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.

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.

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.

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.

Identification of a pituitary ERα-activated enhancer triggering the expression of Nr5a1, the earliest gonadotrope lineage-specific transcription factor.

BACKGROUND: Gonadotrope lineage differentiation is a stepwise process taking place during pituitary development. The early step of gonadotrope lineage specification is characterized by the expression of the Nr5a1 transcription factor, a crucial factor for gonadotrope cell fate determination. Abnormalities affecting Nr5a1 expression lead to hypogonadotropic hypogonadism and infertility. Although significant knowledge has been gained on the signaling and transcriptional events controlling gonadotrope differentiation, epigenetic mechanisms regulating Nr5a1 expression during early gonadotrope lineage specification are still poorly understood. RESULTS: Using ATAC chromatin accessibility analyses on three cell lines recapitulating gradual stages of gonadotrope differentiation and in vivo on developing pituitaries, we demonstrate that a yet undescribed enhancer is transiently recruited during gonadotrope specification. Using CRISPR/Cas9, we show that this enhancer is mandatory for the emergence of Nr5a1 during gonadotrope specification. Furthermore, we identify a highly conserved estrogen-binding element and demonstrate that the enhancer activation is dependent upon estrogen acting through ERα. Lastly, we provide evidence that binding of ERα is crucial for chromatin remodeling of Nr5a1 enhancer and promoter, leading to RNA polymerase recruitment and transcription. CONCLUSION: This study identifies the earliest regulatory sequence involved in gonadotrope lineage specification and highlights the key epigenetic role played by ERα in this differentiation process.

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.

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.

A regulatory loop between miR-132 and miR-125b involved in gonadotrope cells desensitization to GnRH.

The GnRH neurohormone is the main activator of the pituitary gonadotropins, LH and FSH. Here we investigated the contribution of microRNAs in mediating GnRH activation. We first established that miR-125b targets several actors of Gαq/11 signalling pathway, without altering Gαs pathway. We then showed that a Gαs-mediated, PKA-dependent phosphorylation of NSun2 methyltransferase leads to miR-125b methylation and thereby induces its down-regulation. We demonstrated that NSun2 mRNA is a target of miR-132 and that NSun2 may be inactivated by the PP1α phosphatase. Time-course analysis of GnRH treatment revealed an initial NSun2-dependent down-regulation of miR-125b with consecutive up-regulation of LH and FSH expression. Increase of miR-132 and of the catalytic subunit of PP1α then contributed to NSun2 inactivation and to the return of miR-125b to its steady-state level. The Gαq/11-dependent pathway was thus again silenced, provoking the down-regulation of LH, FSH and miR-132. Overall, this study reveals that a regulatory loop that tends to maintain or restore high and low levels of miR-125b and miR-132, respectively, is responsible for gonadotrope cells desensitization to sustained GnRH. A dysregulation of this loop might be responsible for the inverted dynamics of these two miRNAs reported in several neuronal and non-neuronal pathologies.

Epigenetic regulation of alternative promoters and enhancers in progenitor, immature, and mature gonadotrope cell lines.

Gonadotrope cell identity genes emerge in a stepwise process during mouse pituitary development. Cga, encoding for the α-subunit of TSH, LH, and FSH, is initially detected at E11.5 followed by Gnrhr and steroidogenic factor Sf1 at E13.5, specifying cells engaged in a gonadotrope cell fate. Lhb and Fshb appear at E16.5 and 17.5, respectively, typifying differentiated gonadotrope cells. Using the αT1-1, αT3-1 and LßT2 cell lines recapitulating these stages of gonadotrope differentiation, DNA methylation at Gnrhr and Sf1 was investigated. Regulatory regions were found hypermethylated in progenitor αT1-1 cells and hypomethylated in differentiated LßT2 cells. Abundance of RNA polymerase II together with active histone modifications including H3K4me1, H3K4me3, and H3K27ac were strictly correlated with DNA hypomethylation. Analyses of epigenomic modifications and chromatin accessibility were further extended to Isl1, Lhx3, Gata2, and Pitx2, highlighting alternative usages of specific regulatory gene domains in progenitor αT1-1, immature αT3-1, and mature LßT2 gonadotrope cells.

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.

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.

Rapid communication: A microRNA-132/212 pathway mediates GnRH activation of FSH expression.

GnRH plays a key role in the vertebrate reproductive system by stimulating biosynthesis and secretion of pituitary gonadotropins. However, the potential involvement of microRNAs (miRNAs) on this activation has still to be explored. In this study, we investigated the role of miRNA-132 and miRNA-212, two tandemly expressed miRNAs that target the same transcripts, on GnRH-induced FSH expression. We first showed that the GnRH stimulation of FSH secretion was reduced and Fshb mRNA abolished by blocking miR-132/212 action in rat pituitary cells. In mouse LßT2 gonadotrope cells, the GnRH stimulation of Fshb mRNA was also demonstrated to be dependent on miR-132/212 and reproduced by overexpressing one or both miRNAs. We then showed that the miR-132/212-mediated action of GnRH involved a posttranscriptional decrease of sirtuin 1 (SIRT1) deacetylase. The lower level of SIRT1 deacetylase correlated with an increase in the acetylated form of Forkhead Box O1 (FOXO1), a transcriptional repressor of Fshb. Interestingly, we show that the acetylated mimicking mutant of FOXO1 was localized outside the nucleus, thus alleviating its repressive effect on Fshb transcription. Overall, we demonstrate that the GnRH stimulation of Fshb expression is dependent on miR-132/212 and involves a SIRT1-FOXO1 pathway. This is the first demonstration of an obligatory microRNA pathway in the GnRH-regulated expression of a gonadotropin gene.

