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In brief Sex differences in the gut microbiome may impact multiple aspects of human health and disease. In this study, we review the evidence for microbial sex differences in puberty and adulthood and discuss potential mechanisms driving differentiation of the sex-specific gut microbiome. Abstract In humans, the gut microbiome is strongly implicated in numerous sex-specific physiological processes and diseases. Given this, it is important to understand how sex differentiation of the gut microbiome occurs and how these differences contribute to host health and disease. While it is commonly believed that the gut microbiome stabilizes after 3 years of age, our review of the literature found considerable evidence that the gut microbiome continues to mature during and after puberty in a sex-dependent manner. We also review the intriguing, though sparse, literature on potential mechanisms by which host sex may influence the gut microbiome, and vice versa, via sex steroids, bile acids, and the immune system. We conclude that the evidence for the existence of a sex-specific gut microbiome is strong but that there is a dearth of research on how host–microbe interactions lead to this differentiation. Finally, we discuss the types of future studies needed to understand the processes driving the maturation of sex-specific microbial communities and the interplay between gut microbiota, host sex, and human health.

The laboratory mouse gut microbiome has been extensively studied, but our understanding of its diversity remains incomplete. We report the assembly of 51 draft metagenome-assembled genomes (MAGs) from murine fecal samples dominated by uncharacterized bacteria. These MAGs add to our understanding of gut microbial diversity in this critical model organism.

Within pituitary gonadotropes, the gonadotropin-releasing hormone receptor (GnRHR) receives hypothalamic input from GnRH neurons that is critical for reproduction. Previous studies have suggested that androgens may regulate GnRHR, although the mechanisms remain unknown. In this study, we demonstrated that androgens positively regulate Gnrhr mRNA in mice. We then investigated the effects of androgens and androgen receptor (AR) on Gnrhr promoter activity in immortalized mouse LβT2 cells, which represent mature gonadotropes. We found that AR positively regulates the Gnrhr proximal promoter, and that this effect requires a hormone response element (HRE) half site at -159/-153 relative to the transcription start site. We also identified nonconsensus, full-length HREs at -499/-484 and -159/-144, which are both positively regulated by androgens on a heterologous promoter. Furthermore, AR associates with the Gnrhr promoter in ChIP. Altogether, we report that GnRHR is positively regulated by androgens through recruitment of AR to the Gnrhr proximal promoter.

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.

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 LβT2 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.

Acute estrogen deficiency in women can occur due to many conditions including hyperprolactinemia, chemotherapy, GnRH agonist treatment, and removal of hormone replacement therapy. Ovariectomized (OVX) rodent models, often combined with a high-fat diet (HFD), have been used to investigate the effects of decreased estrogen production on metabolism. Since evidence suggests that gut microbes may facilitate the protective effect of estrogen on metabolic dysregulation in an OVX + HFD model, we investigated whether the gut microbiome plays a role in the diet-independent weight gain that occurs after OVX in adult female mice. 16S rRNA gene sequence analysis demonstrated that OVX was not associated with changes in overall gut bacterial biodiversity but was correlated with a shift in beta diversity. Using differential abundance analysis, we observed a difference in the relative abundance of a few bacterial taxa, such as Turicibacter, 3 to 5 weeks after OVX, which was subsequent to the weight gain that occurred 2 weeks postsurgery. A cohousing study was performed to determine whether exposure to a healthy gut microbiome was protective against the development of the metabolic phenotype associated with OVX. Unlike mouse models of obesity, HFD maternal-induced metabolic dysregulation, or polycystic ovary syndrome, cohousing OVX mice with healthy mice did not improve the metabolic phenotype of OVX mice. Altogether, these results indicate that changes in the gut microbiome are unlikely to play a causal role in diet-independent, OVX-induced weight gain (since they occurred after the weight gain) and cohousing with healthy mice did not have a protective effect.

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 LβT2 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.

