Folliculogenesis is a complex process, defined by the growth and development of follicles from the primordial population. Granulosa cells (GCs) play a vital role in every stage of follicular growth through proliferation, acquisition of gonadotropic responsiveness, steroidogenesis and production of autocrine/paracrine factors. A recently discovered hypothalamic neuropeptide phoenixin is involved in the regulation of the reproductive system. Phoenixin acts through its receptor, G protein-coupled receptor 173 (GPR173), to activate the cAMP/PKA pathway leading to the phosphorylation of CREB (pCREB). Here, we demonstrated the expression patterns of phoenixin and GPR173 in human ovary and explored its role in folliculogenesis. Phoenixin and GPR173 were both expressed in the human ovarian follicle, with increased expression in GCs as the follicle grows. Phoenixin treatment at 100 nM for 24 h induced the proliferation of human non-luteinized granulosa cell line, HGrC1 and significantly increased the expression levels of CYP19A1, FSHR, LHR and KITL, but decreased NPPC expression levels. These effects were suppressed by GPR173 siRNA. The expression level of CREB1, pCREB and estradiol (E2) production in the culture medium was significantly enhanced by phoenixin treatment in a concentration-dependent manner. Phoenixin also significantly increased the follicular area in a murine ovarian tissue culture model, leading to an increased number of ovulated oocytes with a higher level of maturation. Taken together, our data demonstrate that phoenixin is an intraovarian factor that promotes follicular growth through its receptor GPR173 by accelerating proliferation of GCs, inducing E2 production and increasing the expression of genes related to follicle development.

We aimed to determine whether a functional link with impact on female ovarian reserve exists between FMR1 expression and expression ratios of AKT/mTOR signaling genes in human granulosa cells in vivo, as suggested from prior in vitro data. Three hundred and nine women, who were classified as normal (NOR; n = 225) and poor (POR; n = 84) responders based on their ovarian reserve, were recruited during stimulation for assisted reproductive techniques. Expressions of FMR1 and of key genes of the AKT/mTOR and AKT/FOXO1/3 signaling pathways were comparatively analyzed in their granulosa cells. FMR1 expression in granulosa cells of NOR and POR correlated significantly with AKT1, TSC2, mTOR, and S6K expression. No correlation was found between FMR1 and FOXO1 in all, and FOXO3 expression in POR, patients. AKT1 expression was significantly higher and FOXO1 expression lower in POR samples, whereas AKT1 expression was lower and FOXO1 expression was higher in NOR samples. In human native granulosa cells, FMR1 expression significantly correlated with the expression of key genes of the AKT/mTOR signaling pathway, but not with the FOXO1/3 signaling pathway. Our data point to a functional link between FMR1 expression and expression of the AKT/mTOR signaling pathway genes controlling human follicular maturation.

Background The protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway regulates early follicular activation and follicular pool maintenance in female germline cells. Fragile X mental retardation 1 (FMR1) regulates folliculogenesis and it is variably expressed in patients with Premature Ovary Insufficiency. FMR1 expression is supposed to be linked to AKT/mTOR signaling in an ovarian response dependent manner as demonstrated in recent invitro and invivo studies in the female germline invitro and invivo. Methods We evaluated changes in the expression of AKT/mTOR signaling pathway genes by real time PCR in the peripheral blood of 74 patients with Premature Ovarian Insufficiency and 56 fertile controls and correlated their expression with FMR1 expression. Results Expression of the genes AKT1, TSC2, mTOR, and S6K was significantly more abundant in patients with POI than in the controls. For AKT1, TSC2 and mTOR, gene expression was not affected by FMR1-CGG repeat number in the 5´-untranslated region. FMR1 and S6K expression levels, however, were significantly upregulated in patients with POI and an FMR1 premutation. Independent of a premutation, expression of mTOR, S6K, and TSC2 was significantly correlated with that of FMR1 in all patients. Furthermore, when grouped according to ovarian reserve, this effect remained significant only for mTOR and S6K, with higher significance note in patients with Premature Ovarian Insufficiency than in the controls. Conclusions In Premature ovarian insufficiency patients, activation of AKT/mTOR signaling pathway is remarkable and putatively pathognomonic. Additionally, it seems to be triggered by an FMR1/mTOR/S6K linkage mechanism, most relevant in premutation carriers.

In humans, FMR1 (Fragile X mental retardation 1) is strongly expressed in granulosa cells (GCs) of the female germline and apparently controls efficiency of folliculogenesis. Major control mechanism(s) of the gene transcription rate seem to be based on the rate of CpG-methylation along the CpG island promoter. Conducting CpG-methylation-specific bisulfite-treated PCR assays and subsequent sequence analyses of both gene alleles, revealed three variably methylated CpG domains (FMR1-VMR (variably methylated region) 1, -2, -3) and one completely unmethylated CpG-region (FMR1-UMR) in this extended FMR1-promoter-region. FMR1-UMR in the core promoter was exclusively present only in female GCs, suggesting expression from both gene alleles, i.e., escaping the female specific X-inactivation mechanism for the second gene allele. Screening for putative target sites of transcription factors binding with CpG methylation dependence, we identified a target site for the transcriptional activator E2F1 in FMR1-VMR3. Using specific electrophoretic mobility shift assays, we found E2F1 binding efficiency to be dependent on CpG-site methylation in its target sequence. Comparative analysis of these CpGs revealed that CpG 94-methylation in primary GCs of women with normal and reduced efficiency of folliculogenesis statistically significant differences. We therefore conclude that E2F1 binding to FMR1-VMR3 in human GCs is part of an epigenetic mechanism regulating the efficiency of human folliculogenesis. Our data indicate that epigenetic mechanisms may control GC FMR1-expression rates.

