Urocortin 3 in the posterodorsal medial amygdala mediates stress-induced suppression of LH pulsatility in female mice

Psychosocial stress disrupts reproduction and interferes with pulsatile LH secretion. The posterodorsal medial amygdala (MePD) is an upstream modulator of the reproductive axis and stress. Corticotropin-releasing factor type-2 receptors (CRFR2) are activated in the presence of psychosocial stress together with increased expression of the CRFR2 ligand Urocortin3 (Ucn3) in the MePD of rodents. We investigate whether Ucn3 signalling in the MePD is involved in mediating the suppressive effect of psychosocial stress on LH pulsatility. Firstly, we administered Ucn3 into the MePD and monitored the effect on LH pulses in ovariectomised mice. Next, we delivered Astressin2B, a selective CRFR2 antagonist, intra-MePD in the presence of predator odor, 2,4,5-Trimethylthiazole (TMT) and examined the effect on LH pulses. Subsequently, we virally infected Ucn3-cre-tdTomato mice with inhibitory DREADDs targeting MePD Ucn3 neurons while exposing mice to TMT or restraint stress and examined the effect on LH pulsatility as well as corticosterone release. Administration of Ucn3 into the MePD dose-dependently inhibited LH pulses and administration of Astressin2B blocked the suppressive effect of TMT on LH pulsatility. Additionally, DREADDs inhibition of MePD Ucn3 neurons blocked TMT and restraint stress-induced inhibition of LH pulses and corticosterone release. These results demonstrate for the first time that Ucn3 neurons in the MePD mediate psychosocial stress-induced suppression of the GnRH pulse generator and corticosterone secretion. Ucn3 signalling in the MePD plays a role in modulating the hypothalamic-pituitary-ganadal and hypothalamic-pituitary-adrenal axes, and this brain locus may represent a nodal centre in the interaction between the reproductive and stress axes.

Deficiency of Arcuate Nucleus Kisspeptin Results in Post-Pubertal Central Hypogonadism

Kisspeptin (encoded by Kiss1), a neuropeptide critically involved in neuroendocrine regulation of reproduction, is primarily synthesized in two hypothalamic nuclei: the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC). AVPV kisspeptin is thought to regulate the estrogen-induced positive feedback control of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH), and the pre-ovulatory LH surge in females. In contrast, ARC kisspeptin neurons, which largely co-express neurokinin B and dynorphin A (collectively named KNDy neurons), are thought to mediate estrogen-induced negative feedback control of GnRH/LH and be the major regulators of pulsatile GnRH/LH release. However, definitive data to delineate the specific roles of AVPV versus ARC kisspeptin neurons in the control of GnRH/LH release is lacking. Therefore, we generated a novel mouse model targeting deletion of Kiss1 to the ARC nucleus (Pdyn-Cre/Kiss1fl/fl KO) to determine the functional differences between ARC and AVPV kisspeptin neurons on the reproductive axis. The efficacy of the knock-out was confirmed at both the mRNA and protein levels. Adult female Pdyn-Cre/Kiss1fl/fl KO mice exhibited persistent diestrus and significantly fewer LH pulses when compared to controls, resulting in arrested folliculogenesis, hypogonadism, and infertility. Pdyn-Cre/Kiss1fl/fl KO males also exhibited disrupted LH pulsatility, hypogonadism, and variable, defective spermatogenesis and subfertility. The timing of pubertal onset in males and females was equivalent to controls. These findings add to the current body of evidence for the critical role of kisspeptin in ARC KNDy neurons in GnRH/LH pulsatility in both sexes, while directly establishing ARC kisspeptin's role in regulating estrous cyclicity in female mice, and gametogenesis in both sexes, and culminating in disrupted fertility. The Pdyn-Cre/Kiss1fl/fl KO mice present a novel mammalian model of post-pubertal central hypogonadism.

