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.

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.

GnRH Transactivates Human AMH Receptor Gene via Egr1 and FOXO1 in Gonadotrope Cells.

BACKGROUND/OBJECTIVES: Anti-Müllerian hormone (AMH) signaling is critical for sexual differentiation and gonadal function. AMH receptor type 2 (AMHR2) is expressed in extragonadal sites such as brain, and pituitary and emerging evidence indicates that AMH biological action is much broader than initially thought. We recently reported that AMH signaling enhances follicle-stimulating hormone synthesis in pituitary gonadotrope cells. However, mechanisms regulating AMHR2 expression in these extragonadal sites remain to be explored. METHOD/RESULTS: Here, we demonstrated in perifused murine LßT2 gonadotrope cells that Amhr2 expression is differentially regulated by GnRH pulse frequency with an induction under high GnRH pulsatility. Furthermore, we showed that GnRH transactivates the human AMHR2 promoter in LßT2 cells. Successive deletions of the promoter revealed the importance of a short proximal region (-53/-37 bp) containing an Egr1 binding site. Using site-directed mutagenesis of Egr1 motif and siRNA mediated-knockdown of Egr1, we demonstrated that Egr1 mediates basal and GnRH-dependent activity of the promoter, identifying Egr1 as a new transcription factor controlling hAMHR2 expression. We also showed that SF1 and ß-catenin are required for basal promoter activity and demonstrated that both factors contribute to the GnRH stimulatory effect, independently of their respective binding sites. Furthermore, using a constitutively active mutant of FOXO1, we identified FOXO1 as a negative regulator of basal and GnRH-dependent AMHR2 expression in gonadotrope cells. CONCLUSIONS: This study identifies GnRH as a regulator of human AMHR2 expression, further highlighting the importance of AMH signaling in the regulation of gonadotrope function.

Frataxin inactivation leads to steroid deficiency in flies and human ovarian cells.

Friedreich ataxia (FA), the most common inherited autosomal-recessive ataxia in Caucasians, is characterized by progressive degeneration of the central and peripheral nervous system, hypertrophic cardiomyopathy and increased incidence of diabetes. FA is caused by a GAA repeat expansion in the first intron of the gene encoding frataxin, an evolutionarily conserved mitochondrial protein, which results in decreased gene expression. Ubiquitous inactivation of the fly frataxin ortholog dfh blocks the transition from larval to pupal stages. In this study, we show that this phenotype is due to ecdysteroid deficiency and that feeding larvae with the 20-hydroxyecdysone steroid hormone rescues this developmental blockage. In mammals, adrenodoxin, the ferredoxin FDX1, is an Fe-S-containing protein essential for the synthesis of various steroid hormones. We show here that the two fly ferredoxins, Fdxh and Fdxh2 (encoded by CG1319), are also involved in steroidogenesis. This provides a potent mechanism by which frataxin, known to be involved in Fe-S cluster biosynthesis, could affect steroidogenesis through reduced ferredoxin activity. Finally, we show that frataxin inactivation decreases progesterone synthesis in human KGN ovarian granulosa cells. Thus, the involvement of frataxin in steroid synthesis appears to be a conserved function of the protein from flies to human and our data suggest that steroidogenesis could be affected in FA patients.

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.

SET protein interacts with intracellular domains of the gonadotropin-releasing hormone receptor and differentially regulates receptor signaling to cAMP and calcium in gonadotrope cells.

