Harnessing the power within: engineering the microbiome for enhanced gynecologic health.

Abstract: Although numerous studies have demonstrated the impact of microbiome manipulation on human health, research on the microbiome's influence on female health remains relatively limited despite substantial disease burden. In light of this, we present a selected review of clinical trials and preclinical studies targeting both the vaginal and gut microbiomes for the prevention or treatment of various gynecologic conditions. Specifically, we explore studies that leverage microbiota transplants, probiotics, prebiotics, diet modifications, and engineered microbial strains. A healthy vaginal microbiome for females of reproductive age consists of lactic acid-producing bacteria predominantly of the Lactobacillus genus, which serves as a protective barrier against pathogens and maintains a balanced ecosystem. The gut microbiota's production of short-chain fatty acids, metabolism of primary bile acids, and modulation of sex steroid levels have significant implications for the interplay between host and microbes throughout the body, ultimately impacting reproductive health. By harnessing interventions that modulate both the vaginal and gut microbiomes, it becomes possible to not only maintain homeostasis but also mitigate pathological conditions. While the field is still working toward making broad clinical recommendations, the current studies demonstrate that manipulating the microbiome holds great potential for addressing diverse gynecologic conditions. Lay summary: Manipulating the microbiome has recently entered popular culture, with various diets thought to aid the microbes that live within us. These microbes live in different locations of our body and accordingly help us digest food, modulate our immune system, and influence reproductive health. The role of the microbes living in and influencing the female reproductive tract remains understudied despite known roles in common conditions such as vulvovaginal candidiasis (affecting 75% of females in their lifetime), bacterial vaginosis (25% of females in their lifetime), cervical HPV infection (80% of females in their lifetime), endometriosis (6-10% of females of reproductive age), and polycystic ovary syndrome (10-12% of females of reproductive age). Here, we review four different approaches used to manipulate the female reproductive tract and gastrointestinal system microbiomes: microbiota transplants, probiotics, prebiotics, and dietary interventions, and the use of engineered microbial strains. In doing so, we aim to stimulate discussion on new ways to understand and treat female reproductive health conditions.

Metabolic dysregulation and gut dysbiosis linked to hyperandrogenism in female mice.

INTRODUCTION: Polycystic ovary syndrome (PCOS) is a common endocrine pathology in women. In addition to infertility, women with PCOS have metabolic dysregulation which predisposes them to Type 2 diabetes, cardiovascular disease and non-alcoholic fatty liver disease. Moreover, women with PCOS have changes in their gut microbial community that may be indicative of dysbiosis. While hyperandrogenism is associated with both the development of metabolic dysfunction and gut dysbiosis in females, the mechanisms involved are not well understood. METHODS: We used dihydrotestosterone (DHT) and ovariectomy (OVX) mouse models coupled with metabolic assessments and 16S rRNA gene sequencing to explore the contributions of hyperandrogenism and oestrogen deficiency to the development of insulin resistance and gut microbial dysbiosis in pubertal female mice. RESULTS: We demonstrated that, while DHT treatment or OVX alone were insufficient to induce insulin resistance during the pubertal-to-adult transition, combining OVX with DHT resulted in insulin resistance similar to that observed in letrozole-treated mice with elevated testosterone and decreased oestrogen levels. In addition, our results showed that OVX and DHT in combination resulted in a distinct shift in the gut microbiome compared to DHT or OVX alone, suggesting that the substantial metabolic dysregulation occurring in the OVX + DHT model was accompanied by unique changes in the abundances of gut bacteria including S24-7, Rikenellaceae and Mucispirillum schaedleri. CONCLUSIONS: While hyperandrogenism plays an important role in the development of metabolic dysregulation in female mice, our results indicate that investigation into additional factors influencing insulin resistance and the gut microbiome during the pubertal-to-adult transition could provide additional insight into the pathophysiology of PCOS.

