Arcuate Kiss1 Research Articles

Involvement of nuclear receptor corepressor 2 (NCOR2) in estrogen-induced repression of arcuate Kiss1 expression in female rats.

Hypothalamic arcuate (ARC) kisspeptin neurons are considered the gonadotropin-releasing hormone pulse generator in rats. In virgin rats, the expression of the ARC kisspeptin gene (Kiss1) is repressed by proestrous levels of estradiol-17β (high E2) but not by diestrous levels of E2 (low E2). In lactating rats, ARC Kiss1 expression is repressed by low E2 during late lactation. This study aimed to investigate whether nuclear receptor corepressor 2 (NCOR2, encoded by Ncor2), an estrogen receptor α corepressor, is involved in the estrogen-induced repression of ARC Kiss1 expression in rats. Double in situ hybridization for Kiss1 and Ncor2 revealed that approximately 80% of ARC Kiss1-expressing cells co-expressed Ncor2 in ovariectomized (OVX) + low E2 virgin rats, while approximately 90% of ARC Kiss1-expressing cells co-expressed Ncor2 in OVX + low E2 lactating rats. To further examine the role of Ncor2, we studied the effects of Kiss1-dependent Ncor2 knockdown on ARC Kiss1 expression and luteinizing hormone (LH) pulses. An adeno-associated virus vector carrying Cre-activated short hairpin RNA (shRNA) for Ncor2 was administered to the ARC in two Kiss1-Cre rat models: OVX + high E2 Kiss1-Cre virgin rats and OVX + low E2 Kiss1-Cre lactating rats. Ncor2-shRNA treatment significantly increased the number of ARC Kiss1-expressing cells and the intensity of Kiss1 signals in OVX + high E2 virgin rats but failed to fully restore low E2-induced Kiss1 repression in lactating rats. The Ncor2-shRNA treatment failed to affect LH pulses in both models. These findings suggest that NCOR2 in ARC kisspeptin neurons mediates high E2-induced repression of ARC Kiss1 expression in virgin rats.

Canonical transient receptor potential channels and hypothalamic control of homeostatic functions.

Recent molecular biological and electrophysiological studies have identified multiple transient receptor potential (TRP) channels in hypothalamic neurons as critical modulators of homeostatic functions. In particular, the canonical transient receptor potential channels (TRPCs) are expressed in hypothalamic neurons that are vital for the control of fertility and energy homeostasis. Classical neurotransmitters such as serotonin and glutamate and peptide neurotransmitters such as kisspeptin, neurokinin B and pituitary adenylyl cyclase-activating polypeptide signal through their cognate G protein-coupled receptors to activate TPRC 4, 5 channels, which are essentially ligand-gated calcium channels. In addition to neurotransmitters, circulating hormones like insulin and leptin signal through insulin receptor (InsR) and leptin receptor (LRb), respectively, to activate TRPC 5 channels in hypothalamic arcuate nucleus pro-opiomelanocortin (POMC) and kisspeptin (arcuate Kiss1 [Kiss1ARH]) neurons to have profound physiological (excitatory) effects. Besides its overt depolarizing effects, TRPC channels conduct calcium ions into the cytoplasm, which has a plethora of downstream effects. Moreover, not only the expression of Trpc5 mRNA but also the coupling of receptors to TRPC 5 channel opening are regulated in different physiological states. In particular, the mRNA expression of Trpc5 is highly regulated in kisspeptin neurons by circulating estrogens, which ultimately dictates the firing pattern of kisspeptin neurons. In obesity states, InsRs are "uncoupled" from opening TRPC 5 channels in POMC neurons, rendering them less excitable. Therefore, in this review, we will focus on the critical role of TRPC 5 channels in regulating the excitability of Kiss1ARH and POMC neurons in different physiological and pathological states.