The transcription factor FOXL2 mobilizes estrogen signaling to maintain the identity of ovarian granulosa cells.

FOXL2 is a lineage determining transcription factor in the ovary, but its direct targets and modes of action are not fully characterized. In this study, we explore the targets of FOXL2 and five nuclear receptors in murine primary follicular cells. We found that FOXL2 is required for normal gene regulation by steroid receptors, and we show that estrogen receptor beta (ESR2) is the main vector of estradiol signaling in these cells. Moreover, we found that FOXL2 directly modulates Esr2 expression through a newly identified intronic element. Interestingly, we found that FOXL2 repressed the testis-determining gene Sox9 both independently of estrogen signaling and through the activation of ESR2 expression. Altogether, we show that FOXL2 mobilizes estrogen signaling to establish a coherent feed-forward loop repressing Sox9. This sheds a new light on the role of FOXL2 in ovarian maintenance and function.

FOXL2, GATA4, and SMAD3 co-operatively modulate gene expression, cell viability and apoptosis in ovarian granulosa cell tumor cells.

Aberrant ovarian granulosa cell proliferation and apoptosis may lead to granulosa cell tumors (GCT), the pathogenesis of which involves transcription factors GATA4, FOXL2, and SMAD3. FOXL2 gene harbors a point mutation (C134W) in a vast majority of GCTs. GATA4 is abundantly expressed in GCTs and its expression correlates with poor prognosis. The TGF-ß mediator SMAD3 promotes GCT cell survival through NF-κB activation, and interacts with FOXL2. Here, we find that the expression patterns of these factors overlap in the normal human ovary and 90 GCTs, and positively correlate with each other and with their mutual target gene CCND2, which is a key factor for granulosa cell proliferation. We have explored the molecular interactions of FOXL2, GATA4, and SMAD3 and their roles in the regulation of CCND2 using co-immunoprecipitation, promoter transactivation, and cell viability assays in human GCT cells. We found that not only SMAD3, but also GATA4 physically interact with both wild type and C134W-mutated FOXL2. GATA4 and SMAD3 synergistically induce a 8-fold increase in CCND2 promoter transactivation, which is 50% reduced by both FOXL2 types. We confirmed that wild type FOXL2 significantly decreases cell viability. Interestingly, GATA4 and SMAD3 caused a marked reduction of GCT cell apoptosis induced by wild type FOXL2. Thus, the effects of GATA4 and SMAD3 on both cell viability and apoptosis are distinct from those of wild type FOXL2; a perturbation of this balance due to the oncogenic FOXL2 mutation is likely to contribute to GCT pathogenesis.

Discovery of novel protein partners of the transcription factor FOXL2 provides insights into its physiopathological roles.

FOXL2 transcription factor is responsible for the Blepharophimosis Ptosis Epicantus inversus Syndrome (BPES), a genetic disease involving craniofacial malformations often associated with ovarian failure. Recently, a somatic FOXL2 mutation (p.C134W) has been reported in >95% of adult-type granulosa cell tumors. Here, we have identified 10 novel FOXL2 partners by yeast-two-hybrid screening and co-immunoprecipitation. Most BPES-inducing mutated FOXL2 proteins display aggregation in cultured cells. Here, we show that two of the partners (NR2C1 and GMEB1) can be sequestered in such aggregates. This co-aggregation can contribute to the pathogenesis of FOXL2 mutations. We have also measured the effects of FOXL2 interactants on the transcriptional regulation of a series of target promoters. Some of the partners (CXXC4, CXXC5, BANF1) were able to repress FOXL2 activity indistinctively of the promoter. Interestingly, CREM-τ2α, which acted as a repressor on most promoters, increased wild-type (WT) FOXL2 activity on two promoters (PTGS2 and CYP19A1), but was unable to increase the activity of the oncogenic mutant p.C134W. Conversely, GMEB1, which also acted as a repressor on most promoters and increased WT FOXL2 activity on the Per2 promoter, increased to a greater extent the activity of the p.C134W variant. Interestingly, partners with intrinsic pro-apoptotic effect were able to increase apoptosis induction by WT FOXL2, but not by the p.C134W mutant, whereas partners with an anti-apoptotic effect decreased apoptosis induction by both FOXL2 versions. Altogether, these results suggest that the p.C134W mutated form fails to integrate signals through protein-protein interactions to regulate target promoter subsets and in particular to induce cell death.

The transcription factor FOXL2: at the crossroads of ovarian physiology and pathology.

FOXL2 is a gene encoding a forkhead transcription factor. Its mutations or misregulation have been shown to cause the blepharophimosis-ptosis-epicanthus inversus (BPES) syndrome and more recently have been associated with the development of Ovarian Granulosa Cell Tumors (OGCT). BPES is a genetic disorder involving mild craniofacial abnormalities often associated with premature ovarian failure. OGCTs are endocrine malignancies, accounting for 2-5% of ovarian cancers, the treatment of which is still challenging. In this review we summarize recent data concerning FOXL2 transcriptional targets and molecular partners, its post-translational modifications, its mutations and its involvement in newly discovered pathophysiological processes. In the ovary, FOXL2 is involved in the regulation of cholesterol and steroid metabolism, apoptosis, reactive oxygen species detoxification and cell proliferation. Interestingly, one of the main roles of FOXL2 is also to preserve the identity of ovarian granulosa cells even at the adult stage and to prevent their transdifferentiation into Sertoli-like cells. All these recent advances indicate that FOXL2 is central to ovarian development and maintenance. The elucidation of the impact of FOXL2 germinal and somatic mutations will allow a better understanding of the pathogenesis of BPES and of OGCTs.