Polycystic ovary syndrome (PCOS) impacts ∼10% of reproductive-aged women worldwide. In addition to infertility, women with PCOS suffer from metabolic dysregulation which increases their risk of developing type 2 diabetes, cardiovascular disease and non-alcoholic fatty liver disease. Studies have shown differences in the gut microbiome of women with PCOS compared to controls, a pattern replicated in mouse models. Recently, using a letrozole-induced mouse model of PCOS, we demonstrated that cohousing was protective against development of metabolic and reproductive phenotypes and showed via 16S amplicon sequencing that this protection correlated with time-dependent shifts in gut bacteria. Here, we applied untargeted metabolomics and shotgun metagenomics approaches to further analyze the longitudinal samples from the cohousing experiment. Analysis of beta diversity found that untargeted metabolites had the strongest correlation to both disease and cohoused states and that shifts in metabolite diversity were detected prior to shifts in bacterial diversity. In addition, log2-fold analyses found numerous metabolite features, particularly bile acids (BA), to be highly differentiated between placebo (P) and letrozole (LET), as well as cohoused LET versus LET. Our results indicate that changes in gut metabolites, particularly BAs, are associated with a PCOS-like phenotype in the LET mouse model as well as the protective effect of cohousing. Our results also suggest that transfer of metabolites via coprophagy occurs rapidly and may precipitate changes in bacterial diversity. This study joins a growing body of research highlighting changes in primary and secondary bile acids that may provide a link between host metabolism and gut microbes relevant to the pathology of PCOS.IMPORTANCEUsing a combination of untargeted metabolomics and metagenomics, we performed a comparative longitudinal analysis of the feces collected in a cohousing study with a PCOS mouse model. Our results showed that gut metabolite composition experienced earlier and more pronounced differentiation in both the disease model and cohoused mice compared with the microbial composition. Notably, statistical and machine learning approaches identified shifts in the relative abundance of primary and secondary BA, which have been implicated as modifiers of gut microbial growth and diversity. Network correlation analysis showed strong associations between particular BA and bacterial species, particularly members of Lactobacillus, and that these correlations were time and treatment dependent. Our results provide novel insights into host/microbe relationships related to hyperandrogenism in females and indicate that focused research into small molecule control of gut microbial diversity and host physiology may provide new therapeutic options for the treatment of PCOS.

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.

Polycystic ovary syndrome (PCOS) is a reproductive disorder in women characterized by hyperandrogenemia, anovulation, cystic ovaries, and LH hyper-pulsatility, but the mechanisms causing the pathophysiology remain incompletely understood. We recently reported a novel mouse model that recapitulates the majority of PCOS phenotypes in adulthood. Females given constant, long-term letrozole to reduce aromatase activity demonstrate PCOS-like phenotypes, including polycystic ovaries, anovulation, elevated circulating testosterone, and increased LH. In vivo LH pulsatile secretion, which is greatly elevated in PCOS women, was not previously studied, nor were possible changes in reproductive neurons known to control GnRH/LH secretion. Here, we used recent technical advances in the field to examine in vivo LH pulse dynamics of freely-moving LET female mice versus control and ovariectomized (OVX) mice. We also studied whether hypothalamic gene expression of several important reproductive regulators, 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. LET mice also had markedly elevated Kiss1, Tac2, and Pdyn expression along with increased Kiss1 neuron activation in the hypothalamic arcuate nucleus. Although elevated, most hyperactive LH pulse parameters and increased arcuate mRNA measures of LET mice were significantly lower than in OVX littermates. Our findings demonstrate that LET mice, like PCOS women, have markedly elevated LH pulsatility which likely drives increased ovarian androgen secretion. Increased arcuate kisspeptin and NKB levels may be fundamental contributors to the enhanced stimulation of LH pulse secretion in this PCOS-like condition, and perhaps, in some PCOS women.

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 (5hours) 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.

