Involvement of JNK/FOXO1 pathway in apoptosis induced by severe hypoxia in porcine granulosa cells. Liu Z et al. (2020) In ovaries, follicles undergo a periodic process of degeneration, namely atresia, during each stage of development. Granulosa cell (GC) apoptosis is believed as the hallmark of follicular atresia. The avascular environment within the granulosa compartment is supposed to cause hypoxic conditions. The effects of hypoxia on organs, tissues, cells can be either positive or negative, depending on the severity and context. The present study aimed to explore whether and how severe hypoxia under in vitro conditions functions in apoptosis of porcine GCs. The current results showed that the apoptosis in porcine GCs exposed to severe hypoxia (1% O2) was correlated with enhanced activation of c-Jun N-terminal kinase (JNK), nuclear accumulation of FOXO1, as well as elevated level of cleaved caspase-3 and decreased ratio of BCL-2/BAX. Further investigations revealed that severe hypoxia-mediated JNK activation was required for the apoptotic death of porcine GCs and the nuclear transport of FOXO1. Moreover, inhibition of FOXO1 reduced GCs apoptosis upon severe hypoxia exposure. Together, these findings suggested that severe hypoxia might act through JNK/FOXO1 axis to induce apoptosis in porcine GCs.////////////////// Protective mechanism of FSH against oxidative damage in mouse ovarian granulosa cells by repressing autophagy. Shen M et al. (2017) Oxidative stress-induced granulosa cell (GCs) death represents a common reason for follicular atresia. Follicle-stimulating hormone (FSH) has been shown to prevent GCs from oxidative injury, although the underlying mechanism remains to be elucidated. Here we first report that the suppression of autophagic cell death via some novel signaling effectors is engaged in FSH-mediated GCs protection against oxidative damage. The decline in GCs viability caused by oxidant injury was remarkably reduced following FSH treatment, along with impaired macroautophagic/autophagic flux under conditions of oxidative stress both in vivo and in vitro. Blocking of autophagy displayed similar levels of suppression in oxidant-induced cell death compared with FSH treatment, but FSH did not further improve survival of GCs pretreated with autophagy inhibitors. Further investigations revealed that activation of the phosphoinositide 3-kinase (PI3K)-AKT-MTOR (mechanistic target of rapamycin serine/threonine kinase]) signaling pathway was required for FSH-mediated GCs survival from oxidative stress-induced autophagy. Additionally, the FSH-PI3K-AKT axis also downregulated the autophagic response by targeting FOXO1, whereas constitutive activation of FOXO1 in GCs not only abolished the protection from FSH, but also emancipated the autophagic process, from the protein level of MAP1LC3B-II to autophagic gene expression. Furthermore, FSH inhibited the production of acetylated FOXO1 and its interaction with Atg proteins, followed by a decreased level of autophagic cell death upon oxidative stress. Taken together, our findings suggest a brand new mechanism involving FSH-FOXO1 signaling in defense against oxidative damage to GCs by restraining autophagy, which may be a potential avenue for the clinical treatment of anovulatory disorders.////////////////// Stress Induces Mouse Follicular Granulosa Cells Apoptosis via JNK/FoxO1 Pathway. [Weng Q et al. (2016) The c-Jun N-terminal protein kinase (JNK) plays an important role in the regulation of cell apoptosis. Forkhead box O (FoxO) transcription factors are involved in diverse biological processes, including cellular metabolism, cell apoptosis, and cell cycle. However, the JNK/FoxO1 pathway involved in the process of apoptosis induced by oxidative stress remains to be elucidated. Here, we demonstrated that the JNK activity significantly increased in response to oxidative stress in mouse follicular granulosa cells (MGCs). SP600125, a selective JNK inhibitor, attenuated the oxidative stress-induced MGCs apoptosis. Oxidative stress enhanced the FoxO1 nuclear translocation by activating the JNK activity. Moreover, JNK mediated the dissociation of FoxO1 from 14-3-3 proteins in MGCs after the treatment with H2O2. Finally, oxidative stress up-regulated the expression of FoxO1 via JNK mediation of FoxO1 self-regulation in MGCs. Taken together, our findings suggest that JNK/FoxO1 is involved in the regulation of oxidative stress-induced cell apoptosis in MGCs.//////////////////