Kisspeptin and lactational anestrus: Current understanding and future prospects

Lactational anestrus, characterized by the suppression of pulsatile gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release, would be a strategic adaptation to ensure survival by avoiding pregnancy during lactation in mammals. In the present article, we first provide a current understanding of the central regulation of reproduction in mammals, i.e., a fundamental role of arcuate kisspeptin neurons in mammalian reproduction by driving GnRH/LH pulses. Second, we discuss the central mechanism inhibiting arcuate Kiss1 (encoding kisspeptin) expression and GnRH/LH pulses during lactation with a focus on suckling stimulus, negative energy balance due to milk production, and the role of circulating estrogen in rats. We also discuss upper regulators that control arcuate kisspeptin neurons in rats during the early and late lactation periods based on the findings obtained by a lactating rat model. Finally, we discuss potential reproductive technology for the improvement of reproductive performance in milking cows.

Intrauterine LPS inhibited arcuate Kiss1 expression, LH pulses, and ovarian function in rats.

Uterine inflammatory diseases are a major cause of infertility in humans and domestic animals. The current findings that intrauterine lipopolysaccharide is absorbed in systemic circulation and attenuates ovarian cyclic activities could provide a basis for developing novel treatments to improve fertility. Uterine inflammatory diseases are a major cause of infertility in humans and domestic animals. Circulating lipopolysaccharide (LPS), a bacterial endotoxin causing uterine inflammation, reportedly downregulates the hypothalamic-pituitary-gonadal axis to mediate ovarian dysfunction. In contrast, the mechanism whereby intrauterine LPS affects ovarian function has not been fully clarified. This study aimed to elucidate whether uterine exposure to LPS downregulates hypothalamic kisspeptin gene (Kiss1) expression, gonadotropin release, and ovarian function. Uterine inflammation was induced by intrauterine LPS administration to ovary-intact and ovariectomized female rats. As a result, plasma LPS concentrations were substantially higher in control rats until 48 h post injection, and the estrous cyclicity was disrupted with a prolonged diestrous phase. Three days post injection, the number of Graafian follicles and plasma estradiol concentration were reduced in LPS-treated rats, while numbers of Kiss1-expressing cells in the anteroventral periventricular nucleus and arcuate nucleus (ARC) were comparable in ovary-intact rats. Four days post injection, ovulation rate and plasma progesterone levels reduced significantly while gene expression of interleukin1β and tumor necrosis factor α was upregulated in the ovaries of LPS-treated rats that failed to ovulate. Furthermore, the number of Kiss1-expressing cells in the ARC and pulsatile luteinizing hormone (LH) release were significantly reduced in ovariectomized rats 24 h post injection. In conclusion, these results indicate that intrauterine LPS is absorbed in systemic circulation and attenuates ovarian function. This detrimental effect might be caused, at least partly, by the inhibition of ARC Kiss1 expression and LH pulses along with an induction of ovarian inflammatory response.

Deletion of Stim1 in Hypothalamic Arcuate Nucleus Kiss1 Neurons Potentiates Synchronous GCaMP Activity and Protects against Diet-Induced Obesity.

