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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.

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.

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. [ABSTRACT FROM AUTHOR]

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 (R 2 = 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, LβT2 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. [ABSTRACT FROM AUTHOR]

Natural epigenetic variation provides a source for the generation of phenotypic diversity, but to understand its contribution to such diversity, its interaction with genetic variation requires further investigation. Here we report population-wide DNA sequencing of genomes, transcriptomes and methylomes of wild Arabidopsis thaliana accessions. Single cytosine methylation polymorphisms are not linked to genotype. However, the rate of linkage disequilibrium decay amongst differentially methylated regions targeted by RNA-directed DNA methylation is similar to the rate for single nucleotide polymorphisms. Association analyses of these RNA-directed DNA methylation regions with genetic variants identified thousands of methylation quantitative trait loci, which revealed the population estimate of genetically dependent methylation variation. Analysis of invariably methylated transposons and genes across this population indicates that loci targeted by RNA-directed DNA methylation are epigenetically activated in pollen and seeds, which facilitates proper development of these structures., DNA methylation is a covalent base modification of plant nuclear genomes that is accurately inherited through both mitotic and meiotic (1) cell divisions. However, similarly to spontaneous mutations in DNA, [...]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The investigation of neuroendocrine systems often requires the delivery of drugs, viruses, or other experimental agents directly into the brains of mice. An intracerebroventricular (ICV) injection allows the widespread delivery of the experimental agent throughout the brain (particularly in the structures near the ventricles). Here, methods for making free-hand ICV injections in adult mice are described. By using visual and tactile landmarks on the heads of mice, injections into the lateral ventricles can be made rapidly and reliably. The injections are made with a glass syringe held in the experimenter's hand and placed at approximate distances from the landmarks. Thus, this technique does not require a stereotaxic frame. Furthermore, this technique requires only brief isoflurane anesthesia, which permits the subsequent assessment of mouse behavior and/or physiology in awake, freely behaving mice. Free-hand ICV injection is a powerful tool for the efficient delivery of experimental agents into the brains of living mice and can be combined with other techniques such as frequent blood sampling, neural circuit manipulation, or in vivo recording to investigate neuroendocrine processes.