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.

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.

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.

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.