FOXO1-Dependent Regulation Of Apoptosis In Mouse Ovarian Granulosa Cells

In female mammalian ovaries, a common phenomenon known as follicular atresia occurs periodically, and it destroys almost all the follicles in the ovary. Although the molecular mechanisms underlying follicular atresia remain largely undetermined, recent studies suggest that granulosa cell (GC) apoptosis is the main marker of follicular atresia.Foxo, a subfamily of the forkhead transcription factors, is known to regulate genes required for diverse processes, including apoptosis, cell cycle arrest, DNA damage repair, oxidative stress, etc. Foxo transcription factors have been shown to regulate follicular growth, development, and maturation through multiple signaling cascades in mammalian ovaries. Recent evidence from several types of mammal cells indicates that oxidative stress induces apoptosis by activating Foxo. However, the relationship among oxidative stress, Foxo, GC apoptosis and follicular atresia is still unclear.Follicle-stimulating Hormone (FSH) acts as the primary survival factor during follicular atresia by restraining apoptosis in GCs. Earlier reports indicated that FSH suppressed the induction of Foxo 1. Therefore, it raises the possibility that FSH-Foxol axis is required for GC survival and atresia prevention. To test this hypothesis, it is necessary to investigate the correlation between Foxol and FSH-mediated GC protection, and mechanisms regarding the regulation modes of Foxol upon FSH stimulation.By conducting most of the experiments in mouse ovary, the major findings of the current research could be presented as follows:1 Foxol is a critical player in oxidative stress-induced GC apoptosis(1) Increased apoptotic signals correlate with elevated expression of Foxol in GCs under oxidative stress derived from the in vivo and in vitro treatmentOxidant 3-NP is used to generate oxidative stress on ovaries in a mouse model, in which ROS specifically increased in ovarian GCs but not other tested tissues. Also, when using 3-NP to inject mice or H2O2 to mimic physiological oxidative stress in cultured GCs, it is found that Foxo1 expression is dramatically up-regulated in the oxidative conditions, followed by elevation of the apoptosis-related gene expressions as well as an increase of apoptotic signals in mouse granulose cells.(2) Overexpression of Foxo1 induces apoptosis in cultured GCsApoptotic signals in cultured MGCs are increased significantly after transfection with Foxo1 expression vector. Enhanced Foxo1 expression promotes the nuclear translocation of Foxo1. The transcripts of apoptosis-related genes, Bim, TRAIL, FasL, and caspase-3 are dramatically increased after overexpression of Foxo1 compared with levels in MGCs transfected with empty vector.(3) Knockdown of Foxo1 expression attenuated H2O2-induced apoptosis in MGCsTransfection of Foxo1 shDNA plasmid into MGCs strikingly inhibits the expression and nuclear distribution of Foxo1, which is elevated by H2O2 treatment. Under the same oxidative stress conditions, mRNA levels of apoptosis-related genes (Bim, TRAIL, FasL, and caspase-3) in MGCs are significantly reduced as a result of Foxo1 knockdown.(4) Insulin inhibits H2O2-induced apoptosis by blocking the activation of Foxo1 in MGCsThe presence of insulin dramatically attenuates H2O2-drived nuclear localization of Foxo1 in MGCs. DNA fragmentation in H2O2-treated MGCs decreases significantly when the cells are cultured with insulin. H2O2 failed to induce mRNA levels of apoptosis-related genes (Bim, TRAIL, FasL, and caspase-3) in MGCs pre-treated with insulin.2 Foxo1 is involved in the effects of FSH on inhibition of apoptosis in MGCs(1) FSH represses Foxo1-dependent apoptosis in MGCs of ovarian dominant folliclesIn atretic dominant follicles, the apoptotic signals are concentrated in GCs. Progress of atresia is associated with an increased apoptosis percentage of GCs. In contrast, when atresia is restrained by FSH, the GCs could be prevented from apoptosis. FSH may antagonize ovarian GCs apoptosis by inhibiting mitochondria-dependent programmed cell death pathway. Among the Foxo family members expressed in GCs of dominant follicles, Foxol is most sensitive to FSH’s regulation. FSH protects MGC from apoptosis in MGCs by suppressing Foxol expression. Bim appeared to be a convergence point of the mitochondrial apoptotic pathway and Foxol-dependent apoptosis that both contribute to FSH-induced GCs survival in the dominant follicles.(2) FSH promotes MGCs survival through the PKA-PI3K-AKT-Foxol axisAmong multiple downstream signaling cascades of FSH in GCs from mouse dominant follicles, it is found that PKA, PI3K, and AKT are activated by FSH to inhibit both the expression and activity of Foxol, which was associated with a suppression of apoptosis in GCs. PKA is identified as the upstream activator of PI3K, which in turn stimulated the activation of AKT. The phosphorylation of Foxol by AKT leads to the nuclear exclusion of phospho-Foxol. Conversely, constitutively active Foxol (all three AKT sites were replaced by alanines) in GCs interrupts the protection from FSH.(3) The self-reinforcing regulation of Foxol in MGCsIn MGCs collected from dominant follicles, Foxol binds directly to the Foxo-recognized element (FRE) site within Foxol promoter, induces the expression of multiple target genes (including Foxol itself and pro-apoptotic genes), as well as the exacerbation of apoptosis in GCs. In contrast, phosphorylation of Foxol by FSH through the PKA-PI3K-AKT axis blocks the nuclear translocation of Foxol, dissociates Foxol from its DNA binding sequence, terminating the positive feedback regulation of Foxol transcription and target genes expression, thus preventing Foxol-dependent apoptosis in GCs.(4) FSH might inhibit Foxol expression through an ERalpha-dependent manner8 transcriptional factors containing TFBS (transcriptional factor binding sites) in Foxol promoter are predicted according to bioinformatics software and literature reports. Six of the 8 genes are expressed in GCs harvested from dominant follicles. A strong correlation between the expression patterns of ERalpha and Foxol (p<0.01) in response to FSH and PKA inhibitor treatment was observed in GCs, indicating that ERalpha might be another molecular switch of Foxo 1 function during FSH mediated GC survival.In summary, the reasearch into the Foxol-dependent regulation of GC apoptosis & follicular atresia provides a potential target for the clinical therapy of ovulation disorders, improvement of the livestock breeding rates, and raising the pregnancy rates in assisted reproductive techniques.