Forkhead box O member FOXO1 regulates the majority of follicle-stimulating hormone responsive genes in ovarian granulosa cells. Herndon MK et al. (2016) FSH promotes maturation of ovarian follicles. One pathway activated by FSH in granulosa cells (GCs) is phosphatidylinositol-3 kinase/AKT. The AKT target FOXO1 is reported to function primarily as a repressor of FSH genes, including Ccnd2 and Inha. Based on its broad functions in other tissues, we hypothesized that FOXO1 may regulate many more GC genes. We transduced GCs with empty adenovirus or constitutively active FOXO1 followed by treatment with FSH for 24 h, and conducted RNA deep sequencing. Results show that FSH regulates 3,772 genes ≥2.0-fold; 60% of these genes are activated or repressed by FOXO1. Pathway Studio Analysis revealed enrichment of genes repressed by FOXO1 in metabolism, signaling, transport, development, and activated by FOXO1 in signaling, cytoskeletal functions, and apoptosis. Gene regulation was verified by q-PCR (eight genes) and ChIP analysis (two genes). We conclude that FOXO1 regulates the majority of FSH target genes in GCs.//////////////////
Expression of PUMA in Follicular Granulosa Cells Regulated by FoxO1 Activation During Oxidative Stress. Liu ZQ et al. (2014) Many studies have demonstrated that oxidative stress-induced apoptosis is a main cause of follicular atresia. Reactive oxygen species (ROS)-induced granulosa cell (GC) apoptosis is regulated by a variety of signaling pathways involving numerous genes and transcription factors. In this study, we found expression of the p53-upregulated modulator of apoptosis (PUMA), a BH3-only Bcl-2 subfamily protein, in ovarian GCs during oxidative stress. By overexpression and knockdown of Forkhead box O1 (FoxO1), we found that FoxO1 regulates PUMA at the protein level. Moreover, as c-Jun N-terminal kinase (JNK) has been shown to activate FoxO1 by promoting its nuclear import, we used a JNK inhibitor to reduce FoxO1 activation and detected decreased PUMA messenger RNA expression and protein levels during oxidative stress. In addition, in vivo oxidative stress-induced upregulation of PUMA was found following injection of 3 nitropropionic acid in mice. In conclusion, oxidative stress increases PUMA expression regulated by FoxO1 in follicular GCs.//////////////////
Involvement of the upregulated FoxO1 expression in follicular granulosa cell apoptosis induced by oxidative stress. Shen M et al. Follicular atresia is common in female mammalian ovaries, where most follicles undergo degeneration at any stage of growth and development. Oxidative stress gives rise to triggering granulosa cell apoptosis that has been suggested as a major cause of follicular atresia. However, the underlying mechanism by which the oxidative stress induces follicular atresia remains unclear. FoxO transcription factors are known as critical mediators in the regulation of oxidative stress and apoptosis. In this study, the involvement of FoxO1 in oxidative stress-induced apoptosis of mouse granulosa cells (MGCs) was investigated in vivo and in vitro. It was observed that increased apoptotic signals correlated with elevated expression of FoxO1 in MGCs when mice were treated with the oxidant. Correspondingly, the expressions of FoxO1 target genes, such as pro-apoptosis genes and anti-oxidative genes, were also upregulated. In primary cultured MGCs, treatment with H2O2 led to FoxO1 nuclear translocation. Further studies with over-expression and knockdown of FoxO1 demonstrated the critical role of FoxO1 in the induction of MGC apoptosis by oxidative stress. Finally, inactivation of FoxO1 by insulin treatment confirmed that FoxO1 induced by oxidative stress played a pivotal role in upregulating the expression of downstream apoptosis related genes in MGCs. Our results suggest that upregulation of FoxO1 by oxidative stress lead to apoptosis of granulosa cells, which eventually results in follicular atresia in mice.
Increased ovarian follicle atresia in obese Zucker rats is associated with enhanced expression of the forkhead transcription factor FOXO1. Kajihara T et al. It is well established that hyperinsulinemia, resulting from insulin resistance, plays a role in the pathophysiology of polycystic ovary syndrome (PCOS). The aim of this study was to investigate if ovarian follicular development and atresia are impaired in obese hyperinsulinemic (fa/fa) Zucker rats. To gain insight into the molecular mechanism of follicular atresia, we also examined the expression and localization of forkhead transcription factor FOXO1, a major regulator of cell fate decisions such as differentiation, cell-cycle arrest, and cell death. Serum insulin but not gonadotropin levels were significantly higher in obese (fa/fa) rats when compared to lean controls. Total ovarian follicle number and the percentage of atretic follicles were also significantly increased in obese (fa/fa) rats. Follicle atresia was associated with nuclear accumulation of FOXO1 transcription factor in TUNEL-positive granulosa cells. These results suggest a role for FOXO1 in granulosa cell apoptosis and increased ovarian follicle atresia associated with hyperinsulinemia.