Distal Enhancer Potentiates Activin- and GnRH-Induced Transcription of FSHB.

FSH is critical for fertility. Transcription of FSHB, the gene encoding the beta subunit, is rate-limiting in FSH production and is regulated by both GnRH and activin. Activin signals through SMAD transcription factors. Although the mechanisms and importance of activin signaling in mouse Fshb transcription are well-established, activin regulation of human FSHB is less well understood. We previously reported a novel enhancer of FSHB that contains a fertility-associated single nucleotide polymorphism (rs10031006) and requires a region resembling a full (8 base-pair) SMAD binding element (SBE). Here, we investigated the role of the putative SBE within the enhancer in activin and GnRH regulation of FSHB. In mouse gonadotrope-derived LßT2 cells, the upstream enhancer potentiated activin induction of both the human and mouse FSHB proximal promoters and conferred activin responsiveness to a minimal promoter. Activin induction of the enhancer required the SBE and was blocked by the inhibitory SMAD7, confirming involvement of the classical SMAD signaling pathway. GnRH induction of FSHB was also potentiated by the enhancer and dependent on the SBE, consistent with known activin/GnRH synergy regulating FSHB transcription. In DNA pull-down, the enhancer SBE bound SMAD4, and chromatin immunoprecipitation demonstrated SMAD4 enrichment at the enhancer in native chromatin. Combined activin/GnRH treatment elevated levels of the active transcriptional histone marker, histone 3 lysine 27 acetylation, at the enhancer. Overall, this study indicates that the enhancer is directly targeted by activin signaling and identifies a novel, evolutionarily conserved mechanism by which activin and GnRH can regulate FSHB transcription.

FSHB Transcription is Regulated by a Novel 5' Distal Enhancer With a Fertility-Associated Single Nucleotide Polymorphism.

The pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone, signal the gonads to regulate male and female fertility. FSH is critical for female fertility as it regulates oocyte maturation, ovulation, and hormone synthesis. Multiple genome-wide association studies (GWAS) link a 130 Kb locus at 11p14.1, which encompasses the FSH beta-subunit (FSHB) gene, with fertility-related traits that include polycystic ovary syndrome, age of natural menopause, and dizygotic twinning. The most statistically significant single nucleotide polymorphism from several GWAS studies (rs11031006) resides within a highly conserved 450 bp region 26 Kb upstream of the human FSHB gene. Given that sequence conservation suggests an important biological function, we hypothesized that the region could regulate FSHB transcription. In luciferase assays, the conserved region enhanced FSHB transcription and gel shifts identified a binding site for Steroidogenic factor 1 (SF1) contributing to its function. Analysis of mouse pituitary single-cell ATAC-seq demonstrated open chromatin at the conserved region exclusive to a gonadotrope cell-type cluster. Additionally, enhancer-associated histone markers were identified by immunoprecipitation of chromatin from mouse whole pituitary and an immortalized mouse gonadotrope-derived LßT2 cell line at the conserved region. Furthermore, we found that the rs11031006 minor allele upregulated FSHB transcription via increased SF1 binding to the enhancer. All together, these results identify a novel upstream regulator of FSHB transcription and indicate that rs11031006 can modulate FSH levels.

Hyperactive LH Pulses and Elevated Kisspeptin and NKB Gene Expression in the Arcuate Nucleus of a PCOS Mouse Model.