Polycystic ovary syndrome (PCOS) is the most common cause of female infertility and is associated with an increased risk of type 2 diabetes and cardiovascular disease. Understanding the etiology of PCOS is crucial to relieve this tremendous health burden. Twin studies estimate that 70% of PCOS etiology can be explained by genetic factors, motivating studies to identify these factors. Recent genome wide association studies identified a PCOS risk locus that encompasses the gene encoding the follicle-stimulating hormone beta (FSHβ) subunit. The most significant single nucleotide polymorphism (SNP) within this locus, rs11031006 (rs06), resides within a region of DNA (~450 base pairs) that is highly conserved among mammals. As FSH supplementation can restore ovulation in some PCOS patients, deficient FSH signaling may be a causative factor of anovulation and potentially other facets of PCOS. FSH transcription and release is stimulated by gonadotropin-releasing hormone (GnRH) produced in the hypothalamus, as well as activin produced by the gonads and pituitary. We previously hypothesized that the conserved region containing rs06 acts as an enhancer of FSHβ transcription, and we determined through luciferase reporter assays that this highly conserved region enhances transcription ~2 fold compared to the minimal FSHβ promoter alone for human and 2.5 fold for mouse. The rs06 SNP minor allele further increased both mouse and human FSHβ luciferase expression. In the present study, we sought to identify interactions between the enhancer and the hormones, GnRH and activin. With DNA constructs from both human and mouse, we identified synergistic interactions between the enhancer and GnRH- or activin-induction of FSHβ. These interactions were maintained after mutation of the rs06 site to the minor allele, suggesting that this SNP alters basal and not hormone-induced FSHβ transcription. We are currently investigating the effect of the enhancer and rs06 on activin/GnRH synergy. Overall, our findings provide evidence that the enhancer functions as a novel mediator of activin and GnRH regulation of FSHβ transcription in both human and mouse.

Approximately 10% of reproductive-aged women worldwide are affected by polycystic ovary syndrome (PCOS). In addition to infertility, many women with PCOS have an increased risk of developing type 2 diabetes, cardiovascular disease, and non-alcoholic fatty liver disease. Studies have shown that the human gut microbiome is altered in humans with metabolic disorders such as obesity and type 2 diabetes and these changes may contribute to metabolic dysfunction. Additionally, studies have reported that changes in the gut microbiome are associated with PCOS in women and in rodent models. These changes include lower alpha diversity (species richness) and changes in specific Bacteroidetes and Firmicutes many of which are also altered in other metabolic diseases. In previous work, we investigated whether the gut microbiome was altered in a PCOS mouse model using letrozole, a nonsteroidal aromatase inhibitor, to increase endogenous testosterone levels and decrease estrogen levels. Letrozole treatment of pubertal female mice results in hallmarks of PCOS including elevated testosterone and LH, acyclicity, polycystic ovaries, and a metabolic phenotype. In the current study, we tested whether manipulation of the gut microbiome in the PCOS mouse model via co-housing improved reproductive and metabolic phenotypes and whether these changes correlated with gut microbial composition. Pubertal female mice were implanted with a placebo or letrozole pellet and divided into 3 groups with two mice per cage: co-housed placebo mice, letrozole mice or placebo and letrozole mice. Our results demonstrated that co-housing letrozole-treated mice with placebo mice resulted in substantial improvement of PCOS reproductive and metabolic phenotypes. Testosterone and LH levels, estrous cycling and ovulation were normalized in letrozole-treated mice co-housed with placebo mice compared to letrozole-treated mice housed together. In addition, weight, fasting blood glucose and insulin levels, and insulin resistance were decreased in letrozole-treated mice co-housed with placebo mice compared to letrozole-treated mice housed together. Using 16S rRNA sequencing, we observed that co-housing letrozole-treated mice with placebo mice did not result in substantial changes in alpha diversity but were associated with changes in specific bacterial genera that may be candidates for pre- or probiotic therapies. Our results suggest that manipulation of the gut microbiome may be a potential treatment option for PCOS.

