Follicle- stimulating hormone (FSH) is a glycoprotein hormone that is an essential regulator of mammalian fertility. Produced within gonadotrope cells in the anterior pituitary, FSH, along with the other pituitary gonadotropin, luteinizing hormone, act on the gonads to regulate pubertal maturation, hormone synthesis, and gametogenesis. Genome-wide association studies (GWAS) link a 130 Kb locus at 11p14.1 that encompasses the FSH beta-subunit gene (FSHB) with fertility-related traits including polycystic ovary syndrome, age of natural menopause, and dizygotic twinning. The rate-limiting step in FSH synthesis is transcription of the beta subunit. Therefore, understanding the transcriptional regulation of FSHB is critical to understand how FSHB dysregulation contributes to infertility. The lead single nucleotide polymorphism from several studies is rs11031006, which resides within a highly evolutionarily conserved, ~450 base pair element located 26 Kb upstream of the FSHB transcriptional start site in humans. We hypothesized that the highly conserved element is an enhancer of FSHB and that the minor allele of the rs11031006 polymorphism would impair FSHB transcription. Chapter 1 describes the conserved element in the context of basal regulation and demonstrates that it is, indeed, an enhancer of FSHB transcription in human and mouse. Counter to our initial hypothesis, the rs11031006 variant increased, rather than decreased, FSHB transcription, likely through creation of a higher affinity binding site for the transcription factor Steroidogenic factor 1. Chapter 2 describes the role of the novel FSHB enhancer in the context of hormone regulation. The enhancer potentiated activin- and gonadotropin-releasing hormone- induction of FSHB, dependent upon a binding site within the enhancer for SMAD transcription factors, which are downstream effectors of activin signaling. Chapter 3 examines the in vivo necessity of the Fshb enhancer in a mouse model and the effect of the rs11031006 minor allele. Neither deletion of the enhancer or the rs11031006 minor allele impaired adult fertility, possibly indicating that other enhancers of Fshb can compensate. Overall, we identified a novel enhancer of basal and hormone-stimulated FSHB transcription and demonstrate that the rs11031006 allele increases the activity of the enhancer, although the role of the enhancer and rs11031006 variant in vivo requires further resolution.

Polycystic ovary syndrome (PCOS) is a complex infertility disorder affecting up to 10% of women in the U.S. and most commonly presents with anovulation and androgen excess. Previous research has shown that PCOS women have significantly higher levels of anti-Müllerian hormone (AMH) and psychiatric comorbidities. However, the effect of AMH on the neuroendocrine reproductive axis has not been fully characterized. Recently, a novel prenatal AMH (pAMH) mouse model of PCOS was developed to recapitulate reproductive phenotypes of the disorder; but non-reproductive aspects, such as stress and anxiety, have not been explored. This project sought to investigate the effect of pAMH exposure on stress reactivity in two generations of offspring.

We found that pAMH treatment led to increased response to psychosocial stress in both generations of female mice. The pAMH group had significantly increased novelty-induced defecation and urination, and also had an overall significant and prolonged increase in corticosterone (CORT) response to restraint stress. Notably, we also observed pAMH-induced transgenerational effects in male mice.

Our findings suggest that in addition to reproductive phenotypes, pAMH mouse model is also a representative model of PCOS on non-reproductive measures. To our knowledge, this is the first study to address psychiatric domains of PCOS in a mouse model. Our results potentially shed light on a previously unknown action of AMH in the brain and could also have implications for psychiatric disorders within and beyond the context of PCOS. Further investigation is required to identify the neural circuitry involved in pAMH-mediated phenotypes.

