated the relation between connexin expression, cell coupling, and apoptosis in avian granulosa cells. Results from qualitative and quantitative immunocytochemical analysis and Western blotting of connexin43 (Cx43) and electron microscopic observations of gap junctions were compared with functional data on gap junctional coupling obtained by fluorescence recovery after photobleaching in four experimental groups: a control group of freshly isolated granulosa cells, 24-hr serum-free cultures as the apoptosis-inducing condition, and two other groups in which apoptosis was inhibited by either hormone substitution or exposure to elevated extracellular calcium. Our work shows that apoptosis induction in granulosa cells is accompanied by an increased level of cell coupling and that decreasing cell coupling with the gap junction blocker alpha-glycyrrhetinic acid dose-dependently inhibits apoptosis. The level of Cx43 expression was inversely related to the apoptotic index, suggesting that Cx43 expression plays a role in granulosa cell survival. Our study supports the hypothesis that gap junctional coupling plays a role in propagating a cell death message and suggests a role for Cx43 expression per se in granulosa cell survival.     

Age-associated up-regulation of EGR1 promotes granulosa cell apoptosis during follicle atresia in mice through the NF-κB pathway

Follicular atresia is the main process responsible for the loss of follicles and oocytes from the ovary, and it is the root cause of ovarian aging. Apoptosis of granulosa cells (GCs) is the cellular mechanism responsible for follicular atresia in mammals. Recent advances have highlighted fundamental roles for EGR1 in age-related diseases via the induction of apoptosis. In the present study, we found that the expression of EGR1 was significantly increased in aged mouse ovaries compared with young ovaries. Immunohistochemical analysis revealed strongly positive EGR1 staining in atretic follicles, especially in apoptotic granulosa cells. We further showed that EGR1 up-regulation in mouse primary granulosa cells inhibited cell proliferation and promoted apoptosis. In addition, the promotion of apoptosis in GCs by EGR1 increases over time and with reactive oxygen species (ROS) stimulation. Our mechanistic study suggested that EGR1 regulates GC apoptosis in a mitochondria-dependent manner and that this mainly occurs through the NF-κB signaling pathway. In conclusion, our results suggested that age-related up-regulation of EGR1 promotes GC apoptosis in follicle atresia during ovarian aging.     

Insulin mitigates apoptosis of porcine follicular granulosa cells by downregulating BimEL

Follicular growth or atresia is governed by the survival and apoptosis of granulosa cells. Increasing evidence shows that follicle growth is influenced by energy intake, which is positively related to insulin levels. However, the function of insulin in granulosa cell survival is poorly understood. This study focused on the effects of insulin on porcine medium follicle granulosa cell survival. In the present study, we showed that insulin markedly mitigated the apoptosis of porcine granulosa cells following serum starvation. Moreover, insulin activated the PI3K/Akt pathway to downregulate bim mRNA expression and simultaneously promoted the phosphorylation of BimEL through activating ERK 1/2, both of which reduced the level of BimEL. The results demonstrate that insulin protected the granulosa cells against apoptosis by reducing levels of the pro-apoptotic protein BimEL. However, the concentration of insulin (1 μg/ml) was relatively high. High levels of insulin partly combined with the IGF-1 receptor to play its roles in granulosa cells. This experiment provides new insight into the role of insulin in granulosa cells and sheds light on nutrition-reproduction interactions.     

Histogenesis of atretic ovarian follicles in a seasonally breeding bird

Histologic examination of ovaries from a non-migratory population of scrub jays (Aphelocoma coerulescens) disclosed a marked annual cycle in the incidence of atresia. Atretic follicles became more common as the nesting season progressed and were most abundant immediately after the cessation of breeding. Atresia involved a dissociation of granulosa cells and movement of these cells into the follicle. Subsequently, granulosa cells showed steatogenesis and ultimately disappeared simultaneously with the invasion of the follicle by ex-thecal gland cells. The data suggest that the diverse histology of avian atretic follicles reflects different stages in the process of atresia rather than multiple origins. Ovarian stromal glands apparently arise both from ex-thecal gland cells of atretic follicles and stromal connective tissue. A possible secretory role of atretic follicles is considered.     

MicroRNA let-7g regulates mouse granulosa cell autophagy by targeting insulin-like growth factor 1 receptor

As an important type of somatic cell, granulosa cells play a major role in deciding the fate of follicles. Therefore, analyses of granulosa cell apoptosis and follicular atresia have become hotspots of animal research. Autophagy is a cellular catabolic mechanism that protects cells from stress conditions, including starvation, hypoxia, and accumulation of misfolded proteins. However, the relationship between autophagy and apoptosis in granulosa cells is not well known. Here, we demonstrate that let-7g regulates the mouse granulosa cell autophagy signaling pathway by inhibiting insulin-like growth factor 1 receptor expression and affecting the phosphorylation of protein kinase B/mammalian target of rapamycin. Small interference-mediated knockdown of insulin-like growth factor 1 receptor significantly promoted autophagy signaling of mouse granulosa cells. In contrast, overexpression of insulin-like growth factor 1 receptor in mouse granulosa cells attenuated autophagy activity in the presence of let-7g. In addition, overexpression of let-7g increased the apoptosis rate, as indicated by an increased number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells. Finally, 3-methyladenine as well as the lysosomal enzyme inhibitor chloroquine partially blocked apoptosis. In summary, this study demonstrates that let-7g regulates autophagy in mouse granulosa cells by targeting insulin-like growth factor 1 receptor and downregulating protein kinase B/mammalian target of rapamycin signaling, and that mouse granulosa cell autophagy induced by let-7g participates in apoptosis.     

Activation of the Akt/protein kinase B signaling pathway is associated with granulosa cell survival

Follicles from the hen ovary that have been selected into the preovulatory hierarchy are committed to ovulation and rarely become atretic under normal physiological conditions. In part, this is attributed to the resistance of the granulosa layer to apoptosis. The present studies were conducted to evaluate the role of the phosphatidylinositol (PI) 3-kinase/Akt signaling pathway in hen granulosa cell survival and, by implication, follicle viability. Cloning of the chicken akt2 homologue revealed a high degree of amino acid homology to its mammalian counterparts within the catalytic domain, plus complete conservation of the putative Thr(308) and Ser(474) phosphorylation sites. Treatment of granulosa cells from the three largest preovulatory follicles with insulin-like growth factor (IGF)-I and, to a lesser extent, transforming growth factor (TGF)-alpha induces rapid phosphorylation of Akt, and such phosphorylation is effectively blocked by the PI 3-kinase-inhibitor LY294006. Serum withdrawal from cultured cells for 33-44 h initiates oligonucleosome formation, an indicator of apoptotic cell death, whereas cotreatment with IGF-I prevents this effect. Moreover, treatment of cultured cells for 20 h with LY294006 induces apoptosis. The potential for nonspecific cell toxicity following LY294006 treatment is considered unlikely because of the ability of either LH or 8-bromo cAMP cotreatment to block LY294006-induced cell death. Finally, both IGF-I and TGF-alpha also activate mitogen-activated protein (MAP) kinase signaling, at least in part, through the phosphorylation of ERK: However, treatment with neither U0126 nor PD98059 (inhibitors of MAP kinase kinase) induced cell death in cultured granulosa cells, despite the ability of each inhibitor to effectively block Erk phosphorylation. Taken together, these results provide evidence for a role of the Akt signaling pathway in promoting cell survival within the preovulatory follicle granulosa layer. In addition, the data indicate the importance of an alternative survival pathway mediated via gonadotropins and protein kinase A independent of Akt signaling.