Microorganisms in the human gut play a role in health and disease, and in adults higher gut biodiversity has been linked to better health. Since gut microorganisms may be pivotal in the development of microbial therapies, understanding the factors that shape gut biodiversity is of utmost interest. We performed large-scale analyses of the relationship of age and sex to gut bacterial diversity in adult cohorts from four geographic regions: the United States, the United Kingdom, Colombia, and China. In the U.S., U.K., and Colombian cohorts, bacterial biodiversity correlated positively with age in young adults but plateaued at about age 40 years, with no positive association being found in middle-aged adults. Young, but not middle-aged, adult women had higher gut bacterial diversity than men, a pattern confirmed via supervised machine learning. Interestingly, in the Chinese cohort, minimal associations were observed between gut biodiversity and age or sex. Our results highlight the patterns of adult gut biodiversity and provide a framework for future research., Gut microbial diversity changes throughout the human life span and is known to be associated with host sex. We investigated the association of age, sex, and gut bacterial alpha diversity in three large cohorts of adults from four geographical regions: subjects from the United States and United Kingdom in the American Gut Project (AGP) citizen-science initiative and two independent cohorts of Colombians and Chinese. In three of the four cohorts, we observed a strong positive association between age and alpha diversity in young adults that plateaued after age 40 years. We also found sex-dependent differences that were more pronounced in younger adults than in middle-aged adults, with women having higher alpha diversity than men. In contrast to the other three cohorts, no association of alpha diversity with age or sex was observed in the Chinese cohort. The association of alpha diversity with age and sex remained after adjusting for cardiometabolic parameters in the Colombian cohort and antibiotic usage in the AGP cohort. We further attempted to predict the microbiota age in individuals using a machine-learning approach for the men and women in each cohort. Consistent with our alpha-diversity-based findings, U.S. and U.K. women had a significantly higher predicted microbiota age than men, with a reduced difference being seen above age 40 years. This difference was not observed in the Colombian cohort and was observed only in middle-aged Chinese adults. Together, our results provide new insights into the influence of age and sex on the biodiversity of the human gut microbiota during adulthood while highlighting similarities and differences across diverse cohorts. IMPORTANCE Microorganisms in the human gut play a role in health and disease, and in adults higher gut biodiversity has been linked to better health. Since gut microorganisms may be pivotal in the development of microbial therapies, understanding the factors that shape gut biodiversity is of utmost interest. We performed large-scale analyses of the relationship of age and sex to gut bacterial diversity in adult cohorts from four geographic regions: the United States, the United Kingdom, Colombia, and China. In the U.S., U.K., and Colombian cohorts, bacterial biodiversity correlated positively with age in young adults but plateaued at about age 40 years, with no positive association being found in middle-aged adults. Young, but not middle-aged, adult women had higher gut bacterial diversity than men, a pattern confirmed via supervised machine learning. Interestingly, in the Chinese cohort, minimal associations were observed between gut biodiversity and age or sex. Our results highlight the patterns of adult gut biodiversity and provide a framework for future research.

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.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting approximately 10% of reproductive-aged women worldwide. Diagnosis requires two of the following: hyperandrogenism, oligo/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 co-housing study was performed using a letrozole-induced PCOS mouse model that recapitulates many reproductive and metabolic characteristics of PCOS. Since mice are coprophagic, co-housing results in repeated, non-invasive inoculation of gut microbes in co-housed mice via the fecal-oral route. In contrast to letrozole-treated mice housed together, letrozole-treated mice co-housed with placebo mice showed significant improvement in both reproductive and metabolic PCOS phenotypes. Using 16S rRNA gene sequencing, we observed that the gut microbial composition of letrozole-treated mice co-housed with placebo mice differed from letrozole mice housed together. In addition, our analyses identified several bacterial taxa including Coprobacillus, Dorea and Adlercreutzia associated with the improved PCOS phenotype in letrozole-treated mice co-housed with placebo mice. These results indicate that disruption of the gut microbiome may play a causal role in PCOS and that manipulation of the gut microbiome may be a potential treatment option for PCOS.SignificancePCOS is a common cause of female infertility and ~80% of women with PCOS have metabolic dysregulation that predisposes them to type 2 diabetes and cardiovascular disease. Since treatment options for the metabolic symptoms of PCOS are limited, there is a need to develop novel therapeutic options. The gut microbiome has emerged as an important player in human health and has been shown to play a causal role in obesity. In this study, we found that exposure to a healthy gut microbiome through co-housing protected against the development of reproductive and metabolic dysregulation in a PCOS mouse model. These results suggest that manipulation of the gut microbiome may be a potential treatment option for women with PCOS.