Polycystic ovary syndrome (PCOS), a common reproductive disorder in women, is characterized by hyperandrogenemia, chronic anovulation, cystic ovarian follicles, and luteinizing hormone (LH) hyper-pulsatility, but the pathophysiology isn't completely understood. We recently reported a novel mouse model of PCOS using chronic letrozole (LET; aromatase inhibitor). Letrozole-treated females demonstrate multiple PCOS-like phenotypes, including polycystic ovaries, anovulation, and elevated circulating testosterone and LH, assayed in "one-off" measures. However, due to technical limitations, in vivo LH pulsatile secretion, which is elevated in PCOS women, was not previously studied, nor were the possible changes in reproductive neurons. Here, we used recent technical advances to examine in vivo LH pulse dynamics of freely moving LET female mice versus control and ovariectomized (OVX) mice. We also determined whether neural gene expression of important reproductive regulators such as kisspeptin, neurokinin B (NKB), and dynorphin, is altered in LET females. Compared to controls, LET females exhibited very rapid, elevated in vivo LH pulsatility, with increased pulse frequency, amplitude, and basal levels, similar to PCOS women. Letrozole-treated mice also had markedly elevated Kiss1, Tac2, and Pdyn expression and increased Kiss1 neuronal activation in the hypothalamic arcuate nucleus. Notably, the hyperactive LH pulses and increased kisspeptin neuron measures of LET mice were not as elevated as OVX females. Our findings indicate that LET mice, like PCOS women, have markedly elevated LH pulsatility, which likely drives increased androgen secretion. Increased hypothalamic kisspeptin and NKB levels may be fundamental contributors to the hyperactive LH pulse secretion in the LET PCOS-like condition and, perhaps, in PCOS women.

Insulin-induced hypoglycaemia suppresses pulsatile luteinising hormone secretion and arcuate Kiss1 cell activation in female mice.

Stress suppresses pulsatile luteinising hormone (LH) secretion in a variety of species, although the mechanism underlying this inhibition of reproductive function remains unclear. Metabolic stress, particularly hypoglycaemia, is a clinically-relevant stress type that is modelled with bolus insulin injection (insulin-induced hypoglycaemia). The present study utilised ovariectomised C57BL/6 mice to test the hypothesis that acute hypoglycaemia suppresses pulsatile LH secretion via central mechanisms. Pulsatile LH secretion was measured in 90-minute sampling periods immediately prior to and following i.p. injection of saline or insulin. The secretion of LH was not altered over time in fed animals or acutely fasted (5 hours) animals following an i.p. saline injection. By contrast, insulin elicited a robust suppression of pulsatile LH secretion in fasted animals, preventing LH pulses in five of six mice. To identify the neuroendocrine site of impairment, a kisspeptin challenge was performed in saline or insulin pre-treated animals in a cross-over design. LH secretion in response to exogenous kisspeptin was not different between animals pre-treated with saline or insulin, indicating normal gonadotrophin-releasing hormone cell and pituitary responses during acute hypoglycaemia. Based on this finding, the effect of insulin-induced hypoglycaemia on arcuate kisspeptin (Kiss1) cell function was determined using c-Fos as a marker of neuronal activation. Insulin caused a significant suppression in the percentage of Kiss1 cells in the arcuate nucleus that contained c-Fos compared to saline-injected controls. Taken together, these data support the hypothesis that insulin-induced hypoglycaemia suppresses pulsatile LH secretion in the female mouse via predominantly central mechanisms, which culminates in the suppression of the arcuate Kiss1 population.

Letrozole treatment of pubertal female mice results in activational effects on reproduction, metabolism and the gut microbiome.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder in reproductive-aged women that is comprised of two out of the following three features: hyperandrogenism, oligo- or amenorrhea, or polycystic ovaries. In addition to infertility, many women with PCOS have metabolic dysregulation that increases the risk of developing type 2 diabetes, hypertension, and non-alcoholic fatty liver disease. Changes in the gut microbiome are associated with PCOS and gut microbes may be involved in the pathology of this disorder. Since PCOS often manifests in the early reproductive years, puberty is considered to be a critical time period for the development of PCOS. Exposure to sex steroid hormones during development results in permanent, organizational effects, while activational effects are transient and require the continued presence of the hormone. Androgens exert organizational effects during prenatal or early post-natal development, but it is unclear whether androgen excess results in organizational or activational effects during puberty. We recently developed a letrozole-induced PCOS mouse model that recapitulates both reproductive and metabolic phenotypes of PCOS. In this study, we investigated whether letrozole treatment of pubertal female mice exerts organizational or activational effects on host physiology and the gut microbiome. Two months after letrozole removal, we observed recovery of reproductive and metabolic parameters, as well as diversity and composition of the gut microbiome, indicating that letrozole treatment of female mice during puberty resulted in predominantly activational effects. These results suggest that if exposure to excess androgens during puberty leads to the development of PCOS, reduction of androgen levels during this time may improve reproductive and metabolic phenotypes in women with PCOS. These results also imply that continuous letrozole exposure is required to model PCOS in pubertal female mice since letrozole exerts activational rather than organizational effects during puberty.