In mammals, the receptor of the neuropeptide gonadotropin-releasing hormone (GnRHR) is unique among the G protein-coupled receptor (GPCR) family because it lacks the carboxyl-terminal tail involved in GPCR desensitization. Therefore, mechanisms involved in the regulation of GnRHR signaling are currently poorly known. Here, using immunoprecipitation and GST pull-down experiments, we demonstrated that SET interacts with GnRHR and targets the first and third intracellular loops. We delineated, by site-directed mutagenesis, SET binding sites to the basic amino acids (66)KRKK(69) and (246)RK(247), located next to sequences required for receptor signaling. The impact of SET on GnRHR signaling was assessed by decreasing endogenous expression of SET with siRNA in gonadotrope cells. Using cAMP and calcium biosensors in gonadotrope living cells, we showed that SET knockdown specifically decreases GnRHR-mediated mobilization of intracellular cAMP, whereas it increases its intracellular calcium signaling. This suggests that SET influences signal transfer between GnRHR and G proteins to enhance GnRHR signaling to cAMP. Accordingly, complexing endogenous SET by introduction of the first intracellular loop of GnRHR in αT3-1 cells significantly reduced GnRHR activation of the cAMP pathway. Furthermore, decreasing SET expression prevented cAMP-mediated GnRH stimulation of Gnrhr promoter activity, highlighting a role of SET in gonadotropin-releasing hormone regulation of gene expression. In conclusion, we identified SET as the first direct interacting partner of mammalian GnRHR and showed that SET contributes to a switch of GnRHR signaling toward the cAMP pathway.

Mechanisms of action of hormone-sensitive lipase in mouse Leydig cells: its role in the regulation of the steroidogenic acute regulatory protein.

Hormone-sensitive lipase (HSL) catalyzes the hydrolysis of cholesteryl esters in steroidogenic tissues and, thus, facilitates cholesterol availability for steroidogenesis. The steroidogenic acute regulatory protein (StAR) controls the rate-limiting step in steroid biosynthesis. However, the modes of action of HSL in the regulation of StAR expression remain obscure. We demonstrate in MA-10 mouse Leydig cells that activation of the protein kinase A (PKA) pathway, by a cAMP analog Bt2cAMP, enhanced expression of HSL and its phosphorylation (P) at Ser-660 and Ser-563, but not at Ser-565, concomitant with increased HSL activity. Phosphorylation and activation of HSL coincided with increases in StAR, P-StAR (Ser-194), and progesterone levels. Inhibition of HSL activity by CAY10499 effectively suppressed Bt2cAMP-induced StAR expression and progesterone synthesis. Targeted silencing of endogenous HSL, with siRNAs, resulted in increased cholesteryl ester levels and decreased cholesterol content in MA-10 cells. Depletion of HSL affected lipoprotein-derived cellular cholesterol influx, diminished the supply of cholesterol to the mitochondria, and resulted in the repression of StAR and P-StAR levels. Cells overexpressing HSL increased the efficacy of liver X receptor (LXR) ligands on StAR expression and steroid synthesis, suggesting HSL-mediated steroidogenesis entails enhanced oxysterol production. Conversely, cells deficient in LXRs exhibited decreased HSL responsiveness. Furthermore, an increase in HSL was correlated with the LXR target genes, steroid receptor element-binding protein 1c and ATP binding cassette transporter A1, demonstrating HSL-dependent regulation of steroidogenesis predominantly involves LXR signaling. LXRs interact/cooperate with RXRs and result in the activation of StAR gene transcription. These findings provide novel insight and demonstrate the molecular events by which HSL acts to drive cAMP/PKA-mediated regulation of StAR expression and steroidogenesis in mouse Leydig cells.

Identification and analysis of two novel sites of rat GnRH receptor gene promoter activity: the pineal gland and retina.