Genes in sterol/steroid and lipid biosynthetic pathways are targets of FSH and FOXO1 regulation in granulosa cells: evidence from cells expressing mutant forms of FOXO1. Liu Z et al. The forkhead box transcription factor FOXO1 is highly expressed in granulosa cells of growing follicles but is down-regulated by FSH in culture or by LH-induced luteinization in vivo. To analyze the function of FOXO1, we infected rat and mouse granulosa cells with adenoviral vectors expressing two FOXO1 mutants: a gain-of-function mutant FOXOA3 that has three serine residues mutated to alanines rendering this protein constitutively active and nuclear and FOXOA3-mDBD in which the DNA binding domain is mutated. The infected cells were then treated with vehicle or FSH for specific time intervals. Infection of the granulosa cells was highly efficient, caused only minimal apoptosis and maintained FOXO1 protein at levels of the endogenous protein observed in cells prior to exposure to FSH. RNA was prepared from control and adenoviral infected cells exposed to vehicle or FSH for 12 and 24h. Affymetrix microarray and data-base analyses identified, and real time RT-PCR verified, that genes within the lipid, sterol and steroidogenic biosynthetic pathways (Hmgcs1, Hmgcr, Mvk, Sqle, Lss, Cyp51, Tm7sf2, Dhcr24 and Star, Cyp11a1 and Cyp19), including two key transcriptional regulators Srebf1 and Srebf2 of cholesterol biosynthesis and steroidogenesis (Nr5a1, Nr5a2) were major targets induced by FSH and suppressed by FOXOA3 and FOXOA3-mDBD in the cultured granulosa cells. By contrast, FOXOA3 and FOXOA3-mDBD induced expression of Cyp27a1 mRNA that encodes an enzyme involved in cholesterol catabolism to oxysterols. The genes up-regulated by FSH in cultured granulosa cells were also induced in granulosa cells of preovulatory follicles and corpora lutea collected from immature mice primed with FSH (eCG) and LH (hCG), respectively. Conversely, Foxo1 and Cyp27a1 mRNAs were reduced by these same treatments. Collectively, these data provide novel evidence that FOXO1 may play a key role in granulosa cells to modulate lipid and sterol biosynthesis, thereby preventing elevated steroidogenesis during early stages of follicle development.
Role of the PI3-Kinase and ERK Pathways in the Induction of HIF-1 Activity and the HIF-1 Target VEGF in Ovarian Granulosa Cells in response to Follicle Stimulating Hormone. Alam H et al. FSH stimulation of granulosa cells (GCs) results in increased hypoxia-inducible factor (HIF)-1alpha protein levels and HIF-1 activity that is necessary for up-regulation of certain FSH target genes including vascular endothelial growth factor (VEGF). We report that the role of the phosphatidylinositol (PI)-3-kinase/AKT pathway in increasing HIF-1alpha protein in FSH-stimulated GCs extends beyond an increase in mammalian target of rapamycin (mTOR)-stimulated translation. FSH increases phosphorylation of the AKT target mouse double minute 2 (MDM2); a phosphomimetic mutation of MDM2 is sufficient to induce HIF-1 activity. The PI3-kinase/AKT target forkhead box-containing protein O subfamily 1 (FOXO1) also effects the accumulation of HIF-1alpha as evidenced by the ability of a constitutively active FOXO1 mutant to inhibit the induction by FSH of HIF-1alpha protein and HIF-1 activity. Activation of the PI3-kinase/AKT pathway in GCs by IGF-1 is sufficient to induce HIF-1alpha protein but surprisingly not HIF-1 activity. HIF-1 activity also appears to require a PD98059-sensitive protein (kinase) activity stimulated by FSH that is both distinct from mitogen-activated extracellular signal-regulated kinase (ERK) kinase1/2 or 5 and independent of the PI3-kinase/AKT pathway. These results indicate that FSH-stimulated HIF-1 activation leading to up-regulation of targets such as VEGF requires not only PI3-kinase/AKT-mediated activation of mTOR as well as phosphorylation of FOXO1 and possibly MDM2 but also a protein (kinase) activity that is inhibited by the classic ERK kinase inhibitor PD98059 but is not ERK1/2 or 5. Thus, regulation of HIF-1 activity in GCs by FSH under normoxic conditions is complex and requires input from multiple signaling pathways.