Exposure to a Healthy Gut Microbiome Protects Against Reproductive and Metabolic Dysregulation in a PCOS Mouse Model.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting ∼10% to 15% of reproductive-aged women worldwide. Diagnosis requires two of the following: hyperandrogenism, oligo-ovulation or anovulation, and polycystic ovaries. In addition to reproductive dysfunction, many women with PCOS display metabolic abnormalities associated with hyperandrogenism. Recent studies have reported that the gut microbiome is altered in women with PCOS and rodent models of the disorder. However, it is unknown whether the gut microbiome plays a causal role in the development and pathology of PCOS. Given its potential role, we hypothesized that exposure to a healthy gut microbiome would protect against development of PCOS. A cohousing study was performed using a letrozole-induced PCOS mouse model that recapitulates many reproductive and metabolic characteristics of PCOS. Because mice are coprophagic, cohousing results in repeated, noninvasive inoculation of gut microbes in cohoused mice via the fecal-oral route. In contrast to letrozole-treated mice housed together, letrozole mice cohoused with placebo mice showed significant improvement in both reproductive and metabolic PCOS phenotypes. Using 16S rRNA gene sequencing, we also observed that the overall composition of the gut microbiome and the relative abundance of Coprobacillus and Lactobacillus differed in letrozole-treated mice cohoused with placebo mice compared with letrozole mice housed together. These results suggest that dysbiosis of the gut microbiome may play a causal role in PCOS and that modulation of the gut microbiome may be a potential treatment option for PCOS.

Letrozole treatment of adult female mice results in a similar reproductive phenotype but distinct changes in metabolism and the gut microbiome compared to pubertal mice.

BACKGROUND: A majority of women with polycystic ovary syndrome (PCOS) have metabolic dysfunction that results in an increased risk of type 2 diabetes. We previously developed a pubertal mouse model using the aromatase inhibitor, letrozole, which recapitulates many of the reproductive and metabolic features of PCOS. To further our understanding of the effects of androgen excess, we compared the effects of letrozole treatment initiated in puberty versus adulthood on reproductive and metabolic phenotypes as well as on the gut microbiome. RESULTS: Letrozole treatment of both pubertal and adult female mice resulted in reproductive hallmarks of PCOS, including hyperandrogenemia, anovulation and polycystic ovaries. However, unlike pubertal mice, treatment of adult female mice resulted in modest weight gain and abdominal adiposity, minimal elevation in fasting blood glucose and insulin levels, and no detectable insulin resistance. In addition, letrozole treatment of adult mice was associated with a distinct shift in gut microbial diversity compared to letrozole treatment of pubertal mice. CONCLUSIONS: Our results indicate that dysregulation of metabolism and the gut microbiome in PCOS may be influenced by the timing of androgen exposure. In addition, the minimal weight gain and lack of insulin resistance in adult female mice after letrozole treatment indicates that this model may be useful for investigating the effects of hyperandrogenemia on the hypothalamic-pituitary-gonadal axis and the periphery without the influence of substantial metabolic dysregulation.

Sex, Microbes, and Polycystic Ovary Syndrome.

