Estradiol promotes cell survival and induces Greb1 expression in granulosa cell tumors of the ovary through an ERα-dependent mechanism.

Granulosa cell tumor (GCT) is a form of ovarian tumor characterized by its tendency to recur years after surgical ablation. Little is known about the mechanisms involved in GCT development and progression. GCTs can produce estradiol (E2), but whether this hormone could play a role in this cancer through its nuclear receptors, i.e. ERα and ERß, remains unknown. Here, we addressed this issue by cell-based and molecular studies on human GCTs and GCT cell lines. Importantly, we observed that E2 significantly increased the growth of GCT cells by promoting cell survival. The use of selective agonists of each type of receptor, together with Esr1 (ERα) or Esr2 (ERß)-deleted GCT cells, revealed that E2 mediated its effects through ERα-dependent genomic mechanisms and ERß/ERα-dependent extra-nuclear mechanisms. Notably, the expression of Greb1, a prototypical ER target gene, was dose-dependently upregulated by E2 specifically through ERα in GCT cells. Accordingly, using GCTs from patients, we found that GREB1 mRNA abundance was positively correlated to intra-tumoral E2 concentrations. Tissue microarray analyses showed that there were various combinations of ER expression in primary and recurrent GCTs, and that ERα expression persisted only in combination with ERß in ~40% of recurrent tumors. Altogether, this study demonstrates that E2 can promote the progression of GCTs, with a clear dependence on ERα. In addition to demonstrating that GCTs can be classified as a hormone-related cancer, our results also highlight that the nature of ER forms present in recurrent GCTs could underlie the variable efficiency of endocrine therapies. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Dysfunction of Human Estrogen Signaling as a Novel Molecular Signature of Polycystic Ovary Syndrome.

Estradiol (E2) is a major hormone-controlling folliculogenesis whose dysfunction may participate in polycystic ovary syndrome (PCOS) infertility. To determine whether both the concentration and action of E2 could be impaired in non-hyperandrogenic overweight PCOS women, we isolated granulosa cells (GCs) and follicular fluid (FF) from follicles of women undergoing ovarian stimulation (27 with PCOS, and 54 without PCOS). An analysis of the transcript abundance of 16 genes in GCs showed that androgen and progesterone receptor expressions were significantly increased in GCs of PCOS (by 2.7-fold and 1.5-fold, respectively), while those of the steroidogenic enzymes CYP11A1 and HSD3B2 were down-regulated (by 56% and 38%, respectively). Remarkably, treatment of GC cultures with E2 revealed its ineffectiveness in regulating the expression of several key endocrine genes (e.g., GREB1 or BCL2) in PCOS. Additionally, a comparison of the steroid concentrations (measured by GC/MS) in GCs with those in FF of matched follicles demonstrated that the significant decline in the E2 concentration (by 23%) in PCOS FF was not the result of the E2 biosynthesis reduction. Overall, our study provides novel hallmarks of PCOS by highlighting the ineffective E2 signaling in GCs as well as the dysregulation in the expression of genes involved in follicular growth, which may contribute to aberrant folliculogenesis in non-hyperandrogenic women with PCOS.

Estradiol Signaling at the Heart of Folliculogenesis: Its Potential Deregulation in Human Ovarian Pathologies.

Estradiol (E2) is a major hormone controlling women fertility, in particular folliculogenesis. This steroid, which is locally produced by granulosa cells (GC) within ovarian follicles, controls the development and selection of dominant preovulatory follicles. E2 effects rely on a complex set of nuclear and extra-nuclear signal transduction pathways principally triggered by its nuclear receptors, ERα and ERß. These transcription factors are differentially expressed within follicles, with ERß being the predominant ER in GC. Several ERß splice isoforms have been identified and display specific structural features, which greatly complicates the nature of ERß-mediated E2 signaling. This review aims at providing a concise overview of the main actions of E2 during follicular growth, maturation, and selection in human. It also describes the current understanding of the various roles of ERß splice isoforms, especially their influence on cell fate. We finally discuss how E2 signaling deregulation could participate in two ovarian pathogeneses characterized by either a follicular arrest, as in polycystic ovary syndrome, or an excess of GC survival and proliferation, leading to granulosa cell tumors. This review emphasizes the need for further research to better understand the molecular basis of E2 signaling throughout folliculogenesis and to improve the efficiency of ovarian-related disease therapies.