{beta}-Catenin (CTNNB1) Promotes Preovulatory Follicular Development but Represses LH-Mediated Ovulation and Luteinization. Fan HY et al. Wingless-type mouse mammary tumor virus integration site family (WNT)/beta-catenin (CTNNB1) pathway components are expressed in ovarian granulosa cells, direct female gonad development, and are regulated by the pituitary gonadotropins. However, the in vivo functions of CTNNB1 during preovulatory follicular development, ovulation, and luteinization remain unclear. Using a mouse model Ctnnb1((Ex3)fl/fl);Cyp19-Cre (Ctnnb1((Ex3)gc-/-)), expressing dominant stable CTNNB1 in granulosa cells of small antral and preovulatory follicles, we show that CTNNB1 facilitates FSH-induced follicular growth and decreases the follicle atresia (granulosa cell apoptosis). At the molecular level, WNT signaling and FSH synergistically promote the expression of genes required for cell proliferation and estrogen biosynthesis, but decrease FOXO1, which negatively regulates proliferation and steroidogenesis. Conversely, dominant stable CTNNB1 represses LH-induced oocyte maturation, ovulation, luteinization, and progesterone biosynthesis. Specifically, granulosa cells in the Ctnnb1((Ex3)gc-/-) mice showed compromised responses to the LH surge and decreased levels of the epidermal growth factor-like factors (Areg and Ereg) that in vivo and in vitro mediate LH action. One underlying mechanism by which CTNNB1 prevents LH responses is by reducing phosphorylation of cAMP-responsive element-binding protein, which is essential for the expression of Areg and Ereg. By contrast, depletion of Ctnnb1 using the Ctnnb1(fl/fl);Cyp19-Cre mice did not alter FSH regulation of preovulatory follicular development or female fertility but dramatically enhanced LH induction of genes in granulosa cells in culture. Thus, CTNNB1 can enhance FSH and LH actions in antral follicles but overactivation of CTNNB1 negatively effects LH-induced ovulation and luteinization, highlighting the cell context-dependent and developmental stage-specific interactions of WNT/CTNNB1 pathway and G protein-coupled gonadotropin receptors in female fertility.
Expression regulated by
FSH, LH, mir183, let7g, mir96, mir181
Comment
miR-181a increases FoxO1 acetylation and promotes granulosa cell apoptosis via SIRT1 downregulation. Zhang M et al. (2017) Oxidative stress impairs follicular development by inducing granulosa cell (GC) apoptosis, which involves enhancement of the transcriptional activity of the pro-apoptotic factor Forkhead box O1 (FoxO1). However, the mechanism by which oxidative stress promotes FoxO1 activity is still unclear. Here, we found that miR-181a was upregulated in hydrogen peroxide (H2O2)-treated GCs and a 3-nitropropionic acid (NP)-induced in vivo model of ovarian oxidative stress. miR-181a overexpression promoted GC apoptosis, whereas knockdown of endogenous miR-181a blocked H2O2-induced cell apoptosis. Moreover, we identified that Sirtuin 1 (SIRT1), a deacetylase that suppresses FoxO1 acetylation in GCs, was downregulated by miR-181a and reversed the promoting effects of H2O2 and miR-181a on FoxO1 acetylation and GC apoptosis. Importantly, decreased miR-181a expression in the in vivo ovarian oxidative stress model inhibited apoptosis by upregulating SIRT1 expression and FoxO1 deacetylation. Together, our results suggest that miR-181a mediates oxidative stress-induced FoxO1 acetylation and GC apoptosis by targeting SIRT1 both in vitro and in vivo.//////////////////
The adequate corpus luteum: miR-96 promotes luteal cell survival and progesterone production. Mohammed BT et al. (2017) Inadequate progesterone production from the corpus luteum is associated with pregnancy loss. Data available in model species suggest important roles of miRNAs in luteal development and maintenance. To comprehensively investigate the involvement of miRNAs during the ovarian follicle-luteal transition. The effects of specific miRNAs on survival and steroid production by human luteinized granulosa cells (hLGCs) were tested using specific miRNA inhibitors. Candidate miRNAs were first identified through microarray analyses of follicular and luteal tissues in a bovine model. UK academic institution associated with teaching hospital. hLGCs were obtained by standard transvaginal follicular fluid aspiration from 35 women undergoing assisted conception. Inhibition of candidate miRNAs in vitro. Levels of miRNAs, mRNAs, FOXO1 protein, apoptosis and steroids were measured in tissues and/or cultured cells. Two specific miRNA clusters, miR-183-96-182 and miR-212-132, were dramatically increased in luteal relative to follicular tissues. miR-96 and miR-132 were the most upregulated miRNAs within each cluster. Database analyses identified FOXO1 as a putative target of both these miRNAs. In cultured hLGCs, inhibition of miR-96 increased apoptosis and FOXO1 protein levels, and decreased progesterone production. These effects were prevented by siRNA-mediated downregulation of FOXO1. In bovine luteal cells, miR-96 inhibition also led to increases in apoptosis and FOXO1 protein levels. miR-96 targets FOXO1 to regulate luteal development through effects on cell survival and steroid production. The miR-183-96-182 cluster could provide a novel target for the manipulation of luteal function.//////////////////