BACKGROUND AND AIMS: In mammals, activation of pituitary GnRH receptor (GnRHR) by hypothalamic GnRH increases the synthesis and secretion of LH and FSH, which, in turn, regulate gonadal functions. However, GnRHR gene (Gnrhr) expression is not restricted to the pituitary. METHODS: To gain insight into the extrapituitary expression of Gnrhr, a transgenic mouse model that expresses the human placental alkaline phosphatase reporter gene driven by the rat Gnrhr promoter was created. RESULTS: This study shows that the rat Gnrhr promoter is operative in two functionally related organs, the pineal gland, as early as embryonic day (E) 13.5, and the retina where activity was only detected at E17.5. Accordingly, Gnrhr mRNA were present in both tissues. Transcription factors known to regulate Gnrhr promoter activity such as the LIM homeodomain factors LHX3 and ISL1 were also detected in the retina. Furthermore, transient transfection studies in CHO and gonadotrope cells revealed that OTX2, a major transcription factor in both pineal and retina cell differentiation, is able to activate the Gnrhr promoter together with either CREB or PROP1, depending on the cell context. CONCLUSION: Rather than using alternate promoters, Gnrhr expression is directed to diverse cell lineages through specific associations of transcription factors acting on distinct response elements along the same promoter. These data open new avenues regarding GnRH-mediated control of seasonal and circadian rhythms in reproductive physiology.

The Neurod1/4-Ntrk3-Src pathway regulates gonadotrope cell adhesion and motility.

Pituitary gonadotrope cells are essential for the endocrine regulation of reproduction in vertebrates. These cells emerge early during embryogenesis, colonize the pituitary glands and organize in tridimensional networks, which are believed to be crucial to ensure proper regulation of fertility. However, the molecular mechanisms regulating the organization of gonadotrope cell population during embryogenesis remain poorly understood. In this work, we characterized the target genes of NEUROD1 and NEUROD4 transcription factors in the immature gonadotrope αT3-1 cell model by in silico functional genomic analyses. We demonstrated that NEUROD1/4 regulate genes belonging to the focal adhesion pathway. Using CRISPR/Cas9 knock-out approaches, we established a double NEUROD1/4 knock-out αT3-1 cell model and demonstrated that NEUROD1/4 regulate cell adhesion and cell motility. We then characterized, by immuno-fluorescence, focal adhesion number and signaling in the context of NEUROD1/4 insufficiency. We demonstrated that NEUROD1/4 knock-out leads to an increase in the number of focal adhesions associated with signaling abnormalities implicating the c-Src kinase. We further showed that the neurotrophin tyrosine kinase receptor 3 NTRK3, a target of NEUROD1/4, interacts physically with c-Src. Furthermore, using motility rescue experiments and time-lapse video microscopy, we demonstrated that NTRK3 is a major regulator of gonadotrope cell motility. Finally, using a Ntrk3 knock-out mouse model, we showed that NTRK3 regulates gonadotrope cells positioning in the developing pituitary, in vivo. Altogether our study demonstrates that the Neurod1/4-Ntrk3-cSrc pathway is a major actor of gonadotrope cell mobility, and thus provides new insights in the regulation of gonadotrope cell organization within the pituitary gland.

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.

Influence of the accessory protein SET on M3 muscarinic receptor phosphorylation and G protein coupling.

The proto-oncogene and inhibitor of protein phosphatase 2A (PP2A), SET, interacts with the third intracellular loop of the M3 muscarinic receptor (M3-MR), and SET knockdown with small interfering RNA (siRNA) in Chinese hamster ovary (CHO) cells augments M3-MR signaling. However, the mechanism of this action of SET on receptor signaling has not been defined, and we initiated studies to address this question. Knockdown of SET by siRNA in CHO cells stably expressing the M3-MR did not alter agonist-induced receptor phosphorylation or receptor internalization. Instead, it increased the extent of receptor dephosphorylation after agonist removal by ∼60%. In competition binding assays, SET knockdown increased high-affinity binding of agonist in intact cells and membrane preparations. Glutathione transferase pull-down assays and site-directed mutagenesis revealed a SET binding site adjacent to and perhaps overlapping the G protein-binding site within the third intracellular loop of the receptor. Mutation of this region in the M3-MR altered receptor coupling to G protein. These data indicate that SET decreases M3-MR dephosphorylation and regulates receptor engagement with G protein, both of which may contribute to the inhibitory action of SET on M3-MR signaling.

Disruption of Pituitary Gonadotrope Activity in Male Rats After Short- or Long-Term High-Fat Diets Is Not Associated With Pituitary Inflammation.