Deciphering the Roles & Regulation of Estradiol Signaling during Female Mini-Puberty: Insights from Mouse Models.

Mini-puberty of infancy is a short developmental phase occurring in humans and other mammals after birth. In females, it corresponds to transient and robust activation of the hypothalamo-pituitary-ovarian (HPO) axis revealed by high levels of gonadotropin hormones, follicular growth, and increased estradiol production by the ovary. The roles of estradiol signaling during this intriguing developmental phase are not yet well known, but accumulating data support the idea that it aids in the implementation of reproductive function. This review aims to provide in-depth information on HPO activity during this particular developmental phase in several mammal species, including humans, and to propose emerging hypotheses on the putative effect of estradiol signaling on the development and function of organs involved in female reproduction.

Estradiol Regulates mRNA Levels of Estrogen Receptor Beta 4 and Beta 5 Isoforms and Modulates Human Granulosa Cell Apoptosis.

Estrogen receptor beta (ERß) plays a critical role in granulosa cell (GC) functions. The existence of four human ERß splice isoforms in the ovary suggests their differential implication in 17ß-estradiol (E2) actions on GC apoptosis causing follicular atresia. In this study, we investigated whether E2 can regulate ERß isoforms expression to fine tune its apoptotic activities in human GC. For this purpose, we measured by RT-qPCR the expression of ERß isoforms in primary culture of human granulosa cells (hGCs) collected from patients undergoing in vitro fertilization, before and after E2 exposure. Besides, we assessed the potential role of ERß isoforms on cell growth and apoptosis after their overexpression in a human GC line (HGrC1 cells). We confirmed that ERß1, ERß2, ERß4, and ERß5 isoform mRNAs were predominant over that of ERα in hGCs, and found that E2 selectively regulates mRNA levels of ERß4 and ERß5 isoforms in these cells. In addition, we demonstrated that overexpression of ERß1 and ERß4 in HGrC1 cells increased cell apoptosis by 225% while ERß5 or ERß2 had no effect. Altogether, our study revealed that E2 may influence GC fate by specifically regulating the relative abundance of ERß isoforms mRNA to modulate the balance between pro-apoptotic and non-apoptotic ERß isoforms.

17ß-estradiol inhibits spreading of metastatic cells from granulosa cell tumors through a non-genomic mechanism involving GPER1.

Granulosa cell tumor (GCT) is a rare and severe form of sex-cord stromal ovarian tumor that is characterized by its long natural history and tendency to recur years after surgical ablation. Because there is no efficient curative treatment beyond surgery, ~20% of patients die of the consequences of their tumor. However, very little is known of the molecular etiology of this pathology. About 70% of GCT patients present with elevated circulating estradiol (E2). Because this hormone is known to increase tumor growth and progression in a number of cancers, we investigated the possible role of E2 in GCTs. Cell-based studies with human GCT metastases and primary tumor-derived cells, ie KGN and COV434 cells, respectively, aimed at evaluating E2 effect on cell growth, migration and invasion. Importantly, we found that E2 did not affect GCT cell growth, but that it significantly decreased the migration and matrix invasion of metastatic GCT cells. Noteworthy, our molecular studies revealed that this effect was accompanied by the inhibition through non-genomic mechanisms of extracellular signal-regulated kinase 1/2 (ERK1/2), which is constitutively activated in GCTs. By using pharmacological and RNA silencing approaches, we found that E2 action was mediated by G protein-coupled estrogen receptor 1 (GPER1) signaling pathway. Analyses of GPER1 expression on tissue microarrays from human GCTs confirmed its expression in ~90% of GCTs. Overall, our study reveals that E2 would act via non-classical pathways to prevent metastasis spreading in GCTs and also reveals GPER1 as a possible target in this disease.

Androgen receptor signaling regulates follicular growth and steroidogenesis in interaction with gonadotropins in the ovary during mini-puberty in mice.

