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

Studies of the cellular and molecular alterations arising in granulosa cell tumors of the ovary

Les tumeurs des cellules de la granulosa (TCG) représentent 7 % des tumeurs ovariennes et peuvent affecter des femmes de tout âge. Bien que peu étudiée, cette maladie n'est pas anodine puisque près de 20 % des patientes en décèdent. Cette morbidité résulte de la grande fréquence des rechutes, survenant en moyenne 10 ans après le diagnostic initial. Hormis le traitement chirurgical, les thérapies adjuvantes (chimio et radiothérapies) donnent des résultats peu satisfaisants. L'étiologie moléculaire de la maladie n'est pas bien connue, exceptée la possible dérégulation de l'action du facteur de transcription FOXL2. Malgré des niveaux circulants anormalement élevés d'oestradiol (E2) retrouvés chez 70 % des patientes, le rôle de cette hormone n'est pas encore clair dans ces tumeurs. D'autre part, la dérégulation des voies de p53 et de Retinoblastoma (Rb) a déjà été décrite, mais son rôle dans ce cancer est inconnu. En l'absence de données claires sur le sujet aboutissant à des traitements efficaces, j'ai mené durant ma thèse des recherches sur les altérations moléculaires et cellulaires associées à cette pathologie. Dans un premier temps, j'ai étudié l'impact de l'inactivation des voies de signalisation p53 et Rb dans le développement de ce cancer. Pour cela, j'ai conduit des études sur un modèle de souris transgéniques (AT83) développant des TCG associées à l'expression de l'antigène T du SV40 dans les cellules de la granulosa (CG). Ces analyses ont mis en évidence que l'inactivation de p53 et de Rb par le SV40 entravait l'élimination physiologique des follicules ovariens par atrésie folliculaire, en empêchant l'apoptose des CG et en permettant leur prolifération excessive. De manière intéressante, nos études biochimiques ont permis de montrer la forte diminution de l'expression de FOXL2 après le développement tumoral, comme observée dans les TCG humaines. Ces résultats inédits ont apporté un éclairage nouveau sur les mécanismes moléculaires qui sous-tendent cette maladie et font l'objet d'une publication dans Oncogene où je suis première auteure. Ces recherches m'ont permis de faire émerger l'hypothèse que SIRT1 serait un acteur essentiel du processus tumoral dans cette maladie. En effet, nous observons une augmentation de son expression au cours de la tumorigenèse des souris AT83. SIRT1 est une histone désacétylase qui participe à la régulation de la prolifération et de la survie cellulaires dont l'expression est augmentée dans de nombreux cancers. J'ai mis en évidence l'augmentation de son expression dans les TCG humaines par rapport aux CG saines. Nos études in vitro montrent que l'inhibition pharmacologique spécifique de SIRT1 par l'EX-527 inhibe la croissance des CG tumorales en agissant sur la survie et sur la prolifération. L'administration de cet inhibiteur sur deux modèles pré-cliniques de la maladie, les souris AT83 et des souris Nude greffées avec des CG tumorales, entraîne la régression tumorale après quatre semaines de traitement. Un article sur ce sujet où je suis première auteure est actuellement en préparation. Parallèlement à ces recherches, j'ai étudié l'effet de l'E2 dans cette pathologie via ses récepteurs nucléaires. Nos études in situ sur des TCG humaines montrent que l'expression des ER (Estrogen Receptor) est conservée dans plus 70% des tumeurs primaires ou métastases avec plusieurs combinaisons d'expression possibles. Les travaux menés in vitro suggèrent que l'E2 stimule la croissance des CG tumorales via ERa selon un mécanisme génomique et via ERb selon un mécanisme non génomique dépendant de ERa. En outre, nos études biochimiques montrent que l'E2 stimule l'expression de GREB1, une cible oncogénique connue des ER, et cela de manière dose-dépendante via ERa uniquement. Ce travail caractérise pour la première fois les effets de l'E2 dans les TCG et la complexité de son action via les ER. Il fait l'objet d'un article soumis dans Cell Death & Disease où je suis première auteure.Granulosa cell tumors (GCT) account for 7% of ovarian tumors and can affect women of all ages. Although poorly studied, about 20% of patients die from this disease. This morbidity results from the high frequency of recurrence, appearing on average 10 years after initial diagnosis. Apart from the surgical treatment, common therapies (chemotherapy and radiotherapy) globally give unsatisfactory results. The molecular aetiology of this disease