Norepinephrine Neurons in the Nucleus of the Solitary Tract Suppress Luteinizing Hormone Secretion in Female Mice.

Stress impairs fertility, at least in part, via inhibition of gonadotropin secretion. Luteinizing hormone (LH) is an important gonadotropin that is released in a pulsatile pattern in males and in females throughout the majority of the ovarian cycle. Several models of stress, including acute metabolic stress, suppress LH pulses via inhibition of neurons in the arcuate nucleus of the hypothalamus that coexpress kisspeptin, neurokinin B, and dynorphin (termed KNDy cells) which form the pulse generator. The mechanism for inhibition of KNDy neurons during stress, however, remains a significant outstanding question. Here, we investigated a population of catecholamine neurons in the nucleus of the solitary tract (NTS), marked by expression of the enzyme dopamine beta-hydroxylase (DBH), in female mice. First, we found that a subpopulation of DBH neurons in the NTS is activated (express c-Fos) during metabolic stress. Then, using chemogenetics, we determined that activation of these cells is sufficient to suppress LH pulses, augment corticosterone secretion, and induce sickness-like behavior. In subsequent studies, we identified evidence for suppression of KNDy cells (rather than downstream signaling pathways) and determined that the suppression of LH pulses was not dependent on the acute rise in glucocorticoids. Together these data support the hypothesis that DBH cells in the NTS are important for regulation of neuroendocrine and behavioral responses to stress.

Allostasis in Neuroendocrine Systems Controlling Reproduction.

Allostasis provides a supporting role to the homeostatic control of biological variables in mammalian species. While the concept of homeostasis is related to the control of variables within a set point or range that are essential to life, allostasis refers to systems that facilitate adaptation to challenges that the organism faces and the new requirements for survival. Essential for such adaptation is the role played by the brain in eliciting neural and neuroendocrine responses. Reproductive function is fundamental for the survival of species but is costly in energetic terms and requires a synchrony with an ever-changing environment. Thus, in many species reproductive function is blocked or delayed over immediate challenges. This review will cover the physiological systems and neuroendocrine pathways that supply allostatic control over reproductive neuroendocrine systems. Light, hypoxia, temperature, nutrition, psychosocial, and immune mediators influence the neuroendocrine control of reproductive functions through pathways that are confluent at the paraventricular nucleus; however, understanding of the integrative responses to these stimuli has not been clarified. Likely, the ultimate consequence of these allostatic mechanisms is the modification of kisspeptin and gonadotropin-releasing hormone neuronal activity, thus compromising reproduction function in the short term, while preserving species survivability.

Extracellular Matrix Influences Gene Expression and Differentiation of Mouse Trophoblast Stem Cells.

Trophoblast stem (TS) cells were first isolated from the mouse placenta; however, little is known about their maintenance and niche in vivo. TS cells, like other stem cells, have a unique microenvironment in which the extracellular matrix (ECM) is a component. Placental pathology is associated with ECM change. However, how these changes and the individual ECM components impact the maintenance or differentiation of TS cells has not been established. This study identified which ECM component(s) maintain the greatest expression of markers associated with undifferentiated mouse trophoblast stem (mTS) cells and which alter the profile of markers of differentiation based on mRNA analysis. mTS cells cultured on individual ECM components and subsequent quantitative polymerase chain reaction analysis revealed that laminin promoted the expression of markers associated with undifferentiated TS cells, fibronectin promoted gene expression associated with syncytiotrophoblast (SynT) layer II cells, and collagen IV promoted the expression of genes associated with differentiated trophoblast. To investigate whether pathological placental ECM influenced the expression of genes associated with different trophoblast subtypes, the mouse model of streptozotocin (STZ)-induced pancreatic ß cell ablation and diabetes was used. Female mice administered STZ (blood glucose ≥300 mg/dL) or control (blood glucose ≤150 mg/dL) were mated. Placental pathology at embryonic day (E)14.5 was confirmed with reduced fetal blood space area, reduced expression of the pericyte marker αSMA, and decreased expression of ECM proteins. mTS cells cultured on ECM isolated from STZ placenta were associated with reduced expression of undifferentiated mTS markers and increased expression of genes associated with terminally differentiated trophoblast [Gcm-1 and SynA (SynT) and junctional zone Tpbpa and Prl2c2]. Altogether, these results support the value of using ECM isolated from the placenta as a tool for understanding trophoblast contribution to placental pathology.