Kisspeptin (Kiss1) neurons are essential for reproduction, but their role in the control of energy balance and other homeostatic functions remains unclear. High-frequency firing of hypothalamic arcuate Kiss1 (Kiss1ARH) neurons releases kisspeptin into the median eminence, and neurokinin B (NKB) and dynorphin onto neighboring Kiss1ARH neurons to generate a slow EPSP mediated by TRPC5 channels that entrains intermittent, synchronous firing of Kiss1ARH neurons. High-frequency optogenetic stimulation of Kiss1ARH neurons also releases glutamate to excite the anorexigenic proopiomelanocortin (POMC) neurons and inhibit the orexigenic neuropeptide Y/agouti-related peptide (AgRP) neurons via metabotropic glutamate receptors. At the molecular level, the endoplasmic reticulum (ER) calcium-sensing protein stromal interaction molecule 1 (STIM1) is critically involved in the regulation of neuronal Ca2+ signaling and neuronal excitability through its interaction with plasma membrane (PM) calcium (e.g., TRPC) channels. Therefore, we hypothesized that deletion of Stim1 in Kiss1ARH neurons would increase neuronal excitability and their synchronous firing, which ultimately would affect energy homeostasis. Using optogenetics in combination with whole-cell recording and GCaMP6 imaging in slices, we discovered that deletion of Stim1 in Kiss1 neurons significantly increased the amplitude and duration of the slow EPSP and augmented synchronous [Ca2+]i oscillations in Kiss1ARH neurons. Deletion of Stim1 in Kiss1ARH neurons amplified the actions of NKB and protected ovariectomized female mice from developing obesity and glucose intolerance with high-fat dieting (HFD). Therefore, STIM1 appears to play a critical role in regulating synchronous firing of Kiss1ARH neurons, which ultimately affects the coordination between energy homeostasis and reproduction.SIGNIFICANCE STATEMENT Hypothalamic arcuate kisspeptin (Kiss1ARH) neurons are essential for stimulating the pulsatile release of gonadotropin-releasing hormone (GnRH) and maintaining fertility. However, Kiss1ARH neurons appear to be a key player in coordinating energy balance with reproduction. The regulation of calcium channels and hence calcium signaling is critically dependent on the endoplasmic reticulum (ER) calcium-sensing protein stromal interaction molecule 1 (STIM1), which interacts with the plasma membrane (PM) calcium channels. We have conditionally deleted Stim1 in Kiss1ARH neurons and found that it significantly increased the excitability of Kiss1ARH neurons and protected ovariectomized female mice from developing obesity and glucose intolerance with high-fat dieting (HFD).

Sex-specific pubertal and metabolic regulation of Kiss1 neurons via Nhlh2.

Hypothalamic Kiss1 neurons control gonadotropin-releasing hormone release through the secretion of kisspeptin. Kiss1 neurons serve as a nodal center that conveys essential regulatory cues for the attainment and maintenance of reproductive function. Despite this critical role, the mechanisms that control kisspeptin synthesis and release remain largely unknown. Using Drop-Seq data from the arcuate nucleus of adult mice and in situ hybridization, we identified Nescient Helix-Loop-Helix 2 (Nhlh2), a transcription factor of the basic helix-loop-helix family, to be enriched in Kiss1 neurons. JASPAR analysis revealed several binding sites for NHLH2 in the Kiss1 and Tac2 (neurokinin B) 5' regulatory regions. In vitro luciferase assays evidenced a robust stimulatory action of NHLH2 on human KISS1 and TAC3 promoters. The recruitment of NHLH2 to the KISS1 and TAC3 promoters was further confirmed through chromatin immunoprecipitation. In vivo conditional ablation of Nhlh2 from Kiss1 neurons using Kiss1Cre:Nhlh2fl/fl mice induced a male-specific delay in puberty onset, in line with a decrease in arcuate Kiss1 expression. Females retained normal reproductive function albeit with irregular estrous cycles. Further analysis of male Kiss1Cre:Nhlh2fl/fl mice revealed higher susceptibility to metabolic challenges in the release of luteinizing hormone and impaired response to leptin. Overall, in Kiss1 neurons, Nhlh2 contributes to the metabolic regulation of kisspeptin and NKB synthesis and release, with implications for the timing of puberty onset and regulation of fertility in male mice.

Central µ-Opioid Receptor Antagonism Blocks Glucoprivic LH Pulse Suppression and Gluconeogenesis/Feeding in Female Rats.