In females, androgens contribute to ovarian diseases such as polycystic ovarian syndrome (PCOS), but their action is also crucial for ovarian physiology, i.e., follicular growth and estradiol (E2) synthesis during reproductive life, in interaction with the gonadotropins LH and FSH. However, it is unclear whether androgens already play a role in the ovary at mini-puberty, a phase of postnatal development with active follicular growth and high E2 levels. Therefore, we analyzed the potential actions of androgens on the ovary and their possible interaction with gonadotropins during this period in mice. We used molecular-based studies and pharmacological approaches in vivo and on cultured ovaries. We found that mini-pubertal ovaries produce significant amounts of testosterone and display androgen receptor (AR) expression in growing follicles, both under the control of LH. By blocking AR signaling either in vivo or in ovarian cultures, we found that this pathway may participate in the regulation of prepubertal E2 synthesis and follicular growth, possibly by regulating the expression of a number of key intra-ovarian regulators, including FSH receptor (Fshr), the aromatase enzyme converting androgens into estrogens (Cyp19a1) and the cell cycle inhibitor p27KIP1 (Cdkn1b). We further showed that AR may stimulate FSH-mediated regulation of Cyp19a1 through its action on Fshr mRNA abundance. Overall, this work supports the idea that AR signaling is already activated in mini-pubertal ovaries to regulate E2 synthesis and follicular growth, at the interplay with LH and FSH signaling. Its early action may, thus, contribute to the implementation of early ovarian function with possible impacts on reproductive function.

Inhibition of mTORC1 signaling reduces tumor growth but does not prevent cancer progression in a mouse model of thyroid cancer.

Selective drugs targeting dysregulated oncogenic pathways are promising cancer therapies. Because the mammalian target of rapamycin complex 1 (mTORC1) pathway is hyperactivated in human follicular thyroid cancer (FTC), we hypothesized that its inhibition could block cancer development and progression. We, therefore, analyzed the effect of a treatment with a specific mTORC1 inhibitor (RAD001) in a faithful mouse model of FTC with constitutive mTORC1 activation (TRbeta(PV/PV)Pten(+/-) mice). The treatment did not prevent capsular and vascular invasion of the thyroid and the occurrence of lung metastasis. However, it substantially decelerated thyroid tumor growth, thereby prolonging TRbeta(PV/PV)Pten(+/-) mouse life span. RAD001 efficiently inhibited mTORC1 activity, as shown by the reduced phosphorylation of its downstream targets involved in the activity of the translation machinery, such as ribosomal S6 kinase (p70(S6K)), eukaryotic translation initiation factor 4E binding protein (4E-BP1) and the eukaryotic translation initiation factors eIF-4B and eIF-4G. Whereas mTORC1 signaling inhibition did not alter cell apoptosis, it induced a significant decrease in cell proliferation that was associated with the reduced abundance and altered activity of key regulators of cell cycle progression. Altogether, our data indicate that mTORC1 signaling plays a major role in the integration of the mitogenic signal in FTC. Therefore, our preclinical study with a relevant mouse model of FTC demonstrates for the first time that RAD001 efficaciously stabilizes cancer growth although it does not prevent its fatal outcome. In conclusion, our work underscores that in the treatment of FTC patients, RAD001 can only be used in combination with drugs and therapies inducing tumor shrinkage and blocking metastasis.

Nuclear receptor corepressor is a novel regulator of phosphatidylinositol 3-kinase signaling.

The nuclear receptor corepressor (NCoR) regulates the activities of DNA-binding transcription factors. Recent observations of its distribution in the extranuclear compartment raised the possibility that it could have other cellular functions in addition to transcription repression. We previously showed that phosphatidylinositol 3-kinase (PI3K) signaling is aberrantly activated by a mutant thyroid hormone beta receptor (TRbetaPV, hereafter referred to as PV) via physical interaction with p85alpha, thus contributing to thyroid carcinogenesis in a mouse model of follicular thyroid carcinoma (TRbetaPV/PV mouse). Since NCoR is known to modulate the actions of TRbeta mutants in vivo and in vitro, we asked whether NCoR regulates PV-activated PI3K signaling. Remarkably, we found that NCoR physically interacted with and competed with PV for binding to the C-terminal SH2 (Src homology 2) domain of p85alpha, the regulatory subunit of PI3K. Confocal fluorescence microscopy showed that both NCoR and p85alpha were localized in the nuclear as well as in the cytoplasmic compartments. Overexpression of NCoR in thyroid tumor cells of TRbetaPV/PV mouse reduced PI3K signaling, as indicated by the decrease in the phosphorylation of its immediate downstream effector, p-AKT. Conversely, lowering cellular NCoR by siRNA knockdown in tumor cells led to overactivated p-AKT and increased cell proliferation and motility. Furthermore, NCoR protein levels were significantly lower in thyroid tumor cells than in wild-type thyrocytes, allowing more effective binding of PV to p85alpha to activate PI3K signaling and thus contributing to tumor progression. Taken together, these results indicate that NCoR, via protein-protein interaction, is a novel regulator of PI3K signaling and could serve to modulate thyroid tumor progression.