Recent studies have shown that sex and sex steroids influence the composition of the gut microbiome. These studies also indicate that steroid regulation of the gut microbiome may play a role in pathological situations of hormonal excess, such as PCOS. Indeed, studies demonstrated that PCOS is associated with decreased alpha diversity and changes in specific Bacteroidetes and Firmicutes, previously associated with metabolic dysregulation. These studies suggest that androgens may regulate the gut microbiome in females and that hyperandrogenism may be linked with a gut 'dysbiosis' in PCOS. Future mechanistic studies will be required to elucidate how sex steroids regulate the composition and function of the gut microbial community and what the consequences of this regulation are for the host.

American Gut: an Open Platform for Citizen Science Microbiome Research.

Although much work has linked the human microbiome to specific phenotypes and lifestyle variables, data from different projects have been challenging to integrate and the extent of microbial and molecular diversity in human stool remains unknown. Using standardized protocols from the Earth Microbiome Project and sample contributions from over 10,000 citizen-scientists, together with an open research network, we compare human microbiome specimens primarily from the United States, United Kingdom, and Australia to one another and to environmental samples. Our results show an unexpected range of beta-diversity in human stool microbiomes compared to environmental samples; demonstrate the utility of procedures for removing the effects of overgrowth during room-temperature shipping for revealing phenotype correlations; uncover new molecules and kinds of molecular communities in the human stool metabolome; and examine emergent associations among the microbiome, metabolome, and the diversity of plants that are consumed (rather than relying on reductive categorical variables such as veganism, which have little or no explanatory power). We also demonstrate the utility of the living data resource and cross-cohort comparison to confirm existing associations between the microbiome and psychiatric illness and to reveal the extent of microbiome change within one individual during surgery, providing a paradigm for open microbiome research and education. IMPORTANCE We show that a citizen science, self-selected cohort shipping samples through the mail at room temperature recaptures many known microbiome results from clinically collected cohorts and reveals new ones. Of particular interest is integrating n = 1 study data with the population data, showing that the extent of microbiome change after events such as surgery can exceed differences between distinct environmental biomes, and the effect of diverse plants in the diet, which we confirm with untargeted metabolomics on hundreds of samples.

Gut Microbial Diversity in Women With Polycystic Ovary Syndrome Correlates With Hyperandrogenism.

Context: A majority of women with polycystic ovary syndrome (PCOS) have metabolic abnormalities that result in an increased risk of developing type 2 diabetes and heart disease. Correlative studies have shown an association between changes in the gut microbiome and metabolic disorders. Two recent studies reported a decrease in α diversity of the gut microbiome in women with PCOS compared with healthy women. Objective: We investigated whether changes in the gut microbiome correlated with specific clinical parameters in women with PCOS compared with healthy women. We also investigated whether there were changes in the gut microbiome in women with polycystic ovarian morphology (PCOM) who lacked the other diagnostic criteria of PCOS. Participants: Subjects were recruited at the Poznan University of Medical Sciences. Fecal microbial diversity profiles of healthy women (n = 48), women with PCOM (n = 42), and women diagnosed with PCOS using the Rotterdam criteria (n = 73) were analyzed using 16S ribosomal RNA gene sequencing. Results: Lower α diversity was observed in women with PCOS compared with healthy women. Women with PCOM had a change in α diversity that was intermediate between that of the other two groups. Regression analyses showed that hyperandrogenism, total testosterone, and hirsutism were negatively correlated with α diversity. Permutational multivariate analysis of variance in UniFrac distances showed that hyperandrogenism was also correlated with ß diversity. A random forest identified bacteria that discriminated between healthy women and women with PCOS. Conclusion: These results suggest that hyperandrogenism may play a critical role in altering the gut microbiome in women with PCOS.

The Gut Microbiome Is Altered in a Letrozole-Induced Mouse Model of Polycystic Ovary Syndrome.