Energetic status often affects reproductive function, glucose homeostasis, and feeding in mammals. Malnutrition suppresses pulsatile release of the gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) and increases gluconeogenesis and feeding. The present study aims to examine whether β-endorphin-μ-opioid receptor (MOR) signaling mediates the suppression of pulsatile GnRH/LH release and an increase in gluconeogenesis/feeding induced by malnutrition. Ovariectomized female rats treated with a negative feedback level of estradiol-17β (OVX + low E2) receiving 2-deoxy-D-glucose (2DG), an inhibitor of glucose utilization, intravenously (iv) were used as a malnutrition model. An administration of D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), a selective MOR antagonist, into the third ventricle blocked the suppression of the LH pulse and increase in gluconeogenesis/feeding induced by iv 2DG administration. Histological analysis revealed that arcuate Kiss1 (kisspeptin gene)-expressing cells and preoptic Gnrh1 (GnRH gene)-expressing cells co-expressed little Oprm1 (MOR gene), while around 10% of arcuate Slc17a6 (glutamatergic marker gene)-expressing cells co-expressed Oprm1. Further, the CTOP treatment decreased the number of fos-positive cells in the paraventricular nucleus (PVN) in OVX + low E2 rats treated with iv 2DG but failed to affect the number of arcuate fos-expressing Slc17a6-positive cells. Taken together, these results suggest that the central β-endorphin-MOR signaling mediates the suppression of pulsatile LH release and that the β-endorphin may indirectly suppress the arcuate kisspeptin neurons, a master regulator for GnRH/LH pulses during malnutrition. Furthermore, the current study suggests that central β-endorphin-MOR signaling is also involved in gluconeogenesis and an increase in food intake by directly or indirectly acting on the PVN neurons during malnutrition in female rats.

Role of CORT Duration and Estradiol Dependence for Stress-level of CORT to Inhibit Pulsatile LH Secretion in Female Mice

Two common responses to stress include elevated circulating glucocorticoids and impaired luteinizing hormone (LH) secretion. We have shown that a chronic stress-level of corticosterone (CORT) can slow LH pulses in female mice and that this reduction required estradiol. In addition, the decrease in LH secretion was associated with a suppression in neuronal activation of arcuate kisspeptin (Kiss1) neurons, regarded as the LH pulse generator. Although the increment of the chronic CORT rise was physiological, the duration of the rise exceeded that of a normal CORT response to an acute stressor. Therefore, the goal of the current study was to investigate whether an acute, exogenous CORT rise is sufficient to inhibit LH pulses and arcuate Kiss1 expression, and whether estradiol is necessary for this inhibition. For this study, adult C57bl6 female mice were ovariectomized and implanted with either an implant filled with oil (OVX) or 100 ng estradiol (OVX+E), which approximates a diestrus level of estradiol. Blood samples to measure pulsatile LH were collected every 6 minutes for 90 minutes prior to and following the initiation of CORT or saline treatment. Animals were randomly assigned to receive one of 4 treatments: a single injection of CORT (0.6 mg/kg, i.p) or saline, or three successive injections separated by 30 minutes of CORT (3xCORT) or saline (3xSaline). This dose of CORT elicited a CORT elevation similar to the endogenous CORT rise induced by restraint stress. One injection of CORT elicited an elevation for 30 minutes; the three-injection paradigm elevated CORT for 90 minutes, the entirety of the post injection LH sampling period. Regardless of estradiol status, LH did not differ between the pre and post injection period in mice administered a single injection of CORT or saline. In contrast, in OVX+E mice, 3xCORT significantly slowed the frequency of LH pulses (pulses/90 min: 2.7±0.2 vs 1.4±0.3; pre vs post, p<0.05) compared to mice given 3xSaline (pulses/90 min: 2.9±0.4 vs 2.8±0.4; pre vs post, p>0.05). Interestingly, in OVX mice, there was no impairment of LH secretion in response to either 3xCORT (pulses/90 min: 4.9±0.4 vs 4.7±0.5; pre vs post, p>0.05) or 3xSaline. Knowing that glucocorticoid receptors are present in arcuate Kiss1 cells, we investigated the effect of CORT on Kiss1 expression. Brains were collected 3 hrs following the initiation of the 3xSaline or 3xCORT treatment, and the arcuate nucleus was micropunched for measurement of Kiss1. In OVX+E mice, Kiss1 expression in the arcuate nucleus was suppressed 47% by 3xCORT compared to 3xSaline treated mice (p<0.05). Collectively these data show that an acute CORT rise is sufficient to impair pulsatile LH secretion and Kiss1 expression, but the rise has to be sustained for more than 30 minutes and requires the presence of estradiol. Whether CORT is acting directly or indirectly on arcuate Kiss1 neurons to impair LH secretion remains to be determined.