Urocortin 3 in the posterodorsal medial amygdala mediates psychosocial stress-induced suppression of LH pulsatility in female mice

Exposure to psychosocial stress disrupts reproductive function and interferes with pulsatile luteinising hormone (LH) secretion in mammals. The posterodorsal sub-nucleus of the medial amygdala (MePD) is part of the limbic brain and is an upstream modulator of the reproductive axis as well as stress and anxiety states. Corticotropin releasing factor type-2 receptors (CRFR2) are activated in the presence of psychosocial stress together with an increased expression of the CRFR2 ligand Urocortin3 (Ucn3) in MePD of rodents. We investigate whether Ucn3 signalling in the MePD is involved in mediating the suppressive effect of psychosocial stress exposure on LH pulsatility. Firstly, we administered Ucn3 into the MePD and monitored the effect on pulsatile LH secretion in ovariectomised mice. Next, we delivered Astressin2B, a highly selective CRFR2 antagonist, intra-MePD in the presence of predator odor, 2,4,5-Trimethylthiazole (TMT) and examined the effect on LH pulses. Subsequently, we virally infected ovariectomised Ucn3-cre-tdTomato mice with inhibitory DREADDs targeting the MePD Ucn3 neurons while exposing the mice to TMT or restraint stress and examined the effect on LH pulsatility as well as corticosterone (CORT) release. Administration of Ucn3 into the MePD dose-dependently inhibited pulsatile LH secretion and intra-MePD administration of Astressin2B blocked the suppressive effect TMT on LH pulsatility. Additionally, DREADDs inhibition of MePD Ucn3 neurons blocked TMT and restraint stress-induced inhibition of LH pulses as well as CORT release in the presence of TMT. These results demonstrate for the first time that Ucn3 neurons in the MePD mediate psychosocial stress-induced suppression of the GnRH pulse generator and psychosocial stress-induced CORT secretion. Ucn3 signalling in the MePD plays a fundamental role in modulating the hypothalamic-pituitary-ganadal and hypothalamic-pituitary-adrenal axes, and this brain locus may represent a nodal centre in the crosstalk between the reproductive and stress axes.

Chronic Excess of Non-Aromatizable Androgens From Puberty Does Not Drive a Neuroendocrine Phenotype Observed in Other Preclinical Rodent Models of PCOS

Polycystic ovary syndrome (PCOS) is the most common form of anovulatory infertility in women of reproductive age, characterised by androgen excess, polycystic appearance of the ovary and irregular menstruation. PCOS is also frequently associated with metabolic abnormalities, including increased adiposity and insulin resistance. The origins of PCOS are unknown, however recent findings in animal models strongly implicate androgen signalling in the brain in the development of PCOS pathophysiology. Exposure to androgen excess, either acutely during prenatal development or chronically from a peripubertal timepoint, can drive the development of PCOS-like features in adulthood. Prenatally androgenized (PNA) mice exhibit the cardinal reproductive features of PCOS and increased luteinizing hormone (LH) pulse frequency. This phenotype is associated with increased GABAergic innervation of gonadotropin-releasing hormone (GnRH) neurons, postulated to drive elevated GnRH/LH release and downstream effects. Chronic exposure to di-hydrotestosterone (DHT) from 3 weeks of age drives both reproductive and metabolic PCOS-like features that are ameliorated by selective AR loss from the brain. Here, we aimed to determine whether chronic exposure to DHT drives a similar increase in LH pulsatility and elevated GABAergic innervation to GnRH neurons as seen following prenatal exposure to androgen excess. GnRH-green fluorescent protein (GFP) female mice received either DHT or blank capsules for 90 days from postnatal day (PND) 21 (N = 6-7/group). Serial tail tip blood sampling was used to measure pulsatile LH and fixed brains were collected and immunolabelled for vesicular GABA transporter (VGAT) to assess putative GABAergic terminals associated with GFP-labelled GnRH neurons. Chronic androgen excess from the peripubertal period resulted in acyclicity and increased body weight as expected. However, LH pulsatility was not different between DHT-treated females and controls. Similarly, the density of VGAT appositions to GnRH neurons was not different between groups. Therefore, the programmed changes in the GnRH neuronal network and hyperactive LH secretion that result from prenatal androgen excess are not affected by chronic DHT exposure initiated at 3 weeks of age. These findings suggest that unique central mechanisms are involved in the reproductive impairments driven by exposure to androgen excess at different developmental stages.