A Modified Ultra-Sensitive ELISA for Measurement of LH in Mice.

A major obstacle to monitoring pulsatile luteinizing hormone (LH) secretion in mice has been an assay with sufficient sensitivity in small blood volumes. In 2013, Steyn and colleagues published a highly sensitive enzyme-linked immunosorbent assay (ELISA) that overcame this barrier by coupling a duo of LH antibodies effective in accurately measuring LH in 4-µL whole-blood aliquots. To address the unavailability of the original detection antibody, AFP240580Rb, we validated a replacement detection antibody, biotinylated-5303 SPRN-5, to be used within the established ELISA. This modified LH ELISA demonstrated a minimum detection limit of 0.0028 ng/mL and a limit of quantification of 0.0333 ng/mL or 0.0666 ng/mL in diluted whole-blood samples of volume 6.4 µL (1:10) or 3.2 µL (1:20), respectively. Detection antibody 5303 SPRN-5 demonstrated parallelism, high precision, and accuracy across the standard curve. LH concentrations in comparison assays, using either 5303 SPRN-5 or AFP240580Rb, were highly correlated (R2 = 0.9829) and demonstrated LH pulse profiles from gonadectomized mice that were nearly superimposable. Pulsatile LH secretion was demonstrated in gonad-intact males and diestrous females and basal LH levels measured with 5303 SPRN-5 were approximately 5-fold higher than the limit of quantification. In addition, we document utility of this new LH ELISA to accurately measure LH in whole blood or serum across multiple sampling sites, as well as in pituitary extracts, LßT2 cells, or media. In summary, the modified LH ELISA described here is highly effective in measuring LH across a range of sample types and small volumes in mice.

Regulation of the gonadotropin-releasing hormone neuron during stress.

The effect of stress on reproduction and gonadal function has captivated investigators for almost 100 years. Following the identification of gonadotropin-releasing hormone (GnRH) 50 years ago, a niche research field emerged fixated on how stress impairs this central node controlling downstream pituitary and gonadal function. It is now clear that both episodic GnRH secretion in males and females and surge GnRH secretion in females are inhibited during a variety of stress types. There has been considerable advancement in our understanding of numerous stress-related signaling molecules and their ability to impair reproductive neuroendocrine activity during stress. Recently, much attention has turned to the effects of stress on two populations of kisspeptin neurons: the stimulatory afferents to GnRH neurons that regulate pulsatile and surge-type gonadotropin secretion. Indeed, future work is still required to fully construct the neuroanatomical framework underlying stress effects, directly or indirectly, on GnRH neuron function. The present review evaluates and synthesizes evidence related to stress-related signaling molecules acting directly on GnRH neurons. Here, we review the evidence for and against the action of a handful of signaling molecules as inhibitors of GnRH neuron function, including corticotropin-releasing hormone, urocortins, norepinephrine, cortisol/corticosterone, calcitonin gene-related peptide and arginine-phenylalanine-amide-related peptide-3.

Neuroendocrine Basis for Disrupted Ovarian Cyclicity in Female Mice During Chronic Undernutrition.