MicroRNA-183~96~182 Cluster Regulate Bovine Granulosa Cell Proliferation and Cell Cycle Transition by Coordinately Targeting FOXO1. Gebremedhn S et al. (2016) Large-scale expression profiling of microRNAs (miRNAs) in bovine granulosa cells from dominant and subordinate follicles on Day 19 of the estrous cycle revealed enriched microRNA-183~96~182 (miR-183~96~182) cluster miRNAs in preovulatory dominant follicles that coordinately regulate the forkhead box protein O1 (FOXO1) gene. However, little is known about the role of this cluster in bovine granulosa cell function. We employed an in vitro granulosa cell culture model to investigate this role. Granulosa cells aspirated from small growing follicles (3-5 mm in diameter) were cultured in DMEM/F-12 medium supplemented with Fetal Bovine Serum (FBS) and transfected with Locked Nucleic Acid (LNA)-based miRNA mimics, inhibitors, and the corresponding negative controls. The overexpression of the miRNA cluster resulted in the suppression of FOXO1 mRNA and protein, while inhibition of the cluster increased the expression of FOXO1 mRNA. Overexpression also increased the relative rate of cell proliferation, while inhibition slowed it down. Similarly, the proportion of cells under G0/G1 arrest declined, while the ratio of cells in the S phase increased in response to miR-183~96~182 overexpression. Selective knockdown of FOXO1 mRNA using anti-FOXO1 small interfering RNA (siRNA) increased the rate of granulosa cell proliferation, decreased the proportion of cells under G0/G1 arrest, and increased the proportion of cells in the S phase of the cell cycle. Our data suggest that miR-183~96~182 cluster miRNAs promote proliferation and G1/S transition of bovine granulosa cells by coordinately targeting FOXO1, suggesting a critical role in granulosa cell function.//////////////////
Let-7g Induces Granulosa Cell Apoptosis by Targeting MAP3K1 in the Porcine Ovary. Rui C et al. (2015) Follicular atresia mainly results from apoptosis of granulosa cells (GCs). Our previous microRNA array data indicated that the miRNA let-7g level increases significantly during porcine ovary follicular atresia. It is uncertain if GCs apoptosis is mediated by microRNA let-7g. In this study, the expression levels of the apoptosis-associated genes CASP3, BAX and BIM were significantly upregulated when let-7g mimic was transfected into porcine GCs, and the anti-apoptotic genes BCL-2 and MCL-1 were significantly downregulated. The apoptosis rate was measured by flow cytometry, and our results indicated that let-7g significantly enhanced GCs apoptosis. In further studies, we found that overexpression of let-7g induced the expression of FoxO1 in GCs and led to nuclear accumulation of dephosphorylated FoxO1. In addition, the effect of let-7g on FoxO1 expression and dephosphorylation resulted from repression of the expression of the MAP3K1 gene in porcine GCs. The site on MAP3K1 mRNA targeted by let-7g was confirmed by luciferase reporter assay. The anti-apoptotic effect of MAP3K1 was validated by silencing MAP3K1 using small interfering RNA technology. In conclusion, our data indicate that let-7g induces porcine GCs apoptosis by inhibiting the MAP3K1 gene, which promotes FoxO1 expression and dephosphorylation with nuclear accumulation.//////////////////
MicroRNA Expression Profile in Bovine Granulosa Cells of Preovulatory Dominant and Subordinate Follicles during the Late Follicular Phase of the Estrous Cycle. Gebremedhn S et al. (2015) In bovine, ovarian follicles grow in a wave-like fashion with commonly 2 or 3 follicular waves emerging per estrous cycle. The dominant follicle of the follicular wave which coincides with the LH-surge becomes ovulatory, leaving the subordinate follicles to undergo atresia. These physiological processes are controlled by timely and spatially expressed genes and gene products, which in turn are regulated by post-transcriptional regulators. MicroRNAs, a class of short non-coding RNA molecules, are one of the important