Chronic Stimulation of Arcuate Kiss1 Neurons Decreases Bone Mass in Female Mice

Loss of peripheral estrogen in postmenopausal women is often associated with decreased physical activity and loss of bone mass, leading to an increased risk of metabolic diseases, osteoporosis, and skeletal fragility. While it is well-established that loss of peripheral estrogen signaling results in bone loss, we previously found that eliminating central estrogen signaling paradoxically results in an unexpected massive increase in bone mass only in female mice. Specifically, deletion of estrogen receptor alpha (ERα) signaling in kisspeptin 1 (Kiss1) expressing neurons of the arcuate nucleus (ARCKiss1) increases bone mass at the expense of reproduction in female mice. Currently, the mechanisms and the neurocircuits that modulate these unexpected responses are unknown. Here, to begin addressing these questions, we asked if changing the neuronal output of ARCKiss1 neurons using chemogenetic manipulation of ARCKiss1 neurons might also alter bone mass and locomotion in female mice. To do this, we delivered stimulatory (AAV2-hM3Dq-mCherry) designer receptors exclusively activated by designer drugs (DREADDs) to the ARC of wild type and Kiss1-Cre+ (Kiss1-CrehM3q-DREADDs) female mice and asked if chronic activation of ARCKiss1 neurons might alter bone mass as analyzed by standard ex-vivo µCT imaging. Clozapine N-oxide (CNO) was delivered for 22 days (0.1 mg/mL). We also leveraged the ANY-Maze system to assess home cage activity over an extensive 96-hour period. Acute activation of ARCKiss1 tended to decrease home cage activity by nearly 40% in Kiss1-CrehM3q-DREADDs mice during the dark period compared to WT females. Interestingly, chronic activation of ARCKiss1 neurons significantly lowered trabecular bone volume by nearly 30%. Current studies are underway to ask if inhibiting ARCKiss1 neurons results in increased bone mass. Our findings collectively suggest that the neuronal activity of ARCKiss1 neurons is sufficient to shift energy allocation away from locomotion and bone-building to maximize reproductive capacity. We speculate that the widely used SERM in breast cancer treatment, Tamoxifen, might exert its bone sparing effect by silencing ARCKiss1 neurons.

Estrogen Differentially Regulates Protein Abundance in Exosomes Released From Immortalized Kisspeptin Neurons in Vitro