Overnutrition is associated with the activation of inflammatory pathways in metabolically linked organs and an early hypothalamic inflammation is now known to disrupt the central control of metabolic function. Because we demonstrated that fatty acids (FA) target the pituitary and affect gonadotropin synthesis, we asked whether overnutrition induces pituitary inflammation that may contribute to obesity-associated disorders in the control of reproduction. We analyzed pituitary inflammation and hypothalamic-pituitary-testicular axis in male rats fed a short- (4 weeks) or long-term (20 weeks) high-fat diet. The effect of diet enrichment with the ω3 polyunsaturated FA, DHA, was also analyzed. After only 4 weeks and before weight gain of rats, high-fat diet caused a significant decrease in pituitary gonadotropin and hypothalamic GnRH transcript levels despite unchanged testosterone and inhibin B levels. Contrasting with the hypothalamus, there was no concomitant increases in gene expression of pituitary inflammatory mediators and even a reduction of prototypical cytokines such as interleukin-1ß and TNF-α. No inflammation was still detected in the pituitary after 20 weeks although gonadotropin transcripts and circulating levels were still altered. Gonadotropins were the only pituitary hormones remaining affected at this stage of the regimen, underlying a differential susceptibility of pituitary lineages to metabolic disorders. DHA enrichment of the diet did not prevent alterations of gonadotrope activity due to either a long- or a short-term high-fat diet although it blocked early hypothalamic inflammation and attenuated several metabolic effects. Taken together, our findings suggest that high-fat diet-induced defects in gonadotrope activity in male rats occurred despite a lack of pituitary inflammation.

Emergence and evolution of the glycoprotein hormone and neurotrophin gene families in vertebrates.

BACKGROUND: The three vertebrate pituitary glycoprotein hormones (GPH) are heterodimers of a common α and a specific ß subunit. In human, they are located on different chromosomes but in a similar genomic environment. We took advantage of the availability of genomic and EST data from two cartilaginous fish species as well as from two lamprey species to identify their repertoire of neurotrophin, lin7 and KCNA gene family members which are in the close environment of gphß. Gphα and gphß are absent outside vertebrates but are related to two genes present in both protostomes and deuterostomes that were named gpa2 and gpb5. Genomic organization and functional characteristics of their protein products suggested that gphα and gphß might have been generated concomitantly by a duplication of gpa2 and gpb5 just prior to the radiation of vertebrates. To have a better insight into this process we used new genomic resources and tools to characterize the ancestral environment before the duplication occurred. RESULTS: An almost similar repertoire of genes was characterized in cartilaginous fishes as in tetrapods. Data in lampreys are either incomplete or the result of specific duplications and/or deletions but a scenario for the evolution of this genomic environment in vertebrates could be proposed. A number of genes were identified in the amphioxus genome that helped in reconstructing the ancestral environment of gpa2 and gpb5 and in describing the evolution of this environment in vertebrates. CONCLUSION: Our model suggests that vertebrate gphα and gphß were generated by a specific local duplication of the ancestral forms of gpa2 and gpb5, followed by a translocation of gphß to a new environment whereas gphα was retained in the gpa2-gpb5 locus. The two rounds of whole genome duplication that occurred early in the evolution of vertebrates generated four paralogues of each gene but secondary gene losses or lineage specific duplications together with genomic rearrangements have resulted in the present organization of these genes, which differs between vertebrate lineages.