posttranscriptional regulators of genes associated with various cellular processes. Here we investigated the expression pattern of miRNAs in granulosa cells of bovine preovulatory dominant and subordinate follicles during the late follicular phase of bovine estrous cycle using Illumina miRNA deep sequencing. In addition to 11 putative novel miRNAs, a total of 315 and 323 known miRNAs were detected in preovulatory dominant and subordinate follicles, respectively. Moreover, in comparison with the subordinate follicles, a total of 64 miRNAs were found to be differentially expressed in preovulatory dominant follicles, of which 34 miRNAs including the miR-132 and miR-183 clusters were significantly enriched, and 30 miRNAs including the miR-17-92 cluster, bta-miR-409a and bta-miR-378 were significantly down regulated in preovulatory dominant follicles. In-silico pathway analysis revealed that canonical pathways related to oncogenesis, cell adhesion, cell proliferation, apoptosis and metabolism were significantly enriched by the predicted target genes of differentially expressed miRNAs. Furthermore, Luciferase reporter assay analysis showed that one of the differentially regulated miRNAs, the miR-183 cluster miRNAs, were validated to target the 3´-UTR of FOXO1 gene. Moreover FOXO1 was highly enriched in granulosa cells of subordinate follicles in comparison with the preovulatory dominant follicles demonstrating reciprocal expression pattern with miR-183 cluster miRNAs. In conclusion, the presence of distinct sets of miRNAs in granulosa cells of preovulatory dominant and subordinate follicles supports the potential role of miRNAs in post-transcriptional regulation of genes involved in bovine follicular development during the late follicular phase of the estrous cycle.//////////////////
Involvement of FoxO1 in the effects of follicle-stimulating hormone on inhibition of apoptosis in mouse granulosa cells. Shen M et al. (2014) In mammalian ovaries, follicular atresia occurs periodically and destroys almost all the follicles in the ovary. Follicle-stimulating hormone (FSH) acts as the primary survival factor during follicular atresia by preventing apoptosis in granulosa cells. FoxO1 is a critical factor in promoting follicular atresia and granulosa cell apoptosis. FSH inhibits the induction of FoxO1. In this report, we investigated the role of FSH-FoxO1 pathway in mouse follicular atresia. FSH dampened stress-induced apoptosis and the expression of FoxO1 and pro-apoptosis genes in mouse granulosa cells (MGCs). In contrast, overexpression of FoxO1 inhibited the viability of MGCs and induced the expression of endogenous FoxO1. The signaling cascades involved in regulating FoxO1 activity upon FSH treatment were identified using FSH signaling antagonists. Blocking protein kinase A (PKA), phosphatidylinositol-3 kinase (PI3K) or protein kinase B (AKT) restored the upregulation of FoxO1 and apoptotic signals, which was suppressed by FSH. Moreover, inhibition of PKA or PI3K impaired FSH-induced AKT activity, but inactivation of PI3K or AKT had little effect on PKA activity in the presence of FSH. Correspondingly, constitutive activation of FoxO1 (all three AKT sites were replaced by alanines) also promoted MGC apoptosis despite FSH administration. Furthermore, both luciferase reporter assays and chromatin immunoprecipitation assays showed that FoxO1 directly bound to a FoxO-recognized element site within the FoxO1 promoter and contributed to the regulation of FoxO1 expression in response to FSH. Taken together, we propose a novel model in which FSH downregulates FoxO1-dependent apoptosis in MGCs by coordinating the PKA-PI3K-AKT-FoxO1 axis and FoxO1-FoxO1 positive feedback.//////////////////
Lhcgr Expression Granulosa Cells: Roles for PKA-Phosphorylated ?catenin, TCF3, and FOXO1. Law NC et al. Ovarian follicles lacking FSH or FSH receptors fail to progress to a preovulatory stage, resulting in infertility. One hallmark of the preovulatory follicle is the presence of luteinizing hormone choriogonadotropin receptors (LHCGR) on granulosa cells (GCs). However, the mechanisms by which FSH induces Lhcgr gene expression are poorly understood. Our results show that protein kinase A (PKA) and phosphoinositide 3-kinase (PI3K)/AKT pathways are required for FSH to activate both the murine Lhcgr-luciferase reporter and expression of Lhcgr mRNA in rat GCs. Based on results showing that an adenovirus (Ad) expressing a steroidogenic factor 1 (SF1) mutant that cannot bind ?catenin abolished FSH-induced Lhcgr mRNA, we evaluated the role of ?catenin in the regulation of Lhcgr gene expression. FSH promoted the PKA-dependent, PI3K-independent