Estrogen (E2) is essential for multiple physiological effects in females, ensuring maximum reproductive fitness and maintaining skeletal homeostasis. E2 has been shown to stimulate cancellous bone formation via activation of estrogen receptor alpha (ERα), an effect widely accepted to be mediated directly at bone. A recent landmark study (Herber et al., Nat Commun 2019) demonstrated bone density increases in female mice harboring ERα-deletions specifically in arcuate Kiss-1 neurons. In this study, bone from transgenic females showed higher osteoblast functioning and increases in the expression of sp7 and runx2, positing a direct neural-bone regulatory axis altered by circulating E2 acting in brain. Our laboratory has used two immortalized Kisspeptin (Kiss1)-expressing and -secreting cell lines, KTaR-1 (representative of female arcuate Kiss-1 neurons) and KTaV-3 cells (representative of female AVPV Kiss-1 neurons) as models to explore the role of Kiss-1 in multiple physiological regulatory contexts. We recently determined that factors in the media of female ARC-derived KTaR-1 cells can affect parameters of osteoblast function in vitro, including increases in sp7 and runx2 expression, and formation of bone matrix (evaluated by Alizarin Red assay). Exposure of canine osteosarcoma cells to conditioned media from KTaR-1 cells led to increases in sp7 expression in an E2-dependent manner, and 24h E2-deprivation of these neurons stimulated secretion of osteogenic factors. In this current study, we have used LCMS-MS proteomic analysis to determine the contents of exosomes isolated from Kisspeptin neurons under varying E2 exposure conditions in vitro. Preliminary results reveal ~150-170 proteins up-regulated by E2 exposure and ~200-220 proteins downregulated by E2 exposure in exosomes of both KTaR-1 and KTaV-3 Kisspeptin neurons. Estrogen-regulated Kiss-1 exosomal proteins include several candidates involved in bone remodeling (pentraxin, osteonectin, osteoclast-stimulating factor-1) and neuronal synaptic plasticity and signaling (annexins, semaphorins, connexins). Current work is exploring the effects of exposure of purified exosomes on morphology and gene expression in immortalized GnRH neurons and osteoblasts. While further study is required, initial results suggest that exosomes may represent additional cellular communication pathways utilized by Kisspeptin neurons to elicit changes in brain and bone.

Characterization of the Action of Tachykinin Signaling on Pulsatile LH Secretion in Male Mice.

The alternation of the stimulatory action of the tachykinin neurokinin B (NKB) and the inhibitory action of dynorphin within arcuate (ARH) Kiss1 neurons has been proposed as the mechanism behind the generation of gonadotropin-releasing hormone (GnRH) pulses through the pulsatile release of kisspeptin. However, we have recently documented that GnRH pulses still exist in gonadectomized mice in the absence of tachykinin signaling. Here, we document an increase in basal frequency and amplitude of luteinizing hormone (LH) pulses in intact male mice deficient in substance P, neurokinin A (NKA) signaling (Tac1KO), and NKB signaling (Tac2KO and Tacr3KO). Moreover, we offer evidence that a single bolus of the NKB receptor agonist senktide to gonad-intact wild-type males increases the basal release of LH without changing its frequency. Altogether, these data support the dispensable role of the individual tachykinin systems in the generation of LH pulses. Moreover, the increased activity of the GnRH pulse generator in intact KO male mice suggests the existence of compensation by additional mechanisms in the generation of kisspeptin/GnRH pulses.

Peripheral interleukin-1β 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-1β 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-1β on luteinizing hormone secretion was enhanced by estradiol. We next utilized serial sampling and showed that interleukin-1β reduced the frequency of luteinizing hormone pulses in ovariectomized female mice treated with estradiol. The interleukin-1β-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-1β upstream of kisspeptin receptor activation. We next tested the hypothesis that estradiol enhances the activation of brainstem nuclei responding to interleukin-1β. We determined that the expression of interleukin-1 receptor was elevated within the brainstem following estradiol. Interleukin-1β 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.

SUN-241 Adult Exposure to Iodoacetic Acid Leads to Abnormal Expression of Key Genes Related to Hypothalamic and Pituitary Control of Reproductive Function