The luteinizing hormone (LH) surge, which prompts ovulation, is dependent on the coincidence of multiple molecular cues. Specifically, estrogen signaling and circadian rhythms work together to gate the LH surge. Although previous studies have shown that the absence of either the estrogen receptor α (ERα) or the circadian clock results in anovulation, the mechanisms behind these regulatory components are not well understood. Kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) initiate the cascade of hormones that ultimately cause the LH surge, and it is thought that these neurons are the location of integration of ERα and circadian rhythm signaling. In this study, we sought to elucidate mechanisms by which ERα signaling and circadian rhythms regulate the expression of the Kiss1 promoter. We found that estrogen induces the Kiss1 promoter after both 12 and 24 hours of treatment, and that this induction is dependent on the expression of ERα. On a finer temporal scale, estrogen-induced Kiss1 promoter expression may be dynamic in vitro, but the expression pattern is not circadian, indicating that estrogen signaling through ERα alone is not sufficient to drive circadian expression of Kiss1. We also found that the molecular clock protein Period 2 (Per2) is capable of inducing the Kiss1 promoter. Taken together, these data indicate that ERα and Per2 regulate the Kiss1 promoter and may be involved in the temporally controlled, estrogen-dependent LH surge.

Gonadotropin-releasing hormone (GnRH) is a primary and essential regulator of vertebrate reproduction. When GnRH neurons are compromised, GnRH expression is attenuated, producing the condition Idiopathic Hypogonadotropic Hypogonadism (IHH). IHH is characterized by infertility and/or delayed or absent puberty. When this condition is accompanied by anosmia, it is termed Kallmann syndrome. Although it is clearly a crucial factor in the reproduction of mammalian species, little is known about the genetic regulators of GnRH neuronal ontogeny. In the work presented herein, we elucidate developmental and genetic mechanisms that control the migration and maturation of GnRH neurons, and therefore illuminate the novel mechanisms responsible for IHH. Furthermore, we have identified new genes influencing the relationship between nasal development and GnRH neuron migration- further elucidating the etiology of Kallmann syndrome. The two genes that are explored in the work presented here are the closely related homeodomain transcription factors, sine oculis-related homeobox 3 (Six3) and Six6. First, we present data demonstrating that Six3 regulates GnRH neuron migration through actions external to the GnRH neuron. Six3 heterozygous mice displayed disrupted migration of GnRH neurons and severe olfactory impairment. Interestingly when the role of Six3 specifically within the GnRH neuron was investigated using a GnRH specific cre-recombinase, we found that Six3 acts within the neuron to upregulate GnRH neuron number. In addition, we investigated the actions of the closely related gene Six6. Six6 was found to be essential for survival of GnRH neurons and maintenance of fertility in a Six6 knock-out mouse. Six6 was found to play a similar role within the GnRH neuron in enabling fertility; however, within the GnRH neuron, Six6 acts to regulate GnRH expression. These investigations have enabled us to garner knowledge concerning the novel regulatory mechanisms of this fascinating hormone; and, has allowed us to shed light on the causes of infertility plaguing ~7% of all couples.

Androgens and androgen receptor (AR) have important roles in both male and female reproductive physiology. In males, androgens are critical for sexual differentiation, the development of secondary sex characteristics, and spermatogenesis. While specific roles of AR within the testis have been studied, whether neuroendocrine AR directly contributes to male fertility remains to be elucidated. Androgens also contribute to female fertility, with global loss of AR in mice resulting in age-dependent subfertility similar to premature ovarian failure in humans. Conversely, excess androgens are a key factor driving the pathophysiology of polycystic ovary syndrome (PCOS), the most common reproductive endocrinopathy in women. In the work presented herein, we demonstrate that AR positively regulates the GnRH receptor (GnRH) by acting at two novel hormone response elements in the GnRHR proximal promoter. We also show that, while AR regulates Gnrhr mRNA in vivo, loss of AR from pituitary gonadotropes (GARKO) does not appear to impair downstream reproductive function. We further investigated neuroendocrine roles of AR by selectively deleting AR from kisspeptin neurons (KARKO) and evaluating effects on male and female fertility. We found that loss of AR from kisspeptin neurons advances puberty and increases pituitary expression of Lhb, suggesting dysregulation of the reproductive neuroendocrine axis. We also utilized the letrozole-induced (LET) mouse model of PCOS to investigate AR contributions to disease pathology in this model. We first demonstrated that treatment with the antiandrogen flutamide ameliorates or reverses many key reproductive and metabolic PCOS phenotypes in LET females. We next treated GARKO females with LET to determine whether selective loss of AR from pituitary gonadotropes affects PCOS phenotypes in this model. We found that deletion of AR from gonadotropes improved reproductive but not metabolic PCOS phenotypes in LET females. Altogether, this work furthers our understanding of the roles of AR in neuroendocrine tissues, both in normal reproductive physiology and in a murine model of PCOS.