phosphorylation of ?catenin on Ser552 and Ser665. FSH activated the ?catenin/T cell factor (TCF) artificial promoter-reporter TOPFlash via a PKA-dependent, PI3K-independent pathway; dominant-negative (DN) TCF abolished FSH-activated Lhcgr-luciferase reporter and induction of Lhcgr mRNA; microarray analysis of GCs treated with Ad-DN-TCF and FSH identified the Lhcgr as the most down-regulated gene. Chromatin immunoprecipitation results placed ?catenin phosphorylated on Ser552 and Ser675 and SF1 on the Lhcgr promoter in FSH-treated GCs; TCF3 was constitutively associated with the Lhcgr promoter. Transduction with an Ad-phospho-?catenin mutant (Ser552/665/Asp) enhanced Lhcgr mRNA expression in FSH-treated cells greater than 3-fold. Finally, we identified a recognized PI3K/AKT target forkhead box O1 as a negative regulator of Lhcgr mRNA expression. These results provide a new understanding of the complex regulation of Lhcgr gene expression in GCs.
Ovarian localization
Oocyte, Granulosa, Theca, Luteal cells
Comment
FOXO1 mediates hypoxia-induced G0/G1 arrest in ovarian somatic granulosa cells via activating the TP53INP1-p53-CDKN1A pathway. Li C et al. (2021) The development of ovarian follicles constitutes the foundation of female reproduction. The proliferation of granulosa cells (GCs) is a basic process required to ensure normal follicular development. However, the mechanisms involved in controlling GC cell cycle are not fully understood. Here, by performing gene expression profiling, we showed that cell cycle arrest at G0/G1 phase is highly correlated with pathways associated with hypoxic stress and FOXO signalling. Specifically, the elevated proportion of GCs at the arrested G0/G1 phase was accompanied by increased nuclear translocation of FOXO1 under conditions of hypoxia both in vivo and in vitro. Actually, phosphorylation of 14-3-3 by the JNK kinase is required for hypoxia-mediated FOXO1 activation and the resultant G0/G1 arrest. Notably, FOXO1 mutant without DNA-binding activity failed to induce G0/G1 arrest of GCs during hypoxia. Importantly, we identified a new target gene of FOXO1, namely TP53INP1, which contributed to the suppression of the G1-S cell cycle transition in response to hypoxia. Furthermore, we demonstrated that the inhibitory effect of the FOXO1-TP53INP1 axis on GC cell cycle is mediated through a p53-CDKN1A-dependent mechanism. These findings might provide avenues for the clinical treatment of human infertility caused by impaired follicular development.//////////////////Characterization of FOXO1, 3 and 4 transcription factors in ovaries of fetal, prepubertal and adult rhesus macaques. Ting AY et al. (2017) The phosphoinositide 3-kinase/AKT signaling pathway negatively regulates follicle activation via the forkhead box O (FOXO) transcription factor in rodents. FOXO3 knockout mice exhibit global activation of primordial follicles leading to early depletion of ovarian follicles and subsequent infertility. Whether a similar mechanism for follicle activation exists in the primate ovary is unclear. In the current study, protein localization of FOXO1, 3 and 4 as well as their upstream regulator, AKT/p-AKT, were examined in rhesus macaque ovaries of 3 developmental stages: fetal, prepubertal and adult. FOXO1 protein is expressed in granulosa cells of fetal, prepubertal and adult ovaries. FOXO3 is distributed sparsely in the mitotically active germ cells, but its expression decreases following follicle formation in the macaque fetal ovary. In addition, FOXO3 is seldom with inter-animal variation in the prepubertal ovary and is absent in the adult ovary. FOXO4 is nondetectable in fetal ovaries, although it is expressed in some theca cells of antral follicles and some stromal cells in prepubertal and adult ovaries. Our results suggest that the regulation and/or function of FOXO3 in the primate primordial follicle may differ than that of the rodent. Nevertheless, AKT/p-AKT is expressed in macaque primordial oocytes, suggesting that similar upstream events, but different downstream effects may regulate primordial follicle activation in nonhuman primates compared to rodents. Elucidation of the mechanism responsible for follicle activation in primates will be crucial for understanding primary ovarian insufficiency, improving female fertility, and applying techniques for in vitro maturation of follicles for fertility preservation in cancer survivors.//////////////////