Mice With Targeted Deletion of ARC Kisspeptin Exhibit Immature Gametogenesis and Impaired Fertility

Hypothalamic kisspeptin is primarily synthesized in two discrete nuclei - the anteroventral periventricular (AVPV) and the arcuate (ARC) nuclei. We have previously developed a selective, conditional ARC kisspeptin knock-out (KO) mouse line, namely the Pdyn-Cre/Kissfl/fl KO mice, that exhibited normal puberty onset in both sexes, but impaired estrous cyclicity and LH pulsatility in Pdyn-Cre/Kissfl/fl KO females. To examine the end-organ effect of the lack of ARC kisspeptin, we examined gametogenesis, gonad morphology, and fertility. Hematoxylin and eosin (H&E) staining of serial-sectioned whole ovaries demonstrated that Pdyn-Cre/Kissfl/fl KO female mice lacked corpora lutea - their ovarian folliculogenesis did not progress beyond antral follicle development, suggesting an ovulatory defect in Pdyn-Cre/Kissfl/fl KO females. 75% of the Pdyn-Cre/Kissfl/fl KO male mice had testes exhibiting a striking decrease in mature sperm in the seminiferous tubules. The remaining 25% showed evidence of mature sperm. Further evidence of a hypogonadal phenotype of the Pdyn-Cre/Kissfl/fl KO mice included the significantly low weight and small size of the ovaries, uteri, and testes when compared to control littermates. In a controlled, continuous mating paradigm with proven WT males, 2-4-month-old Pdyn-Cre/Kissfl/fl KO female mice failed to become pregnant or produce any pups, whereas age-matched WT females exhibited normal pregnancies to term. Thus, Pdyn-Cre/Kissfl/fl KO females have complete infertility. Ongoing studies of male fertility data suggest that Pdyn-Cre/Kissfl/fl KO males are subfertile, in accordance with their variable spermatogenesis phenotype - some KO males sired pups when paired with proven, WT females, whereas other KO males are infertile. Future experiments include assessing the capability of Pdyn-Cre/Kissfl/fl KO mice to respond to chronic, exogenous kisspeptin and GnRH administration to rescue abnormal LH pulsatility and estrous cyclicity in females, as well as the impaired fertility in both sexes.

Androgen Suppresses In Vivo and In Vitro LH Pulse Secretion and Neural Kiss1 and Tac2 Gene Expression in Female Mice

Androgens can affect the reproductive axis of both sexes. In healthy women, as in men, elevated exogenous androgens decrease gonad function and lower gonadotropin levels; such circumstances occur with anabolic steroid abuse or in transgender men (genetic XX individuals) taking androgen supplements. The neuroendocrine mechanisms by which endogenous or exogenous androgens regulate gonadotropin release, including aspects of pulsatile luteinizing hormone (LH) secretion, remain unknown. Because animal models are valuable for interrogating neural and pituitary mechanisms, we studied effects of androgens in the normal male physiological range on in vivo LH secretion parameters in female mice and in vitro LH secretion patterns from isolated female pituitaries. We also assessed androgen effects on hypothalamic and gonadotrope gene expression in female mice, which may contribute to altered LH secretion profiles. We used a nonaromatizable androgen, dihydrotestosterone (DHT), to isolate effects occurring specifically via androgen receptor (AR) signaling. Compared with control females, DHT-treated females exhibited markedly reduced in vivo LH pulsatility, with decreases in pulse frequency, amplitude, peak, and basal LH levels. Correlating with reduced LH pulsatility, DHT-treated females also exhibited suppressed arcuate nucleus Kiss1 and Tac2 expression. Separate from these neural effects, we determined in vitro that the female pituitary is directly inhibited by AR signaling, resulting in lower basal LH levels and reduced LH secretory responses to gonadotropin-releasing hormone pulses, along with lower gonadotropin gene expression. Thus, in normal adult females, male levels of androgen acting via AR can strongly inhibit the reproductive axis at both the neural and pituitary levels.