Water disinfection byproducts (DBPs) are formed when chemicals used to decontaminate water come into contact with natural or synthetic organic material. DBPs have been linked to a range of health concerns including reproductive disfunction. One such DBP, the monohalogenated iodoacetic acid (IAA), is formed when iodide reacts with a disinfectant, for example, chlorine. IAA is of particular health concern; not only is iodide widely present in the water supply, especially in coastal communities and those near fracking sites, but IAA has been found to be one of the most cyto- and genotoxic DBPs. Further, a previous study has indicated that in vitro IAA exposure significantly inhibits antral follicle growth and reduces estradiol levels in ovaries. However, little is known about how IAA affects the other major components of the reproductive axis: the hypothalamus and pituitary. The reproductive axis relies on homeostatic release of hormones to communicate from one organ to another and alterations at any level may impact reproduction. So, we set out to test the hypothesis that exposure to IAA would lead to disrupted expression of key hypothalamic and pituitary genes related to reproductive function. We continually exposed female adult CD1 mice to 0.5, 10, 100, or 500 mg/L IAA in their drinking water for approximately 35 days (postnatal day 40 (P40) to their first day in diestrus following P75.) Whole pituitaries and hypothalamic punches containing the arcuate nucleus (ARC), anteroventral periventricular zone (AVPV), and medial preoptic nucleus (mPOA) were collected and processed for qPCR analysis. We find that while kisspeptin (Kiss1) expression in the AVPV - the population responsible for generating the LH surge - is unchanged, 0.5 mg/L IAA exposure significantly increases Kiss1 in the ARC, which controls pulsatile GnRH release, and there is a trending increase (p=.056) at 10mg/L. We also measured ARC expression of Neurokinin B (NKB; Tac2), a neuropeptide secreted by kisspeptin co-expressing neurons to autosynaptically stimulate Kiss1 release. We found no change in mRNA levels of Tac2. We also saw no significant changes in GnRH (Gnrh1) mRNA expression. At the level of the pituitary, there is no change in Lhb mRNA levels. Exposure to 10 mg/L IAA leads to significantly reduced Fshb expression, however FSH serum levels are not significantly changed. These data, taken together with previous findings in the ovary, indicate that IAA has the potential to disrupt each major level of the reproductive axis: ovarian follicle development and steroid synthesis, hypothalamic arcuate Kiss1 synthesis, and Fshb synthesis from the pituitary. Further research is necessary to elucidate at which levels IAA acts directly and at which it acts through action on another component of the axis. Additionally, future studies can clarify the mechanism through which IAA has these effects.

SUN-263 In Vitro Modeling of Brain-Bone Communication: Factors Secreted from Immortalized Female Kisspeptin Neurons Modulate Gene Expression in Osteoblasts

While the sex steroid hormone estrogen (E2) is essential for maximum reproductive fitness, circulating E2 is also involved in maintaining skeletal homeostasis in females. E2 in male and female animals has been shown to stimulate cancellous bone formation via activation of estrogen receptor alpha (ERα), and it was widely thought that this effect was mediated directly at the level of bone. A recent study, however, demonstrated a large increase in bone density in female mice in which ERα was deleted from specific neuroendocrine neurons in the arcuate nucleus of the hypothalamus, specifically those expressing kiss1, a population required for fertility and pubertal progression. Bone from transgenic Kiss1-cre X ERα floxed females showed higher osteoblast functioning, accompanied by increases in the expression of sp7 and runx2, positing the existence of a direct neural-bone regulatory axis that is altered by circulating E2 at the level of the brain (Herber et al., Nat Commun 2019).Our laboratory recently published a study demonstrating that GnRH and Kisspeptin, typically thought to act primarily within the neuroendocrine reproductive axis, are synthesized and secreted in an autocrine fashion by canine osteosarcoma cells in vitro, and that these neuropeptides can stimulate tumor cell proliferation (BMC Cancer 2019). Separately, our lab has also recently generated two immortalized Kiss1-expressing and –secreting cell lines, KTaR-1 (representative of female arcuate Kiss-1 neurons) and KTaV-3 cells (representative of female AVPV Kiss-1 neurons) (Endocrinology 2017). In the current study, we have combined these two in vitro models to explore if factors secreted by female ARC-derived KTaR-1 cells may affect multiple parameters of osteoblast function, including sp7 and runx2 expression (evaluated by qPCR), and ability to form bone matrix (evaluated by Alizarin Red assay). Preliminary results suggest that exposure canine osteosarcoma (COS) cells to conditioned media from KTaR-1 cells leads to increases in sp7 expression in an E2-dependent manner, such that 24h E2-deprivation of these neurons stimulates secretion of osteogenic factors. Additionally, media from both KTaR-1 and KTaV-3 cells stimulated Ca2+ production from cultured osteoblasts, as evaluated by Alizarin Red. We are continuing to explore these in vitro interactions using COS cells, as well as normal osteoblasts (cNOB) and immortalized cNOBs, and are evaluating media and exosomal proteomics to further characterize putative factors. While further study is required, these initial results suggest that our immortalized neuronal KP cell models may provide useful molecular tools to explore the regulation of this newly-proposed neural-bone axis.