Women with polycystic ovary syndrome (PCOS) have reproductive and metabolic abnormalities that result in an increased risk of infertility, diabetes and cardiovascular disease. The large intestine contains a complex community of microorganisms (the gut microbiome) that is dysregulated in humans with obesity and type 2 diabetes. Using a letrozole-induced PCOS mouse model, we demonstrated significant diet-independent changes in the gut microbial community, suggesting that gut microbiome dysbiosis may also occur in PCOS women. Letrozole treatment was associated with a time-dependent shift in the gut microbiome and a substantial reduction in overall species and phylogenetic richness. Letrozole treatment also correlated with significant changes in the abundance of specific Bacteroidetes and Firmicutes previously implicated in other mouse models of metabolic disease in a time-dependent manner. Our results suggest that the hyperandrogenemia observed in PCOS may significantly alter the gut microbiome independently of diet.

A Novel Letrozole Model Recapitulates Both the Reproductive and Metabolic Phenotypes of Polycystic Ovary Syndrome in Female Mice.

Polycystic ovary syndrome (PCOS) pathophysiology is poorly understood, due partly to lack of PCOS animal models fully recapitulating this complex disorder. Recently, a PCOS rat model using letrozole (LET), a nonsteroidal aromatase inhibitor, mimicked multiple PCOS phenotypes, including metabolic features absent in other models. Given the advantages of using genetic and transgenic mouse models, we investigated whether LET produces a similar PCOS phenotype in mice. Pubertal female C57BL/6N mice were treated for 5 wk with LET, which resulted in increased serum testosterone and normal diestrus levels of estradiol, similar to the hyperandrogenemia and follicular phase estrogen levels of PCOS women. As in PCOS, ovaries from LET mice were larger, polycystic, and lacked corpora lutea versus controls. Most LET females were acyclic, and all were infertile. LET females displayed elevated serum LH levels and higher Lhb mRNA in the pituitary. In contrast, serum FSH and Fshb were significantly reduced in LET females, demonstrating differential effects on gonadotropins, as in PCOS. Within the ovary, LET females had higher Cyp17, Cyp19, and Fsh receptor mRNA expression. In the hypothalamus, LET females had higher kisspeptin receptor mRNA expression but lower progesterone receptor mRNA levels. LET females also gained more weight than controls, had increased abdominal adiposity and adipocyte size, elevated adipose inflammatory mRNA levels, and impaired glucose tolerance, mirroring the metabolic phenotype in PCOS women. This is the first report of a LET paradigm in mice that recapitulates both reproductive and metabolic PCOS phenotypes and will be useful to genetically probe the PCOS condition.

FOXO1 is regulated by insulin and IGF1 in pituitary gonadotropes.

The FOXO1 transcription factor is important for multiple aspects of reproductive function. We previously reported that FOXO1 functions as a repressor of gonadotropin hormone synthesis, but how FOXO1 is regulated in pituitary gonadotropes is unknown. The growth factors, insulin and insulin-like growth factor I (IGF1), function as key regulators of cell proliferation, metabolism and apoptosis in multiple cell types through the PI3K/AKT signaling pathway. In this study, we found that insulin and IGF1 signaling in gonadotropes induced FOXO1 phosphorylation through the PI3K/AKT pathway in immortalized and primary cells, resulting in FOXO1 relocation from the nucleus to the cytoplasm. Furthermore, insulin administration in vivo induced phosphorylation of FOXO1 and AKT in the pituitary. Thus, insulin and IGF1 act as negative regulators of FOXO1 activity and may serve to fine-tune gonadotropin expression.

Constitutively active FOXO1 diminishes activin induction of Fshb transcription in immortalized gonadotropes.