Gonadal Expression of Foxo1, but Not Foxo3, Is Conserved in Diverse Mammalian Species. Tarnawa ED et al. The Foxos are key effectors of the PI3K/Akt signaling pathway and regulate diverse physiologic processes. Two of these factors, Foxo1 and Foxo3, serve specific roles in reproduction in the mouse. Foxo3 is required for suppression of primordial follicle activation in females while Foxo1 regulates spermatogonial stem cell maintenance in males. In the mouse ovary, Foxo1 is highly expressed in somatic cells (but not in oocytes), suggesting an important functional role for Foxo1 in these cells. Given that invertebrate model species such as C. elegans and D. melanogaster harbor a single ancestral Foxo homolog, these observations suggest that gene duplication conferred a selective advantage by permitting the Foxos to adopt distinct roles in oogenesis and spermatogenesis. Our objective was to determine if the remarkably specific expression patterns of Foxo1 and Foxo3 in mouse gonads (and by inference, Foxo function) are conserved in diverse mammalian species. Western blotting was used to validate isoform-specific antibodies in rodents, companion animals, farm animals, nonhuman primates, and humans. Following validation of each antibody, immunohistochemistry (IHC) was performed to ascertain Foxo1 and Foxo3 gonadal expression patterns. While Foxo1 expression in spermatogonia and granulosa cells was conserved in each species evaluated, Foxo3 expression in oocytes was not. Our findings suggest that Foxo3 is not uniquely required for primordial follicle maintenance in nonrodent species, and that other Foxos, particularly Foxo1, may contribute to oocyte maintenance in a functionally-redundant manner.
Differential expression of genes for transcription factors in theca and granulosa cells following selection of a dominant follicle in cattle. Zielak AE et al. Transcription factors inhibit or assist RNA polymerases in the initiation and maintenance of transcription; however, the cell specific expression and roles of transcription factors within bovine ovarian follicles during development are unknown. The aim of present study was to determine if the expression of transcription factors in theca and granulosa cells differ between the dominant and the largest subordinate follicles at different stages of the follicle wave. We used a bovine cDNA microarray to screen granulosa and theca cells from dominant and subordinate follicles for differential expression of genes coding for transcription factors. Expression was confirmed using reverse transcription polymerase chain reaction and differences in mRNA abundance further examined at Emergence, Selection and Dominance stages of the follicle wave. We have identified five genes encoding for transcription factors that have not been previously described in developing follicles with greater mRNA abundance in subordinate compared to dominant follicles. The genes (and their putative roles) are CEBP-beta (responsible for luteinization), SRF (cell survival), FKHRL1 (stimulates apoptosis), NCOR1 (modulation of the actions of the oestradiol receptor) and Midnolin (control of development via regulation of mRNA transport in cells). Mol. Reprod. Dev. (c) 2007 Wiley-Liss, Inc.
Follicle stages
Comment
FOXO1, FOXO3, AND FOXO4 are differently expressed during mouse oocyte maturation and preimplantation embryo development. Kuscu N et al. (2015) Preimplantation embryo development is affected by its environment. FoxO transcription factors are regulated by PI3K/Akt signaling pathway that essentially supports growth and development. FoxO transcription factors are at the interface of crucial cellular processes, orchestrating programs of gene expression that regulate apoptosis, cell-cycle arrest, oxidative stress resistance, DNA repair, glucose metabolism, and differentiation. In the presence of growth factors, FoxO transcription factors are localized in the cytoplasm, whereas under stress conditions they move to the nucleus and trigger transcriptional activities of their target genes. The aim of the present study is to investigate whether FoxO transcription factors are present during in vivo oocyte maturation and preimplantation embryo development. Presence and localizations of FoxO1, FoxO3 and FoxO4 proteins have been determined with immunofluorescence staining. Our results have confirmed that FoxO1, FoxO3 and FoxO4 proteins are differentially expressed in prophase I, metaphase I, metaphase II oocytes, as well as in fertilized oocyte, 2-cell embryo, 4-cell embryo, 8-cell embryo, morula, and blastocyst. FoxOs translocate to nucleus in embryos with developmental delay. Our findings indicate that FoxO transcription factors are present during both oocyte and embryo in vivo maturation and provide fundamental knowledge that FoxOs may regulate in vitro embryo development under stress conditions.//////////////////