R-spondin2 signaling is required for oocyte-driven intercellular communication and follicular growth.

R-spondin2 (RSPO2) is a member of the R-spondin family, which are secreted activators of the WNT/ß-catenin (CTNNB1) signaling pathway. In the mouse postnatal ovary, WNT/CTNNB1 signaling is active in the oocyte and in the neighboring supporting cells, the granulosa cells. Although the role of Rspo2 has been previously studied using in vitro experiments, the results are conflicting and the in vivo ovarian function of Rspo2 remains unclear. In the present study, we found that RSPO2/Rspo2 expression is restricted to the oocyte of developing follicles in both human and mouse ovaries from the beginning of the follicular growth. In mice, genetic deletion of Rspo2 does not impair oocyte growth, but instead prevents cell cycle progression of neighboring granulosa cells, thus resulting in an arrest of follicular growth. We further show this cell cycle arrest to be independent of growth promoting GDF9 signaling, but rather associated with a downregulation of WNT/CTNNB1 signaling in granulosa cells. To confirm the contribution of WNT/CTNNB1 signaling in granulosa cell proliferation, we induced cell type specific deletion of Ctnnb1 postnatally. Strikingly, follicles lacking Ctnnb1 failed to develop beyond the primary stage. These results show that RSPO2 acts in a paracrine manner to sustain granulosa cell proliferation in early developing follicles. Taken together, our data demonstrate that the activation of WNT/CTNNB1 signaling by RSPO2 is essential for oocyte-granulosa cell interactions that drive maturation of the ovarian follicles and eventually female fertility.

Aberrant granulosa cell-fate related to inactivated p53/Rb signaling contributes to granulosa cell tumors and to FOXL2 downregulation in the mouse ovary.

Ovarian granulosa cell tumors (GCTs) are indolent tumors of the ovary affecting women at all ages and potentially displaying late recurrence. Even if there is still little information regarding the mechanisms involved in GCT development and progression, FOXL2 would be a major tumor suppressor gene in granulosa cells. We analyzed the mechanisms underlying GCT initiation and progression by using mice with targeted expression of SV40 large T-antigen in granulosa cells (AT mouse), which develop GCTs. Consistent with patients, AT mice with developing GCTs displayed increased levels in circulating anti-Müllerian hormone (AMH), estradiol and androgens, as well as decreased FOXL2 protein abundance. Very few mice developed metastases (1 out of 30). In situ analyses revealed that GCT initiation resulted from both increased granulosa cell survival and proliferation in large antral follicles. Tumorigenesis was associated with the combined inactivation of p53 and Rb pathways, as shown by the impaired expression of respective downstream targets regulating cell apoptosis and proliferation, i.e., Bax, Bak, Gadd45a, Ccna2, Ccne1, E2f1, and Orc1. Importantly, the expression of FOXL2 was still present in newly developed GCTs and its downregulation only started during GCT growth. Collectively, our experiments provide evidence that disrupted p53/Rb signaling can drive tumor initiation and growth. This model challenges the current paradigm that impaired FOXL2 signaling is a major switch of granulosa cell tumorigenesis, albeit possibly contributing to tumor growth.

Age-dependent vulnerability of the ovary to AhR-mediated TCDD action before puberty: Evidence from mouse models.