The Goto-Kakizaki rat is a spontaneous prototypical rodent model of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is characterized by an oligo-anovulation, hyperandrogenism and polycystic ovarian morphology combined with major metabolic disturbances. However, despite the high prevalence and the human and economic consequences of this syndrome, its etiology remains unknown. In this study, we show that female Goto-Kakizaki (GK) rats, a type 2 diabetes mellitus model, encapsulate naturally all the reproductive and metabolic hallmarks of lean women with PCOS at puberty and in adulthood. The analysis of their gestation and of their fetuses demonstrates that this PCOS-like phenotype is developmentally programmed. GK rats also develop features of ovarian hyperstimulation syndrome. Lastly, a comparison between GK rats and a cohort of women with PCOS reveals a similar reproductive signature. Thus, this spontaneous rodent model of PCOS represents an original tool for the identification of the mechanisms involved in its pathogenesis and for the development of novel strategies for its treatment.

Carbon Black Nanoparticles Selectively Alter Follicle-Stimulating Hormone Expression in vitro and in vivo in Female Mice.

Toxic effects of nanoparticles on female reproductive health have been documented but the underlying mechanisms still need to be clarified. Here, we investigated the effect of carbon black nanoparticles (CB NPs) on the pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which are key regulators of gonadal gametogenesis and steroidogenesis. To that purpose, we subjected adult female mice to a weekly non-surgical intratracheal administration of CB NPs at an occupationally relevant dose over 4 weeks. We also analyzed the effects of CB NPs in vitro, using both primary cultures of pituitary cells and the LßT2 gonadotrope cell line. We report here that exposure to CB NPs does not disrupt estrous cyclicity but increases both circulating FSH levels and pituitary FSH ß-subunit gene (Fshb) expression in female mice without altering circulating LH levels. Similarly, treatment of anterior pituitary or gonadotrope LßT2 cells with increasing concentrations of CB NPs dose-dependently up-regulates FSH but not LH gene expression or release. Moreover, CB NPs enhance the stimulatory effect of GnRH on Fshb expression in LßT2 cells without interfering with LH regulation. We provide evidence that CB NPs are internalized by LßT2 cells and rapidly activate the cAMP/PKA pathway. We further show that pharmacological inhibition of PKA significantly attenuates the stimulatory effect of CB NPs on Fshb expression. Altogether, our study demonstrates that exposure to CB NPs alters FSH but not LH expression and may thus lead to gonadotropin imbalance.

Sustained gonadotropin-releasing hormone stimulation mobilizes the cAMP/PKA pathway to induce nitric oxide synthase type 1 expression in rat pituitary cells in vitro and in vivo at proestrus.

Previous in vivo studies have established that pituitary nitric oxide synthase type 1 (NOS1) is regulated by gonadotropin-releasing hormone (GnRH). The aim of our study was to elucidate the mechanisms of NOS1 regulation by GnRH in rat pituitary cells. Using a perifused cell system, we demonstrated that NOS1 induction was sensitive to GnRH pulse frequency and was maximally induced under continuous GnRH stimulation. In primary cultures of rat pituitary cells, sustained stimulation with the GnRH agonist triptorelin (GnRHa) increased NOS1 protein levels, whereas NOS2 and NOS3 levels were unaffected. NOS1 up-regulation occurred in gonadotroph cells only, in a time-dependent and concentration-dependent manner (maximum increase, 2.5-fold; half-maximal concentration, 0.17 nM). GnRHa effect was mimicked by cAMP pathway activators and, most importantly, was blocked by disruption of the protein kinase A (PKA) pathway using pharmacological inhibitors such as Rp-cAMP or drug phosphatase technology-protein kinase inhibitor (DPT-PKI), a cell-permeant PKI peptide. In contrast, modulation of the PKC pathway and inhibition of the MAPK cascade were ineffective. Overall, these experiments demonstrated that GnRH-induced up-regulation of pituitary NOS1 is mediated notably by the cAMP/PKA pathway. Last, in vivo administration of a GnRH antagonist markedly inhibited the pituitary cAMP rise at proestrus in addition to suppressing NOS1 increase. Altogether, our data suggest that the cAMP/PKA signaling pathway is preferentially recruited under sustained GnRH stimulation in vivo during proestrus, allowing the expression of a specific set of PKA-regulated proteins, including NOS1, in gonadotroph cells.

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.

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.