Chronic undernutrition is a type of metabolic stress that impairs reproduction in multiple species. Although energy balance and female reproductive capacity is recognized as tightly coupled, the neuroendocrine loci and molecular mechanisms that mediate ovarian cycle dysfunction during chronic undernutrition in adult females remain poorly understood. Here, we present a series of studies in which we tested the hypothesis that inhibition of kisspeptin (Kiss1) neurons, which are critical for controlling luteinizing hormone (LH) pulses and the preovulatory LH surge in females, underlies the impairment of the ovarian cycle by undernutrition. We first investigated the effect of chronic undernutrition (70% of unrestricted feed intake) on estrous cyclicity in intact female c57bl6 mice. Undernutrition caused a rapid cessation of ovarian cyclicity during the 2-week treatment, suppressing ovarian steroidogenesis and inhibiting ovulation. Using 2 well-defined estradiol-replacement paradigms, we directly tested the hypothesis that undernutrition inhibits Kiss1 neurons in the arcuate nucleus (ARCKiss1), which are required for LH pulses and in the anteroventral periventricular nucleus (AVPVKiss1), which are necessary for LH surge secretion. Undernutrition prevented LH pulses and impaired ARCKiss1 neuronal activation, using c-Fos as a marker, in ovariectomized females subcutaneously implanted with a pellet containing a diestrus-like level of estradiol. In addition, undernutrition completely blocked the estradiol-induced LH surge and diminished Kiss1 messenger RNA abundance, without decreasing estradiol receptor α (Erα), in micropunches of the AVPV. Collectively, these studies demonstrate that undernutrition disrupts ovarian cyclicity in females via impairment both of ARCKiss1 control of LH pulses and AVPVKiss1 induction of the LH surge.

Peripheral interleukin-1ß inhibits arcuate kiss1 cells and LH pulses in female mice.

Peripheral immune/inflammatory challenges rapidly disrupt reproductive neuroendocrine function. This inhibition is considered to be centrally mediated via suppression of gonadotropin-releasing hormone secretion, yet the neural pathway(s) for this effect remains unclear. We tested the hypothesis that interleukin-1ß inhibits pulsatile luteinizing hormone secretion in female mice via inhibition of arcuate kisspeptin cell activation, a population of neurons considered to be the gonadotropin-releasing hormone pulse generator. In the first experiment, we determined that the inhibitory effect of peripheral interleukin-1ß on luteinizing hormone secretion was enhanced by estradiol. We next utilized serial sampling and showed that interleukin-1ß reduced the frequency of luteinizing hormone pulses in ovariectomized female mice treated with estradiol. The interleukin-1ß-induced suppression of pulse frequency was associated with reduced kisspeptin cell activation, as determined by c-Fos coexpression, but not as a result of impaired responsiveness to kisspeptin challenge. Together, these data suggest an inhibitory action of interleukin-1ß upstream of kisspeptin receptor activation. We next tested the hypothesis that estradiol enhances the activation of brainstem nuclei responding to interleukin-1ß. We determined that the expression of interleukin-1 receptor was elevated within the brainstem following estradiol. Interleukin-1ß induced c-Fos in the area postrema, ventrolateral medulla, and nucleus of the solitary tract; however, the response was not increased by estradiol. Collectively, these data support a neural mechanism whereby peripheral immune/inflammatory stress impairs reproductive neuroendocrine function via inhibition of kisspeptin cell activation and reduced pulsatile luteinizing hormone secretion. Furthermore, these findings implicate the influence of estradiol on peripherally mediated neural pathways such as those activated by peripheral cytokines.

Estradiol Enables Chronic Corticosterone to Inhibit Pulsatile Luteinizing Hormone Secretion and Suppress Kiss1 Neuronal Activation in Female Mice.

INTRODUCTION: Two common responses to stress include elevated circulating glucocorticoids and impaired luteinizing hormone (LH) secretion. We have previously shown that a chronic stress level of corticosterone can impair ovarian cyclicity in intact mice by preventing follicular-phase endocrine events. OBJECTIVE: This study is aimed at investigating if corticosterone can disrupt LH pulses and whether estradiol is necessary for this inhibition. METHODS: Our approach was to measure LH pulses prior to and following the administration of chronic corticosterone or cholesterol in ovariectomized (OVX) mice treated with or without estradiol, as well as assess changes in arcuate kisspeptin (Kiss1) neuronal activation, as determined by co-expression with c-Fos. RESULTS: In OVX mice, a chronic 48 h elevation in corticosterone did not alter the pulsatile pattern of LH. In contrast, corticosterone induced a robust suppression of pulsatile LH secretion in mice treated with estradiol. This suppression represented a decrease in pulse frequency without a change in amplitude. We show that the majority of arcuate Kiss1 neurons contain glucocorticoid receptor, revealing a potential site of corticosterone action. Although arcuate Kiss1 and Tac2 gene expression did not change in response to corticosterone, arcuate Kiss1 neuronal activation was significantly reduced by chronic corticosterone, but only in mice treated with estradiol. CONCLUSIONS: Collectively, these data demonstrate that chronic corticosterone inhibits LH pulse frequency and reduces Kiss1 neuronal activation in female mice, both in an estradiol-dependent manner. Our findings support the possibility that enhanced sensitivity to glucocorticoids, due to ovarian steroid milieu, may contribute to reproductive impairment associated with stress or pathophysiologic conditions of elevated glucocorticoids.