In female mammals, puberty and fertility are regulated by the synthesis of estradiol (E2) by the ovaries at the infantile stage and at the approach of puberty, a process which may be affected by endocrine disrupting chemicals (EDC)s acting through the Aryl hydrocarbon receptor (AhR). However, there is no information on AhR-mediated regulation of ovarian estrogenic activity during these developmental periods. Here, we assessed in mouse models, the intrinsic and exogenous ligand-induced AhR action on E2 synthesis at the infantile stage (14 days postnatal (dpn)) and at the approach of puberty (28 dpn). Intrinsic AhR pathway became activated in the ovary at the approach of puberty, as suggested by the decreased intra-ovarian expression in prototypical and steroidogenesis-related AhR targets and E2 contents in Ahr knockout (Ahr-/-) mice versus Ahr+/+ mice exclusively at 28 dpn. Accordingly, AhR nuclear localization in granulosa cells, reflecting its activity in cells responsible for E2 synthesis, was much lower at 14 dpn than at 28 dpn in C57BL/6 mice. However, AhR signaling could be activated by exogenous ligands at both ages, as revealed by FICZ- and TCDD-induced Ahrr and Cyp1a1 expression in C57BL/6 mice. Nevertheless, TCDD impacted ovarian estrogenic activity only at 28 dpn. This age-related AhR action may be ligand-dependent, since FICZ had no effect on E2 synthesis at 28 dpn. In conclusion, AhR would not regulate ovarian estrogenic activity before the approach of puberty. Its activation by EDCs may be more detrimental to reproductive health at this stage than during infancy.

Novel oncogenic actions of TRbeta mutants in tumorigenesis.

The thyroid hormone, T3, plays important roles in metabolism, growth, and differentiation. Germline mutations in thyroid hormone receptor beta (TRbeta) have been identified in many individuals with resistance to thyroid hormone, a syndrome of reduced sensitivity to T3. A close association of somatic mutations of TRbeta with several human cancers has become increasingly apparent, but how TRbeta mutants could be involved in the carcinogenesis in vivo has not been addressed. The creation of a mouse model (TRbeta(PV/PV) mouse) that harbors a knockin mutation of TRbeta (denoted TRbetaPV) has facilitated the study of the molecular actions of TRbeta mutants in vivo. The striking phenotype of thyroid cancer and the development of pituitary tumors exhibited by TRbeta(PV/PV) mice have uncovered novel functions of a TRbeta mutant in tumorigenesis. It led to the important findings that the oncogenic action of TRbetaPV is mediated by both genomic and nongenomic actions to alter gene expression and signaling pathways activity.

Nongenomic activation of phosphatidylinositol 3-kinase signaling by thyroid hormone receptors.

Thyroid hormone (T3) is critical in growth, development, differentiation, and maintenance of metabolic homeostasis. Recent studies suggest that thyroid hormone receptors (TRs) not only mediate the biological activities of T3 via nucleus-initiated transcription, but also could act via nongenomic pathways. The striking phenotype of thyroid cancer exhibited by a knockin mutant mouse that harbors a dominant negative TRbeta mutant (TRbeta(PV/PV) mouse) allows the elucidation of novel oncogenic activity of a TRbeta mutant (PV) via extra-nuclear actions. PV physically interacts with the regulatory p85alpha subunit of phosphatidylinositol 3-kinase (PI3K) to activate the downstream AKT-mammalian target of rapamycin (mTOR) and p70(S6K) and PI3K-integrin-linked kinase-matrix metalloproteinase-2 signaling pathways. The PV-mediated PI3K activation results in increased cell proliferation, motility, migration, and metastasis. Remarkably, a nuclear receptor corepressor (NCoR) was found to regulate the PV-activated PI3K signaling by competing with PV for binding to the C-terminal SH2 domain of p85alpha. Over-expression of NCoR in thyroid tumor cells of TRbeta(PV/PV) mice reduces AKT-mTOR-p70(S6K) signaling. Conversely, lowering cellular NCoR by siRNA knockdown in tumor cells leads to over-activated PI3K-AKT signaling to increase cell proliferation and motility. Furthermore, NCoR protein levels are significantly lower in thyroid tumor cells than in wild type thyrocytes, allowing more effective binding of PV to p85alpha to activate PI3K signaling, thereby contributing to tumor progression. Thus, PV, an apo-TRbeta, could act via direct protein-protein interaction to mediate critical oncogenic actions. These studies also uncovered a novel extra-nuclear role of NCoR in modulating the nongenomic actions of a mutated TRbeta in controlling thyroid carcinogenesis.

FSH inhibits AMH to support ovarian estradiol synthesis in infantile mice.