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 USA, UK, and Australia to one another and to environmental samples. Our results show an unexpected range of beta-diversity in human stool microbiomes as 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.ImportanceWe 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.

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.

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 β 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.

Synthesis of the gonadotropin β-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 LβT2 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.

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.

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 β-subunit (LHβ) 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 LHβ expression in immortalized mouse gonadotrope cells. Analyses revealed that glucocorticoid repression localizes to a region of the LHβ 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 LHβ 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 LHβ promoter during GnRH induction and GR repression.

Differential regulation of gonadotropin hormone production in the pituitary is critical for fertility. Activin and progesterone signaling in gonadotrope cells is important for Fshb gene expression. Previously, we reported that synergy between activin and progestins required the binding of SMAD proteins and the progesterone receptor (PR) to the murine Fshb promoter. In this study, we demonstrate that the FOXL2 transcription factor is also necessary for the full synergistic response between activin and progestins. We show that this synergy occurs in a species-specific manner and that multiple elements in the Fshb promoter that bind forkhead box L2 (FOXL2), SMA/mothers against decapentaplegic homologs (SMAD), and PR are required. Furthermore, we demonstrate that FOXL2 can physically interact with PR and SMAD3. Thus, it is likely that protein-protein interactions among FOXL2, SMAD, and PR recruited to the Fshb promoter play a key role in facilitating Fshb transcription before the secondary FSH surge in rodents.

Follicle-stimulating hormone (FSH), produced by pituitary gonadotrope cells, is required for maturation of ovarian follicles. The FSHbeta subunit is the limiting factor for production of mature hormone and provides biological specificity. Activin dramatically induces FSHbeta transcription and the secondary rise in FSH, important for follicular development, is dependent on this induction. Thus, regulation of FSHbeta levels by activin is crucial for female reproductive fitness. This review discusses activin signaling pathways, transcription factors and FSHbeta promoter elements required for activin responsiveness. Because FoxL2, a forkhead transcription factor, was recently shown to be instrumental in relaying activin signaling to the FSHbeta promoter, we focus in this paper on its role and the inter-relatedness of several key players in activin responsiveness on the FSHbeta promoter.

Synthesis of the FSH beta-subunit (FSHbeta) is critical for normal reproduction in mammals, and its expression within the pituitary gonadotrope is tightly regulated by activin. Here we show that Runt-related (RUNX) proteins, transcriptional regulators known to interact with TGFbeta signaling pathways, suppress activin induction of FSHbeta gene expression. Runx2 is expressed within the murine pituitary gland and dramatically represses activin-induced FSHbeta promoter activity, without affecting basal expression in LbetaT2 cells, an immortalized mouse gonadotrope cell line. This repressive effect is specific, because RUNX2 induces LHbeta transcription (with or without activin) and does not interfere with GnRH induction of either gonadotropin beta-subunit gene. Analysis of the murine FSHbeta promoter by transfection and gel shift assays reveals that RUNX2 repression localizes to a Runx-binding element at -159/-153, which is adjacent to a previously recognized region critical for activin induction. Mutation of this -153 activin-response element or, indeed, any of the five activin-responsive regions prevents activin induction and, in fact, RUNX2 suppression, instead converting RUNX2 to an activator of the FSHbeta gene. Although the Runx-binding element is required for RUNX2-mediated repression of FSHbeta induction by either activin or Smad3, confirming a functional role of this novel site, protein interactions in addition to those between RUNX2 and Smads are necessary to account for full repression of activin induction. In summary, the present study provides evidence for Runx2-mediated repression of activin-induced FSHbeta gene expression and reveals the context dependence of Runx2 action in hormonal regulation of the gonadotropin genes.

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.