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.

Neural and endocrine mechanisms underlying stress-induced suppression of pulsatile LH secretion.

Stress is well-known to inhibit a variety of reproductive processes, including the suppression of episodic Gonadotropin releasing hormone (GnRH) secretion, typically measured via downstream luteinizing hormone (LH) secretion. Since pulsatile secretion of GnRH and LH are necessary for proper reproductive function in both males and females, and stress is common for both human and animals, understanding the fundamental mechanisms by which stress impairs LH pulses is of critical importance. Activation of the hypothalamic-pituitary-adrenal axis, and its corresponding endocrine factors, is a key feature of the stress response, so dissecting the role of stress hormones, including corticotrophin releasing hormone (CRH) and corticosterone, in the inhibition of LH secretion has been one key research focus. However, some evidence suggests that these stress hormones alone are not sufficient for the full inhibition of LH caused by stress, implicating the additional involvement of other hormonal or neural signaling pathways in this process (including inputs from the brainstem, amygdala, parabrachial nucleus, and dorsomedial nucleus). Moreover, different stress types, such as metabolic stress (hypoglycemia), immune stress, and psychosocial stress, appear to suppress LH secretion via partially unique neural and endocrine pathways. The mechanisms underlying the suppression of LH pulses in these models offer interesting comparisons and contrasts, including the specific roles of amygdaloid nuclei and CRH receptor types. This review focuses on the most recent and emerging insights into endocrine and neural mechanisms responsible for the suppression of pulsatile LH secretion in mammals, and offers insights in important gaps in knowledge.

Frequent Tail-tip Blood Sampling in Mice for the Assessment of Pulsatile Luteinizing Hormone Secretion.

In many endocrine systems, circulating factors or hormones are not released continuously, but are secreted as a discrete pulse in response to a releasing factor. Single-point sampling measures are inadequate to fully understand the biological significance of the secretory pattern of pulsatile hormones either under normal physiologic conditions or during conditions of dysregulation. Luteinizing hormone (LH) is synthesized by the anterior pituitary gonadotrope cells and secreted in a pulsatile pattern which requires frequent collection of blood samples for pulse assessment. This has not been possible in mice until recently, due to the development of a high-sensitivity LH assay and advancement in a technique for frequent low-volume sample collection, initially described by Steyn and colleagues.1 Here we describe a protocol for the frequent peripheral blood sample collection from mice with sufficient handling acclimatization to detect pulsatile secretion of LH. The current protocol details an expanded acclimatization period that allows assessment of robust and continuous pulses of LH over multiple hours. In this protocol, the tip of the tail is clipped and blood is collected from the tail using a hand-held pipette. For assessment of pulsatile LH in gonadectomized mice, serial samples are collected every 5-6 min for 90-180 min. Importantly, the collection of blood and measurement of robust pulses of LH can be accomplished in awake, freely behaving mice, given adequate handling acclimatization and effort to minimize environmental stressors. Sufficient acclimatization can be achieved within 4-5 weeks prior to blood collection. This protocol highlights advances in the methodology to ensure collection of whole blood samples for assessment of pulsatile LH secretion patterns over multiple hours in the mouse, a powerful animal model for neuroendocrine research.

Acute Psychosocial Stress Inhibits LH Pulsatility and Kiss1 Neuronal Activation in Female Mice.