Anti-Müllerian hormone (AMH) regulates ovarian function in cyclic females, notably by preventing premature follicle-stimulating hormone (FSH)-mediated follicular growth and steroidogenesis. Its expression in growing follicles is controlled by FSH and by estradiol (E2). In infantile females, there is a transient increase in the activity of the gonadotrope axis, as reflected by elevated levels of both gonadotropins and E2. We previously demonstrated in mice that elevated FSH concentrations are necessary to induce E2 production by preantral/early antral follicles through the stimulation of aromatase expression without supporting their growth. However, whether this action of FSH could involve AMH is unknown. Here, we show that Amh mRNA and protein abundance and serum AMH levels are elevated in infantile mouse females, compared with those in adults. By experimentally manipulating FSH and E2 levels in infantile mice, we demonstrate that high FSH concentrations lower Amh expression specifically in preantral/early antral follicles, whereas E2 has no effect. Importantly, treatment of infantile ovaries in organotypic cultures with AMH decreases FSH-mediated expression of Cyp19a1 aromatase, but it does not alter the expression of cyclin D2-mediating granulosa cell proliferation. Overall, our data indicate that the infantile elevation in FSH levels suppresses Amh expression in preantral/early antral follicles, thereby favoring Cyp19a1 aromatase expression and E2 production. Together with recent discoveries that AMH can act on both the hypothalamus and the pituitary to increase gonadotropin levels, this work suggests that AMH is a critical regulator of the gonadotrope axis during the infantile period, thereby contributing to adult reproductive function programming.

GnRH regulates the expression of its receptor accessory protein SET in pituitary gonadotropes.

Reproductive function is under the control of the neurohormone GnRH, which activates a G-protein-coupled receptor (GnRHR) expressed in pituitary gonadotrope cells. GnRHR activates a complex signaling network to regulate synthesis and secretion of the two gonadotropin hormones, luteinizing hormone and follicle-stimulating hormone, both regulating gametogenesis and steroidogenesis in gonads. Recently, in an attempt to identify the mechanisms underlying GnRHR signaling plasticity, we identified the first interacting partner of GnRHR, the proto-oncogene SET. We showed that SET binds to intracellular domains of GnRHR to enhance its coupling to cAMP pathway in αT3-1 gonadotrope cells. Here, we demonstrate that SET protein is rapidly regulated by GnRH, which increases SET phosphorylation state and decreases dose-dependently SET protein level. Our results highlight a post-translational regulation of SET protein involving the proteasome pathway. We determined that SET phosphorylation upon GnRH stimulation is mediated by PKC and that PKC mediates GnRH-induced SET down-regulation. Phosphorylation on serine 9 targets SET for degradation into the proteasome. Furthermore, a non-phosphorylatable SET mutant on serine 9 is resistant to GnRH-induced down-regulation. Altogether, these data suggest that GnRH-induced SET phosphorylation on serine 9 mediates SET protein down-regulation through the proteasome pathway. Noteworthy, SET down-regulation was also observed in response to pulsatile GnRH stimulation in LßT2 gonadotrope cells as well as in vivo in prepubertal female mice supporting its physiological relevance. In conclusion, this study highlights a regulation of SET protein by the neurohormone GnRH and identifies some of the mechanisms involved.

Regulation of fibroblast growth factor receptor-1 (FGFR1) by thyroid hormone: identification of a thyroid hormone response element in the murine Fgfr1 promoter.

T(3) is essential for normal skeletal development, acting mainly via the TRalpha1 nuclear receptor. Nevertheless, the mechanisms of T(3) action in bone are poorly defined. Fibroblast growth factor receptor-1 (FGFR1) is also essential for bone formation. Fgfr1 expression and activity are positively regulated by T(3) in osteoblasts, and in mice that harbor a dominant negative PV mutation targeted to TRalpha1 or TRbeta, Fgfr1 expression is sensitive to skeletal thyroid status. To investigate mechanisms underlying T(3) regulation of FGFR1, we obtained primary calvarial osteoblasts from wild-type and TRbeta(PV/PV) littermate mice. T(3) treatment increased Fgfr1 expression 2-fold in wild-type cells, but 8-fold in TRbeta(PV/PV) osteoblasts. The 4-fold increased T(3) sensitivity of TRbeta(PV/PV) osteoblasts was associated with a markedly increased ratio of TRalpha1:TRbeta1 expression that resulted from reduced TRbeta1 expression in TRbeta(PV/PV) osteoblasts compared with wild-type. Bioinformatics and gel shift studies, and mutational analysis, identified a specific TR binding site 279-264 nucleotides upstream of the murine Fgfr1 promoter transcription start site. Transient transfection analysis of a series of Fgfr1 promoter 5'-deletion constructs, of a mutant reporter construct, and a series of heterologous promoter constructs, confirmed that this region of the promoter mediates a TR-dependent transcriptional response to T(3). Thus, in addition to indirect regulation of FGFR1 expression by T(3) reported previously, T(3) also activates the Fgfr1 promoter directly via a thyroid hormone response element located at positions -279/-264.