In the present study, we investigate whether the FOXO1 transcription factor modulates activin signaling in pituitary gonadotropes. Our studies show that overexpression of constitutively active FOXO1 decreases activin induction of murine Fshb gene expression in immortalized LßT2 cells. We demonstrate that FOXO1 suppression of activin induction maps to the -304/-95 region of the Fshb promoter containing multiple activin response elements and that the suppression requires the FOXO1 DNA-binding domain (DBD). FOXO1 binds weakly to the -125/-91 region of the Fshb promoter in a gel-shift assay. Since this region of the promoter contains a composite SMAD/FOXL2 binding element necessary for activin induction of Fshb transcription, it is possible that FOXO1 DNA binding interferes with SMAD and/or FOXL2 function. In addition, our studies demonstrate that FOXO1 directly interacts with SMAD3/4 but not SMAD2 in a FOXO1 DBD-dependent manner. Moreover, we show that SMAD3/4 induction of Fshb-luc and activin induction of a multimerized SMAD-binding element-luc are suppressed by FOXO1 in a DBD-dependent manner. These results suggest that FOXO1 binding to the proximal Fshb promoter as well as FOXO1 interaction with SMAD3/4 proteins may result in decreased activin induction of Fshb in gonadotropes.

Forkhead box O1 is a repressor of basal and GnRH-induced Fshb transcription in gonadotropes.

Synthesis of the gonadotropin ß-subunits is tightly controlled by a complex network of hormonal signaling pathways that may be modulated by metabolic cues. Recently, we reported that insulin regulates FOXO1 phosphorylation and cellular localization in pituitary gonadotropes and that FOXO1 overexpression inhibits Lhb transcription. In the current study, we investigated whether FOXO1 modulates Fshb synthesis. Here, we demonstrate that FOXO1 represses basal and GnRH-induced Fshb transcription in LßT2 cells. In addition, we show that PI3K inhibition, which increases FOXO1 nuclear localization, results in decreased Fshb mRNA levels in murine primary pituitary cells. FOXO1 also decreases transcription from the human FSHB promoter, suggesting that FOXO1 regulation of FSHB transcription may be conserved between rodents and humans. Although the FOXO1 DNA-binding domain is necessary for suppression of Fshb, we do not observe direct binding of FOXO1 to the Fshb promoter, suggesting that FOXO1 exerts its effect through protein-protein interactions with transcription factors required for Fshb synthesis. FOXO1 suppression of basal Fshb transcription may involve PITX1 because PITX1 interacts with FOXO1, FOXO1 repression maps to the proximal Fshb promoter containing a PITX1-binding site, PITX1 induction of Fshb or a PITX1 binding element in CV-1 cells is decreased by FOXO1, and FOXO1 suppresses Pitx1 mRNA and protein levels. GnRH induction of an Fshb promoter containing a deletion at -50/-41 or -30/-21 is not repressed by FOXO1, suggesting that these two regions may be involved in FOXO1 suppression of GnRH-induced Fshb synthesis. In summary, our data demonstrate that FOXO1 can negatively regulate Fshb transcription and suggest that FOXO1 may relay metabolic hormonal signals to modulate gonadotropin production.

A human FSHB promoter SNP associated with low FSH levels in men impairs LHX3 binding and basal FSHB transcription.

FSH production is important for human gametogenesis. In addition to inactivating mutations in the FSHB gene, which result in infertility in both sexes, a G/T single-nucleotide polymorphism (SNP) at -211 relative to the transcription start site of the 5' untranslated region of FSHB has been reported to be associated with reduced serum FSH levels in men. In this study, we sought to identify the potential mechanism by which the -211 SNP reduces FSH levels. Although the SNP resides in a putative hormone response element, we showed that, unlike the murine gene, human FSHB was not induced by androgens or progestins in gonadotropes. On the other hand, we found that the LHX3 homeodomain transcription factor bound to an 11-bp element in the human FSHB promoter that includes the -211 nucleotide. Furthermore, we also demonstrated that LHX3 bound with greater affinity to the wild-type human FSHB promoter compared with the -211 G/T mutation and that LHX3 binding was more effectively competed with excess wild-type oligonucleotide than with the SNP. Finally, we showed that FSHB transcription was decreased in gonadotrope cells with the -211 G/T mutation compared with the wild-type FSHB promoter. Altogether, our results suggest that decreased serum FSH levels in men with the SNP likely result from reduced LHX3 binding and induction of FSHB transcription.