Psychosocial stress, such as isolation and restraint, disrupts reproductive neuroendocrine activity. Here we investigate the impact of psychosocial stress on luteinizing hormone (LH) pulses and gene expression and neuronal activation within Rfrp and Kiss1 cells in female mice. Mice were ovariectomized (OVX) and handled daily to habituate to the tail-tip blood collection procedure. Blood was collected every 5 minutes for 180 minutes for measurement of LH. After 90 minutes, stress animals were placed into restraint devices and isolated to new cages. No-stress control animals remained in their home cages. LH pulses occurred at regular intervals during the entire 180-minute sampling period in controls. In contrast, stress induced a rapid and robust suppression of pulsatile LH secretion. Stress reduced the frequency of pulses by 60% and diminished basal LH levels by 40%; pulse amplitude was unaffected. In a separate cohort of OVX females, brains were collected after 45, 90, or 180 minutes of stress or in no-stress controls. At all time points, stress induced a potent decrease in arcuate Kiss1 neuronal activation, using cfos induction as a marker, with a 50% to 60% suppression vs control levels, whereas Rfrp and cfos coexpression in the dorsal-medial nucleus was elevated after 45 minutes of stress. Although arcuate Kiss1 gene expression remained stable, Rfrp expression was elevated 20% after 180 minutes of stress. These findings demonstrate rapid suppression of LH pulsatile secretion by psychosocial stress, associated with reduced cfos induction in Kiss1 neurons and time-dependent increases in Rfrp neuronal activation and messenger RNA.

Androgens Mediate Sex-Dependent Gonadotropin Expression During Late Prenatal Development in the Mouse.

Central organization of the hypothalamic-pituitary-gonadal axis is initiated during fetal life. At this critical time, gonadal hormones mediate sex-specific development of the hypothalamic-pituitary axis, which then dictates reproductive physiology and behavior in adulthood. Although studies have investigated the effects of prenatal androgens on central factors influencing gonadotropin-releasing hormone (GnRH) release, the impact of fetal androgens on gonadotrope function has been overlooked. In the current study, we demonstrated that gonadotropin gene expression and protein production were robustly elevated in female mice compared with males during late fetal development and that this sex difference was dependent on fetal androgens. Treatment of dams from embryonic day (E)15.5 to E17.5 with testosterone, dihydrotestosterone (DHT), or the androgen antagonist flutamide eliminated the sex difference at E18.5. Specifically, flutamide relieved the suppression in male gene expression, elevating the level to that of females, whereas testosterone or DHT attenuated female gene expression to male levels. The gonadotrope population is equivalent in males and females, and gonadotropic cells in both sexes express androgen receptors, suggesting that androgen-dependent transcriptional regulation can occur in these cells in either sex. Studies using mouse models lacking GnRH signaling show that GnRH is necessary for enhanced gonadotropin expression in females and is therefore required to observe the sex difference. Collectively, these data suggest that circuits controlling GnRH input to the fetal pituitary are unrestrained in females yet robustly inhibited in males via circulating androgens and demonstrate plasticity in gonadotropin synthesis and secretion in both sexes depending on the androgen milieu during late prenatal development.

Corticosterone Blocks Ovarian Cyclicity and the LH Surge via Decreased Kisspeptin Neuron Activation in Female Mice.

Stress elicits activation of the hypothalamic-pituitary-adrenal axis, which leads to enhanced circulating glucocorticoids, as well as impaired gonadotropin secretion and ovarian cyclicity. Here, we tested the hypothesis that elevated, stress-levels of glucocorticoids disrupt ovarian cyclicity by interfering with the preovulatory sequence of endocrine events necessary for the LH surge. Ovarian cyclicity was monitored in female mice implanted with a cholesterol or corticosterone (Cort) pellet. Cort, but not cholesterol, arrested cyclicity in diestrus. Subsequent studies focused on the mechanism whereby Cort stalled the preovulatory sequence by assessing responsiveness to the positive feedback estradiol signal. Ovariectomized mice were treated with an LH surge-inducing estradiol implant, as well as Cort or cholesterol, and assessed several days later for LH levels on the evening of the anticipated surge. All cholesterol females showed a clear LH surge. At the time of the anticipated surge, LH levels were undetectable in Cort-treated females. In situ hybridization analyses the anteroventral periventricular nucleus revealed that Cort robustly suppressed the percentage of Kiss1 cells coexpressing cfos, as well as reduced the number of Kiss1 cells and amount of Kiss1 mRNA per cell, compared with expression in control brains. In addition, Cort blunted pituitary expression of the genes encoding the GnRH receptor and LHß, indicating inhibition of gonadotropes during the blockage of the LH surge. Collectively, our findings support the hypothesis that physiological stress-levels of Cort disrupts ovarian cyclicity, in part, through disruption of positive feedback mechanisms at both the hypothalamic and pituitary levels which are necessary for generation of the preovulatory LH surge.