Fragile X-associated primary ovarian insufficiency (FXPOI) is characterized by oligo/amenorrhea and hypergonadotropic hypogonadism and is caused by the expansion of the CGG repeat in the 5′UTR of Fragile X Mental Retardation 1 (FMR1). Approximately 20% of women carrying an FMR1 premutation (PM) allele (55–200 CGG repeat) develop FXPOI. Repeat Associated Non-AUG (RAN)-translation dependent on the variable CGG-repeat length is thought to cause FXPOI, due to the production of a polyglycine-containing FMR1 protein, FMRpolyG. Peripheral blood monocyte cells (PBMCs) and granulosa cells (GCs) were collected to detect FMRpolyG and its cell type-specific expression in FMR1 PM carriers by immunofluorescence staining (IF), Western blotting (WB), and flow cytometric analysis (FACS). For the first time, FMRpolyG aggregates were detected as ubiquitin-positive inclusions in PBMCs from PM carriers, whereas only a weak signal without inclusions was detected in the controls. The expression pattern of FMRpolyG in GCs was comparable to that in the lymphocytes. We detected FMRpolyG as a 15- to 25-kDa protein in the PBMCs from two FMR1 PM carriers, with 124 and 81 CGG repeats. Flow cytometric analysis revealed that FMRpolyG was significantly higher in the T cells from PM carriers than in those from non-PM carriers. The detection of FMRpolyG aggregates in the peripheral blood and granulosa cells of PM carriers suggests that it may have a toxic potential and an immunological role in ovarian damage in the development of FXPOI.

Study question Does repeat-associated non-AUG (RAN) translation lead to accumulation of polyglycine- containing protein (FMRpolyG) in human lymphocytes and mural granulosa cells of FMR1 premutation carriers? Summary answer Lymphocytes and granulosa cells from FMR1 premutation carriers contain intracellular inclusions that stain positive for both FMRpolyG and ubiquitin. What is known already: Fragile-X-associated-Primary-Ovarian-Insufficiency (FXPOI) is characterized by oligo/amenorrhea and hypergonadotropic hypogonadism associated with the expansion of CGG-repeats in the 5’UTR of FMR1, called premutation (PM) (n: 55–200). Approximately 20% of women carrying a FMR1-premutation (PM) allele develop FXPOI. RAN-translation dependent on variable CGG-repeat length is hypothesized to cause FXPOI due to the production of a polyglycine-containing FMR1-protein, FMRpolyG. Recently, FMRpolyG inclusions were found in neuronal brain cells of FXTAS patients and stromal cells of the ovary of an FXPOI patient. Study design, size, duration: Lymphocytes and granulosa cells (GCs) from women with PM (6) and women without PM (10) (controls) were analyzed by immunofluorescence (IF) staining for the presence of inclusions positive for ubiquitin and FMRpolyG. Cell lysis and protein extraction samples were subjected to Fluorescent Western Blot (WB) analysis to detect FMRP and FMRpolyG Participants/materials, setting, methods Human GCs were obtained from follicular fluid after oocyte retrieval and lymphocytes were isolated from peripheral blood using Ficoll-Paque. Cells suspended in PBS were adhered to a glass-coverslip placed at the bottom of the 6-well culture plate, via gravity sedimentation. Adhered cells were fixed, IF staining for FMRpolyG and ubiquitin was performed and analyzed by fluorescence microscopy. Fluorescent WB was used to demonstrate the expression of FMRP, FMRpolyG in extracted protein from lymphocytes and GCs. Main results and the role of chance FMRP was successfully detected by fluorescence WB in both lymphocytes and GCs. FMRP is mainly present in cytoplasm and was expressed in greater amount in GCs than in leukocytes. Moreover, FMRP expression was significantly decreased in GCs from FMR1-PM compared with controls. Lymphocytes from PM-carriers and controls were immunostained for FMRpolyG and ubiquitin. In PM-carriers, FMRpolyG was present as aggregates, whereas in controls only a weak signal without inclusions was detectable. The expression pattern of FMRpolyG in GCs was similar to that in lymphocytes with a significant increase in PM-carriers. There, the FMRpolyG-aggregates additionally demonstrated as ubiquitin-positive inclusions. These may resemble the toxic potential of these protein fractions involved the ovarian damage in developing FXPOI. Limitations, reasons for caution More patients are needed to support the present findings. Further investigation into the possible consequences of these FMRpolyG-positive inclusions in PM-carriers is also advisable. Wider implications of the findings: We found for the first time FMRpolyG-accumulation in lymphocytes and GCs from FMR1-PM-carriers in ubiquitin-positive inclusions. Future experiments evaluating consistency in more patients and elucidating the impact on fertility and prospective value for individual ovarian reserve are therefore in preparation. Trial registration number Not applicable

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