FOXO1 transcription factor inhibits luteinizing hormone ß gene expression in pituitary gonadotrope cells.

Synthesis of luteinizing hormone (LH) is tightly controlled by a complex network of hormonal signaling pathways that can be modulated by metabolic cues, such as insulin. One group of candidate genes that may be regulated by insulin signaling in pituitary gonadotrope cells is the FOXO subfamily of forkhead transcription factors. In this study we investigated whether FOXO1 is expressed in gonadotropes and if it can modulate LH ß-subunit (Lhb) gene expression. We demonstrated that FOXO1 is expressed in murine gonadotrope cells and that insulin signaling increased FOXO1 phosphorylation and cytoplasmic localization in a PI3K-dependent manner. We also showed that FOXO1 repressed basal transcription and gonadotropin-releasing hormone (GnRH) induction of both the murine and human LHB genes in LßT2 cells, suggesting that FOXO1 regulation of LHB transcription may be conserved between rodents and humans. Although we did not detect FOXO1 binding to the proximal Lhb promoter, the FOXO1 DNA binding domain was necessary for the suppression, suggesting that FOXO1 exerts its effect through protein-protein interactions with transcription factors/cofactors required for Lhb gene expression. FOXO1 repression mapped to the proximal Lhb promoter containing steroidogenic factor 1 (SF1), pituitary homeobox 1 (PTX1), and early growth response protein 1 (EGR1) binding elements. Additionally, FOXO1 blocked induction of the Lhb promoter with overexpressed SF1, PTX1, and EGR1, indicating that FOXO1 repression occurs via these transcription factors but not through regulation of their promoters. In summary, we demonstrate that FOXO1 phosphorylation and cellular localization is regulated by insulin signaling in gonadotropes and that FOXO1 inhibits Lhb transcription. Our study also suggests that FOXO1 may play an important role in controlling LH levels in response to metabolic cues.

Stress levels of glucocorticoids inhibit LHß-subunit gene expression in gonadotrope cells.

Increased glucocorticoid secretion is a common response to stress and has been implicated as a mediator of reproductive suppression upon the pituitary gland. We utilized complementary in vitro and in vivo approaches in the mouse to investigate the role of glucocorticoids as a stress-induced intermediate capable of gonadotrope suppression. Repeated daily restraint stress lengthened the ovulatory cycle of female mice and acutely reduced GnRH-induced LH secretion and synthesis of LH ß-subunit (LHß) mRNA, coincident with increased circulating glucocorticoids. Administration of a stress level of glucocorticoid, in the absence of stress, blunted LH secretion in ovariectomized female mice, demonstrating direct impairment of reproductive function by glucocorticoids. Supporting a pituitary action, glucocorticoid receptor (GR) is expressed in mouse gonadotropes and treatment with glucocorticoids reduces GnRH-induced LHß expression in immortalized mouse gonadotrope cells. Analyses revealed that glucocorticoid repression localizes to a region of the LHß proximal promoter, which contains early growth response factor 1 (Egr1) and steroidogenic factor 1 sites critical for GnRH induction. GR is recruited to this promoter region in the presence of GnRH, but not by dexamethasone alone, confirming the necessity of the GnRH response for GR repression. In lieu of GnRH, Egr1 induction is sufficient for glucocorticoid repression of LHß expression, which occurs via GR acting in a DNA- and dimerization-independent manner. Collectively, these results expose the gonadotrope as an important neuroendocrine site impaired during stress, by revealing a molecular mechanism involving Egr1 as a critical integrator of complex formation on the LHß promoter during GnRH induction and GR repression.