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.

Roles of binding elements, FOXL2 domains, and interactions with cJUN and SMADs in regulation of FSHß.

We previously identified FOXL2 as a critical component in FSHß gene transcription. Here, we show that mice deficient in FOXL2 have lower levels of gonadotropin gene expression and fewer LH- and FSH-containing cells, but the same level of other pituitary hormones compared to wild-type littermates, highlighting a role of FOXL2 in the pituitary gonadotrope. Further, we investigate the function of FOXL2 in the gonadotrope cell and determine which domains of the FOXL2 protein are necessary for induction of FSHß transcription. There is a stronger induction of FSHß reporter transcription by truncated FOXL2 proteins, but no induction with the mutant lacking the forkhead domain. Specifically, FOXL2 plays a role in activin induction of FSHß, functioning in concert with activin-induced SMAD proteins. Activin acts through multiple promoter elements to induce FSHß expression, some of which bind FOXL2. Each of these FOXL2-binding sites is either juxtaposed or overlapping with a SMAD-binding element. We determined that FOXL2 and SMAD4 proteins form a higher order complex on the most proximal FOXL2 site. Surprisingly, two other sites important for activin induction bind neither SMADs nor FOXL2, suggesting additional factors at work. Furthermore, we show that FOXL2 plays a role in synergistic induction of FSHß by GnRH and activin through interactions with the cJUN component of the AP1 complex that is necessary for GnRH responsiveness. Collectively, our results demonstrate the necessity of FOXL2 for proper FSH production in mice and implicate FOXL2 in integration of transcription factors at the level of the FSHß promoter.

Influence of stress-induced intermediates on gonadotropin gene expression in gonadotrope cells.

Despite extensive investigation, a comprehensive understanding of the mechanisms whereby stress impacts fertility remains elusive. Since the 1930s, when Hans Selye popularized studying adaptations to stress (Selye, 1937), we have learned that compensatory mechanisms involve a complex interplay of neural and hormonal processes that allow various body functions to adjust to stress, in a coordinated manner. In terms of reproduction, the adjustment to a stressor interferes with integrated functioning at multiple levels of regulation--the hypothalamus, anterior pituitary gland, gonads, and neural centers coordinating behavior. Various mediators are postulated to participate in reproductive suppression. These include catecholamines, cytokines, prostaglandins, endogenous opioid peptides, and hormones of the hypothalamic-pituitary-adrenal axis. This review focuses on one class of mediators, the glucocorticoids, and provides our views on the relevance and mode of action of this inhibitory intermediate within the anterior pituitary gonadotrope, as a potential cellular site whereby glucocorticoids contribute to stress-induced reproductive suppression.

Phosphorylation of ATF2 and interaction with NFY induces c-Jun in the gonadotrope.

Induction of c-Jun and c-Fos, partners that comprise the AP1 transcription factor, is critical for GnRH regulation of FSHß gene expression. The signaling pathways that are necessary for regulation of AP1 in the gonadotrope cell are not known. Here, we investigate the mechanism of c-Jun induction by GnRH, the sole regulator of c-Jun in the gonadotrope. We identify that GnRH phosphorylates ATF2 via p38 and JNK, the same pathways responsible for GnRH induction of c-Jun. Upon phosphorylation, ATF2 binds the CRE element within the c-Jun proximal promoter and interacts with NFY. Functional ATF2 is necessary for both GnRH induction of c-Jun and FSHß. Taken together, these studies elucidate the specificity of c-Jun induction by GnRH in the gonadotrope by demonstrating GnRH activation of the p38 and JNK signaling pathways that lead to phosphorylation of ATF2, providing critical insight into GnRH regulation of its target gene, the gonadotropin subunit FSHß.

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.

Runt-related transcription factors impair activin induction of the follicle-stimulating hormone {beta}-subunit gene.

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.