FSH is produced by the pituitary gonadotrope to regulate gametogenesis. Steroid hormones, including androgens, progestins, and glucocorticoids, have all been shown to stimulate expression of the FSHβ subunit in primary pituitary cells and rodent models. Understanding the molecular mechanisms of steroid induction of FSHβ has been difficult due to the heterogeneity of the anterior pituitary. Immortalized LβT2 cells are a model of a mature gonadotrope cell and express the endogenous steroid receptor for each of the three hormones. Transient transfection of each receptor, along with ligand treatment, stimulates the mouse FSHβ promoter, but induction is severely diminished using receptors that lack the ability to bind DNA, indicating that induction is likely through direct DNA binding. All three steroid hormones act within the first 500 bp of the FSHβ promoter where six putative hormone response elements exist. The −381 site is critical for FSHβ induction by all three steroid hormones, whereas the −197 and −139 sites contribute to maximal induction. Interestingly, the −273 and −230 sites are also necessary for androgen and progestin induction of FSHβ, but not for glucocorticoid induction. Additionally, we find that all three receptors bind the endogenous FSHβ promoter, in vivo, and specifically bind the −381 site in vitro, suggesting that the binding of the receptors to this element is critical for the induction of FSHβ by these 3-keto steroid hormones. Our data indicate that androgens, glucocorticoids, and progestins act via their receptors to directly activate FSHβ gene expression in the pituitary gonadotrope.

LH beta-subunit (LHbeta), which is essential for ovulation and reproductive fitness, is synthesized specifically in pituitary gonadotropes. In this study, we show that LHbeta gene expression is induced by activin in mouse primary pituitary cells if the cells are treated within 24 h after dispersion in culture. Furthermore, male mice deficient in Smad3, and therefore in activin signaling, have lower expression of both LHbeta and FSHbeta mRNAs compared with their wild-type littermates. Using the LbetaT2 immortalized mouse gonadotrope cell line that endogenously expresses LH, we identify specific elements in the regulatory region of the rat LHbeta gene necessary for its induction by activin. Activin responsiveness is conferred by a promoter-proximal region located -121/-86 from the transcriptional start site. Maximal LHbeta induction by activin requires a homeobox element (HB) and a 5'-early growth response (Egr) site found in this region of the promoter. Juxtaposed to the HB are three Smad-binding elements (SBEs), which are essential for LHbeta induction. Interestingly, two of the SBEs are also critical for basal expression of the LHbeta gene. We demonstrate that Smad proteins are necessary and sufficient for activin induction of the LHbeta gene. Furthermore, Smad proteins can bind one of the identified SBEs. In addition to binding this SBE, Smad proteins interact with pituitary homeobox 1 (Ptx-1) and orthodenticle homeobox 1 (Otx-1), which can bind the HB located close to the Smad-binding site. Thus, activin induction of LHbeta gene expression requires a combination of several transcription factors, both basal and activin induced, as well as cooperation between multiple DNA elements.

Little is known about the molecular mechanisms of androgen regulation of the FSHbeta gene; however, studies suggest that it consists of a complex feedback loop that involves multiple mechanisms acting at both the level of the hypothalamus and the pituitary. In the present study, we address androgen regulation of the FSHbeta gene in immortalized gonadotrope cells and investigate the roles of activin and GnRH in androgen action. Using transient transfection assays in the FSHbeta-expressing mouse gonadotrope cell line, LbetaT2, we demonstrate that androgens stimulate expression of an ovine FSHbeta reporter gene in a dose-dependent manner. Mutation of either of two conserved androgen response elements at -245/-231 and -153/-139 within the proximal region of the ovine FSHbeta gene promoter abolishes this stimulation, and androgen receptor binds directly to the -244 ARE in vitro. Androgen induction of the FSHbeta reporter gene is also dependent upon the activin autocrine loop present in the LbetaT2 cells, as well as an activin-response element at -138/-124 of the FSHbeta gene. However, activin regulation of other genes remains unaffected by androgens. In addition, androgens stimulate expression of a mouse GnRH receptor reporter gene, and thus may indirectly augment the response of the FSHbeta gene to GnRH. Taken together, these data demonstrate that, in mouse gonadotropes, androgens act directly on the ovine FSHbeta gene to stimulate expression by a mechanism that is dependent upon activin, as well as acting indirectly, potentially through a second mechanism that may be dependent upon induction of GnRH receptor.