Gonadotropin-releasing hormone (GnRH), a neuropeptide released from a small population of neurons in the hypothalamus, is the central mediator of the hypothalamic-pituitary-gonadal axis, and is required for normal reproductive development and function. Evolutionarily conserved regulatory elements in the mouse, rat, and human Gnrh1 gene include three enhancers and the proximal promoter, which confer Gnrh1 gene expression specifically in GnRH neurons. In immortalized mouse hypothalamic GnRH (GT1-7) neurons, which show pulsatile release of GnRH in culture, RNA sequencing and RT-qPCR revealed that expression of a novel long noncoding RNA at Gnrh1 enhancer 1 correlates with high levels of Gnrh1 mRNA expression. In GT1-7 neurons, which contain a transgene carrying 3 kb of the rat Gnrh1 regulatory region, both the mouse and rat GnRH-E1 RNAs are expressed. I investigated the characteristics and function of the mouse Gnrh1 enhancer-derived noncoding RNA (GnRH-E1 RNA). Strand-specific RT-PCR analysis of GnRH-E1 RNA in GT1-7 cells revealed sense and antisense GnRH-E1 RNAs that are transcribed from distinct 5’ start sites, are 3’ polyadenylated, and are over 2 kb in length. GnRH-E1 RNAs are long intergenic noncoding RNAs localized in the nucleus with a half-life of 8 hours. In GT1-7 neurons, siRNA knockdown of mouse GnRH-E1 RNA resulted in a significant decrease in the expression of the Gnrh1 primary transcript and Gnrh1 mRNA. Over-expression of either the mouse or rat GnRH-E1 RNA in GT1-7 cells caused a decrease in the transcriptional activity of co-transfected rat Gnrh1 regulatory elements, which suggests a critical role of balanced GnRH-E1 RNA abundance. The action of GnRH-E1 RNA appears to require critical locations in the Gnrh1 regulatory region. However, the over-expression of either the sense or antisense variants of the mouse GnRH-E1 RNA in immature, migratory GnRH (GN11) neurons, which do not express GnRH-E1 RNA or Gnrh1 mRNA, induced the transcriptional activity of co-transfected rat Gnrh1 gene regulatory elements, where the induction requires the presence of the rat Gnrh1 promoter. Together, these data indicate that GnRH-E1 RNA functions as an inducer of Gnrh1 gene transcriptional activity. GnRH-E1 RNA may play an important role in the development and maturation of GnRH neurons.

Infertility is becoming increasingly prevalent in our society, where one in six couples are faced with difficulties in conceiving. Fertility is tightly regulated by the hypothalamic-pituitary-gonadal axis, where the hypothalamic gonadotropin-releasing hormone (GnRH) neurons and the suprachiasmatic nucleus (SCN) are most vital. Here we identify a novel transcription factor, Ventral anterior homeobox 1 (VAX1), as a critical regulator of GnRH neuron development and SCN function. By the use of transgenic mouse models, we deleted Vax1 in GnRH neurons early in development leading to a 99% reduction in GnRH expression, resulting in delayed pubertal onset, hypogonadism, infertility in both sexes, and atypical estrous cyclicity in females. Furthermore, the deletion of Vax1 in the mature SCN significantly reduced expression of the key SCN peptide, VIP, which was associated with disrupted wheel-running activity. The weakened SCN output led to female subfertility, while the males were unaffected. These data together highlight the importance of VAX1 in both the developmental stage of fertility and its maintenance through adulthood.