is not well-known, except the possible deregulation in the action of the transcription factor FOXL2. Despite abnormally high circulating levels of estradiol (E2) found in 70% of patients, the role of this hormone is not clear in this pathology. In addition, deregulation of the p53 and Retinoblastoma (Rb) pathways has already been described in this form of cancer, but its effect on tumor development and progression remained unknown. In this context, during my PhD I conducted researches on the molecular and cellular alterations possibly associated with this pathology. First, I investigated the effect of inactivated p53 and Rb signaling pathways in the development and progression of this cancer. For that purpose, I used a transgenic mouse model (AT83) developing GCT associated with the expression of the large T SV40 antigen in granulosa cells (GC). These analyses showed that inactivation of p53 and Rb induced by SV40 inhibited the physiological elimination of ovarian follicles through the process of follicular atresia, by both preventing apoptosis and driving excessive proliferation of GC. Interestingly, our biochemical studies showed the strong decrease in FOXL2 expression after tumor initiation, suggesting that its deregulation arises as a consequence of tumor development. These results shed light on novel molecular mechanisms underlying the development of this disease. They are published in Oncogene where I appear as first author. These studies led us to investigate the possibility that SIRT1, a histone deacetylase which contributes to cell proliferation and survival found overexpressed in many cancers, could have an essential role in this pathology. Indeed, we observed an increase in its expression during tumorigenesis in our mouse model. I have demonstrated the increase of its expression in human GCT as compared to healthy GC. Our in vitro studies showed that pharmacological inhibition of SIRT1 by EX-527 inhibits the growth of tumoral GC by acting on their survival and proliferation. Administration of this inhibitor in two preclinical mouse models of the disease, i.e. the AT83 mice and nude mice grafted with tumoral GC, led to the regression of the tumor after four weeks of treatment. An article in which I am the first author is currently in preparation. I also questioned the role of E2 in this pathology via its nuclear receptors. Our in situ studies on human GCT showed that either Estrogen Receptor (ER) a or b were expressed in more than 70% of primary or recurrent tumors, with a strong variability in ER equipment between GCT. Our in vitro studies suggested that E2 stimulates the growth of tumoral GC via ERa by a genomic mechanism and via ERb probably by a non-genomic mechanism ERa-dependent. In addition, our biochemical studies showed that E2 stimulates GREB1 expression, a known oncogenic target of ER in other cancers, in a dose-dependent manner specifically via ERa. These studies characterized for the first time the effects of E2 in GCT and the complexity of its action via its different nuclear receptors. They are submitted to Cell Death & Disease in a paper where I appear as first author

Production d’œstradiol par l’ovaire lors de la mini-puberté

International audienceNo abstract availabl

FSH inhibits AMH to support ovarian estradiol synthesis in infantile mice

International audienceAnti-Mullerian 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

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

International audienceOvarian 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-Mullerian 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

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

International audienceGranulosa 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. ER alpha and ER beta, 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 (ER alpha) or Esr2 (ER beta)-deleted GCT cells, revealed that E2 mediated its effects through ER alpha-dependent genomic mechanisms and ER beta/ER alpha-dependent extra-nuclear mechanisms. Notably, the expression of Greb1, a prototypical ER target gene, was dose-dependently upregulated by E2 specifically through ER alpha 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 ER alpha expression persisted only in combination with ER beta in similar to 40% of recurrent tumors. Altogether, this study demonstrates that E2 can promote the progression of GCTs, with a clear dependence on ER alpha. 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. (C) 2022 The Pathological Society of Great Britain and Ireland