To elucidate the earliest molecular steps in the activation of transcription by the progesterone receptor (PR), we investigated its activity in a cell-free transcription system utilizing chromatin templates. PR prepared as a ligand-free, recombinant protein failed to induce transcription on chromatin templates. However, transcriptional competence could be restored by coincubation with rabbit reticulocyte lysate (RRL). The interaction of PR with chaperones results in a receptor conformation competent to bind ligand and RRL contains abundant chaperone-mediated protein folding activity. Blocking this activity with the specific inhibitor geldanamycin inhibited receptor-dependent transcriptional activity. However, recombinant chaperones could not replace RRL in the restoration of transcriptional activity on chromatin templates, suggesting the presence of an additional activity in the lysate. Under chromatin assembly conditions, PR could bind naked DNA and RRL did not increase that binding. In contrast, PR bound to a chromatin template only poorly. Interestingly, RRL stimulated sequence-specific binding by PR to target sites in chromatin and the concomitant recruitment of the steroid receptor coactivator 1 to the promoter. Thus, our results indicate that a novel protein-mediated activity in RRL is involved in an additional, heretofore unrecognized, activation step required for PR to become transcriptionally competent on chromatin templates.

The transcription factor FOXO1 is important for several aspects of reproductive function, including endometrial decidualization, ovarian follicular maturation, and spermatogenesis. We have previously reported that in pituitary gonadotropes FOXO1 functions as a repressor of both luteinizing hormone beta (Lhb) and follicle stimulating hormone beta (Fshb) transcription, which are critical for reproduction. Insulin-like growth factor I (IGFI) is a negative regulator of FOXO1 transcriptional activity. Given that FOXO1 can repress Lhb and Fshb transcription and that IGFI stimulation of gonadotrope cells enhances Lhb and Fshb synthesis, we investigated how IGFI regulates FOXO1 activity in gonadotropes in the current study. In LβT2 gonadotrope cells, we determined by western analyses that IGFI induced Ser256 FOXO1 phosphorylation, causing FOXO1 inhibition and cytoplasmic localization as identified by immunofluorescence microscopy. In addition, inhibition of components of the IGFI signaling pathway, such as PI3K o...

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 LβT2 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.

Glucocorticoid receptor (GR) and progesterone receptor (PR) are closely related members of the steroid receptor family of transcription factors. The two receptors share a similar domain structure, substantial sequence identity, DNA binding specificity, and the ability to induce many of the same genes. Despite these similarities, the unliganded GR is localized predominantly in the cytoplasm, while unliganded PR is found predominantly in the nucleus. By expressing green fluorescent protein (GFP)-tagged receptors and assessing subcellular localization in living cells by confocal microscopy, we have investigated the structural basis for the differential localization of GR and PR. By constructing a series of GFP-tagged receptor chimeras between GR and PR, we have shown that multiple features in the N-terminal half of the ligand-binding domain (LBD) are the critical determinants that mandate the differential localization of GR and PR. Replacement of residues encompassing helices 1-5 of GR with those of PR yields a receptor that is nuclear. However, this domain is unable to mediate nuclear import by itself when removed from the context of the receptor. The chimeric receptors also indicate that regions encompassing helices 6 and 7 are key determinants of the ligand binding potential and the transactivation potential of receptors. Thus, the determinants specifying localization of hormone-free receptors are separable from those governing ligand binding character.

The estrogen receptor (ER) regulates the expression of genes involved in the growth, proliferation and differentiation of skeletal, cardiovascular, neural and reproductive tissues. A basic scheme for the mechanism for ER action has been developed, but precise details on the interactions between ER and the cellular signaling and transcription machinery required for receptor-mediated regulation of specific target genes are still lacking. We have developed a genetic approach to explore the functional interactions of ER. In this work, we describe the development of an estrogen responsive system in the fruit fly, Drosophila melanogaster. Transgenic flies carrying the human ER alpha and an estrogen responsive green fluorescent protein (GFP) reporter gene were constructed. In vivo expression of the GFP reporter gene was observed when larvae were grown on a food source containing steroidal or nonsteroidal estrogens. The induction of the reporter gene by estrogens was blocked upon treatment with tamoxifen, an estrogen antagonist. However, we failed to recapitulate ligand-independent activation of the receptor in vivo or in cultured Drosophila cells. An estrogen responsive Drosophila system could be used to identify and characterize the complex functional interactions between ER and the other components of the cellular transcriptional apparatus.