Using Aptamers as a Novel Method for Determining GnRH/LH Pulsatility

Aptamers are a novel technology enabling the continuous measurement of analytes in blood and other body compartments, without the need for repeated sampling and the associated reagent costs of traditional antibody-based methodologies. Aptamers are short single-stranded synthetic RNA or DNA that recognise and bind to specific targets. The conformational changes that can occur upon aptamer–ligand binding are transformed into chemical, fluorescent, colour changes and other readouts. Aptamers have been developed to detect and measure a variety of targets in vitro and in vivo. Gonadotropin-releasing hormone (GnRH) is a pulsatile hypothalamic hormone that is essential for normal fertility but difficult to measure in the peripheral circulation. However, pulsatile GnRH release results in pulsatile luteinizing hormone (LH) release from the pituitary gland. As such, LH pulsatility is the clinical gold standard method to determine GnRH pulsatility in humans. Aptamers have recently been shown to successfully bind to and measure GnRH and LH, and this review will focus on this specific area. However, due to the adaptability of aptamers, and their suitability for incorporation into portable devices, aptamer-based technology is likely to be used more widely in the future.

Kisspeptin and LH pulsatility in patients with functional hypothalamic amenorrhea

Purpose Functional hypothalamic amenorrhea (FHA) occurs in response to exaggerated stressors with or without body weight loss. Various hormones, neurotransmitters, and neuromodulators are involved in the control of GnRH and kisspeptin is one of them. Our study aimed to evaluate the putative temporal coupling between kisspeptin and GnRH-induced LH pulsatile secretion. Methods In total, 71 patients with FHA were selected for this study. All patients undergo to a pulsatility study for LH and kisspeptin evaluation (120 min, sampling every 10 min), and to an endocrine evaluation for prolactin (PRL), estradiol (E2), androstenedione (A), 17-hydroxy-progesterone (17OHP), TSH, fT3, fT4, insulin, cortisol and testosterone (T), glucose, total cholesterol, triglycerides. Results Our data demonstrated kisspeptin and LH pulsatile secretions and that both hormones are co-secreted and temporally coupled at time 0 (p < 0.05). When patients were subdivided in hypo-LH (≤3 mIU/ml, n = 58) and normo-LH (>3 mIU/ml, n = 13), more insights were observed on the specific correlations of metabolic and hormone profiles with pulsatility indexes of LH and kisspeptin. Conclusions Our study demonstrated the presence of a distinct kisspeptin episodic secretion in patients with FHA, and showed the temporally coupling of kisspeptin with LH secretory episodes thus supporting that though in amenorrhea, the reproductive axis is still relying on kisspeptin to drive GnRH discharge. In addition, correlations among hormonal data sustain the hypothesis that stress-induced compensatory events are the main direct and indirect promoters of the reproductive blockade in patients affected by FHA.

Different dendritic domains of the GnRH neuron underlie the pulse and surge modes of GnRH secretion in female mice

The gonadotropin-releasing hormone (GnRH) neurons exhibit pulse and surge modes of activity to control fertility. They also exhibit an unusual bipolar morphology comprised of a classical soma-proximal dendritic zone and an elongated secretory process that can operate as both a dendrite and an axon, termed a ‘dendron’. We show using expansion microscopy that the highest density of synaptic inputs to a GnRH neuron exists at its distal dendron. In vivo, selective chemogenetic inhibition of the GnRH neuron distal dendron abolishes the luteinizing hormone (LH) surge and markedly dampens LH pulses. In contrast, inhibitory chemogenetic and optogenetic strategies targeting the GnRH neuron soma-proximal dendritic zone abolish the LH surge but have no effect upon LH pulsatility. These observations indicate that electrical activity at the soma-proximal dendrites of the GnRH neuron is only essential for the LH surge while the distal dendron represents an autonomous zone where synaptic integration drives pulsatile GnRH secretion.

SUN-LB50 Increased In Vivo Pulsatile LH Secretion and Hypothalamic Kisspeptin, NKB, and Dynorphin RNA Levels in a PCOS-Like Mouse Model

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.