Polycystic ovary syndrome (PCOS) is a common reproductive disorder characterized by elevated androgens and anti-Mullerian hormone (AMH). These hormones remain elevated throughout pregnancy, and potential effects of hormone exposure on offspring from women with PCOS remain largely unexplored. Expanding on recent reports of prenatal AMH exposure in mice, we have fully characterized the reproductive consequences of prenatal AMH (pAMH) exposure throughout the lifespan of first- and second-generation offspring of both sexes. We also sought to elucidate mechanisms underlying pAMH-induced reproductive effects. There is a known reciprocal relationship between AMH and androgens, and in PCOS and PCOS-like animal models, androgen feedback is dysregulated at the level the hypothalamus. Kisspeptin neurons express androgen receptors and play a critical role in sexual development and function. We therefore hypothesized that pAMH-induced reproductive phenotypes would be mediated by androgen signaling at the level of kisspeptin cells. We tested the pAMH model in kisspeptin-specific androgen receptor knockout (KARKO) mice and found that virtually all pAMH-induced phenotypes assayed are eliminated in KARKO offspring compared to littermate controls. By demonstrating the necessity of androgen receptor in kisspeptin cells to induce pAMH phenotypes, we have advanced understanding of the interactions between AMH and androgens in the context of prenatal exposure, which could have significant implications for children of women with PCOS.

Kisspeptin neurons, located in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) of the hypothalamus, serve as critical regulators of reproduction, mediating puberty onset, ovulation, and pulsatile gonadotropin-releasing hormone/luteinizing hormone (LH) secretion. Homeodomain transcription factors, such as VAX1 and SIX3, have been implicated in fertility, although the role of these proteins in regulating kisspeptin neuron activity and Kiss1 gene expression is unclear. I have demonstrated that VAX1 differentially regulates Kiss1 gene expression in the ARC of male versus female mice and that the loss of Vax1 from kisspeptin neurons results in a reduction of Kiss1-positive cells at the time of the pre-ovulatory LH surge. I have also found that kisspeptin-specific SIX3 is necessary for estrous cyclicity and maximal fecundity in female mice and that SIX3 differentially regulates the mouse and human Kiss1/KISS1 promoters in vitro and can act as repressor of cFos transcription in vitro and in vivo. This work provides context for the specific roles that homeodomain transcription factors play within kisspeptin neurons and their implications in fertility.

Circadian rhythms control many aspects of our daily lives: from waking and sleeping, to cell cycling within the body’s tissues. The suprachiasmatic nucleus (SCN) is often referred to as the body’s master pacemaker due to its ability to receive light information from the optic nerve and modulate a transcriptional-translational feedback loop, known as the molecular clock, and project circadian information from the SCN to the rest of the body via neuropeptide regulation. VAX1 and SIX3 are two homeodomain transcription factors, known to regulate gene expression, that are highly expressed in the mature mouse SCN. Through the use of transgenic mouse models, we developed conditional Vax1 and Six3 knockout mice bred with Per2-luciferase reporters, a molecular clock gene, to monitor the effects of VAX1 and SIX3 deletion on SCN circadian output. We find that deletion of Vax1 or Six3 from the adult mouse SCN results in shorter and longer circadian period, respectively. Additionally, we determine the ability of VAX1 and SIX3 to increase the expression of Per2-luciferase in vitro, and additionally the ability of VAX1 to drive Vip-luciferase expression, the primary SCN neuropeptide responsible for projecting circadian information from the SCN to the body. Homeodomain transcription factors are known to regulate gene expression by directly binding to ATTA sites within the gene’s promoter. We identified ATTA sites within regulatory regions of Per2 and Vip genes as potential targets for direct VAX1 interaction and regulation of gene expression. Overall, these data collectively identify VAX1 and SIX3 as novel proteins that are responsible for maintaining proper circadian rhythms during adulthood.