A novel action of follicle-stimulating hormone in the ovary promotes estradiol production without inducing excessive follicular growth before puberty.

In cyclic females, FSH stimulates ovarian estradiol (E2) production and follicular growth up to the terminal stage. A transient elevation in circulating FSH and E2 levels occurs shortly after birth. But what could be the action of FSH on the ovary during this period, and in particular how it stimulates ovarian steroidogenesis without supporting terminal follicular maturation is intriguing. By experimentally manipulating FSH levels, we demonstrate in mice that the mid-infantile elevation in FSH is mandatory for E2 production by the immature ovary, but that it does not stimulate follicle growth. Importantly, FSH increases aromatase expression to stimulate E2 synthesis, however it becomes unable to induce cyclin D2, a major driver of granulosa cell proliferation. Besides, although FSH prematurely induces luteinizing hormone (LH) receptor expression in granulosa cells, LH pathway is not functional in these cells to induce their terminal differentiation. In line with these results, supplying infantile mice with a superovulation regimen exacerbates E2 production, but it does not stimulate the growth of follicles and it does not induce ovulation. Overall, our findings unveil a regulation whereby high postnatal FSH concentrations ensure the supply of E2 required for programming adult reproductive function without inducing follicular maturation before puberty.

A homozygous FANCM mutation underlies a familial case of non-syndromic primary ovarian insufficiency.

Primary Ovarian Insufficiency (POI) affects ~1% of women under forty. Exome sequencing of two Finnish sisters with non-syndromic POI revealed a homozygous mutation in FANCM, leading to a truncated protein (p.Gln1701*). FANCM is a DNA-damage response gene whose heterozygous mutations predispose to breast cancer. Compared to the mother's cells, the patients' lymphocytes displayed higher levels of basal and mitomycin C (MMC)-induced chromosomal abnormalities. Their lymphoblasts were hypersensitive to MMC and MMC-induced monoubiquitination of FANCD2 was impaired. Genetic complementation of patient's cells with wild-type FANCM improved their resistance to MMC re-establishing FANCD2 monoubiquitination. FANCM was more strongly expressed in human fetal germ cells than in somatic cells. FANCM protein was preferentially expressed along the chromosomes in pachytene cells, which undergo meiotic recombination. This mutation may provoke meiotic defects leading to a depleted follicular stock, as in Fancm-/- mice. Our findings document the first Mendelian phenotype due to a biallelic FANCM mutation.

Anti-Müllerian hormone: a new actor of sexual dimorphism in pituitary gonadotrope activity before puberty.

Anti-Müllerian hormone (AMH) contributes to male sexual differentiation and acts on gonads of both sexes. Identification of AMH receptivity in both pituitary and brain has led to the intriguing idea that AMH participates to the hypothalamic-pituitary control of reproduction, however in vivo experimental evidence is still lacking. We show that AMH stimulates secretion and pituitary gene expression of the gonadotropin FSH in vivo in rats. AMH action is sex-dependent, being restricted to females and occurring before puberty. Accordingly, we report higher levels of pituitary AMH receptor transcripts in immature females. We show that AMH is functionally coupled to the Smad pathway in LßT2 gonadotrope cells and dose-dependently increases Fshb transcript levels. Furthermore, AMH was shown to establish complex interrelations with canonical FSH regulators as it cooperates with activin to induce Fshb expression whereas it reduces BMP2 action. We report that GnRH interferes with AMH by decreasing AMH receptivity in vivo in females. Moreover, AMH specifically regulates FSH and not LH, indicating that AMH is a factor contributing to the differential regulation of gonadotropins. Overall, our study uncovers a new role for AMH in regulating gonadotrope function and suggests that AMH participates in the postnatal elevation of FSH secretion in females.