To determine the origin and evolutionary significance of a recently discovered isoform of the estrogen receptor (ERbeta), we examined the phylogenetic relationship of ERbeta to the well-known alpha isoform (ERalpha) and other steroid receptors. Our phylogenetic analyses traced the origin of ERbeta to a single duplication event at least 450 million years ago. Since this duplication, the evolution of both ER isoforms has apparently been constrained such that 80% of the amino acid positions in the DNA binding domain (DBD) and 53% of the ligand binding domain (LBD) have remained unchanged. Using the phylogenetic tree, we determined the amount of evolutionary change that had occurred in two ER isoforms. The DBD and the LBD had lower rates of evolutionary change compared to the NH(2) terminal domain. However, even with strong selective constraints on the DBD and LBD, our phylogenetic analyses demonstrate two clearly separate phylogenetic histories for ERalpha and ERbeta dating back several hundred million years. The ancient duplication of ER and the parallel evolution of the two ER isoforms suggest that, although ERalpha and ERbeta share a substantial degree of sequence identity, they play unique roles in vertebrate physiology and reproduction.

Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are produced by pituitary gonadotrope cells and are required for steroidogenesis, the maturation of ovarian follicles, ovulation, and spermatogenesis. Synthesis of LH and FSH is tightly regulated by a complex network of signaling pathways activated by hormones including gonadotropin-releasing hormone, activin and sex steroids. Members of the forkhead box (FOX) transcription factor family have been shown to act as important regulators of development, homeostasis and reproduction. In this review, we focus on the role of four specific FOX factors (FOXD1, FOXL2, FOXO1 and FOXP3) in gonadotropin hormone production and discuss our current understanding of the molecular function of these factors derived from studies in mouse genetic and cell culture models.

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.

A systematic study of selectively modified, 36-mer hammerhead ribozymes has resulted in the identification of a generic, catalytically active and nuclease stable ribozyme motif containing 5 ribose residues, 29-30 2'-O-Me nucleotides, 1-2 other 2'-modified nucleotides at positions U4 and U7, and a 3'-3'-linked nucleotide "cap." Eight 2'-modified uridine residues were introduced at positions U4 and U7. From the resulting set of ribozymes, several have almost wild-type catalytic activity and significantly improved stability. Specifically, ribozymes containing 2'-NH2 substitutions at U4 and U7, or 2'-C-allyl substitutions at U4, retain most of their catalytic activity when compared to the all-RNA parent. Their serum half-lives were 5-8 h in a variety of biological fluids, including human serum, while the all-RNA parent ribozyme exhibits a stability half-life of only approximately 0.1 min. The addition of a 3'-3'-linked nucleotide "cap" (inverted T) did not affect catalysis but increased the serum half-lives of these two ribozymes to > 260 h at nanomolar concentrations. This represents an overall increase in stability/activity of 53,000-80,000-fold compared to the all-RNA parent ribozyme.

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 β-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 LβT2 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.

The precise interplay of hormonal influences that governs gonadotropin hormone production by the pituitary includes endocrine, paracrine and autocrine actions of hypothalamic gonadotropin-releasing hormone (GnRH), activin and steroids. However, most studies of hormonal regulation of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the pituitary gonadotrope have been limited to analyses of the isolated actions of individual hormones. LHbeta and FSHbeta subunits have distinct patterns of expression during the menstrual/estrous cycle as a result of the integration of activin, GnRH, and steroid hormone action. In this review, we focus on studies that delineate the interplay among these hormones in the regulation of LHbeta and FSHbeta gene expression in gonadotrope cells and discuss how signaling cross-talk contributes to differential expression. We also discuss how recent technological advances will help identify additional factors involved in the differential hormonal regulation of LH and FSH.