Sexually dimorphic gene expression and neurite sensitivity to estradiol in fetal arcuate Kiss1 cells.

Kiss1 neurons of the arcuate (ARC) nucleus form an interconnected network of cells that communicate via neurokinin B (encoded by Tac2) and its receptor (encoded by Tacr3) and play key roles in the control of the reproductive axis through sex hormone-regulated synthesis and release of kisspeptin peptides (Kp, encoded by Kiss1). The aim of this study was to determine whether the Kiss1 cell population of the ARC already displays sexually dimorphic features at embryonic age E16.5 in mice. At this time of development, Kiss1-GFP- and Kp-immunoreactive cell bodies were restricted to the ARC and not found in the pre-optic area (POA). The Kiss1-GFP cell population was identical in size between sexes but had significantly lower Kiss1, Tac2, and Tacr3 mRNA levels and lower Kp-ir fiber density in the POA in male compared to female fetuses. Receptors for androgen (Ar) and estrogen (Esr1, Esr2, Gpr30) and the Cyp19a1 gene (encoding the estradiol-producing enzyme aromatase) transcripts were also detected in fetal ARC Kiss1-GFP cells with significant sex differences for Ar (higher in males) and Esr1 (higher in females). Functional studies on primary cultures of sorted fetal Kiss1-GFP cells revealed a significant negative effect of estradiol treatment on neurite outgrowth on the fourth day of culture in the female group specifically. We conclude that the ARC Kiss1 cell population is already sexually differentiated at E16.5 and that its morphogenetic development may be particularly vulnerable to estradiol exposure at this early developmental time.

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 (5hours) 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.

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.

SAT-407 Evidence for Impaired Kisspeptin Signaling During Metabolic Stress in Female Mice

Stress impairs reproduction, at least in part, by suppressing gonadotropin secretion. One common metabolic stressor is hypoglycemia, which occurs in a variety of pathologies and in healthy people, particularly athletes. Insulin-induced hypoglycemia (IIH) is a repeatable, reliable, and quantifiable acute model of metabolic stress that inhibits pulsatile luteinizing hormone (LH) secretion, though little is known about the underlying mechanism. We have observed that IIH robustly suppresses LH pulses in female mice, which could be mediated by either a pituitary or hypothalamic effect. In the present study, we performed a kisspeptin challenge to determine whether downstream cells involved in LH pulsatility are impaired during IIH. Adult mice (C57/BL6, n = 8/group) were ovariectomized and acclimated to handling for tail-tip blood collection and i.p. injection. On the day of each challenge, mice were fasted for 5 hr and then received an i.p. injection of either saline or insulin (0.75mU/kg). All animals then received an injection of kisspeptin (2μg/g), 45 min later. This time point represents a consistent period of LH pulse suppression following IIH. Frequent blood samples were collected for 30 min before and after kisspeptin injection. Two-weeks later, the experiment was repeated with each mouse receiving the alternate treatment (i.e. insulin or saline) in a cross-over design. Mean LH concentrations were significantly increased after kisspeptin injection in animals treated with saline (saline, pre: 3.8 ± 0.6 vs. post: 7.8 ± 0.5 ng/ml). Interestingly, the LH response to kisspeptin in animals treated with insulin was also markedly increased (insulin, pre: 3.2 ± 0.6 vs. post: 6.4 ± 0.3 ng/ml), but not significantly different from animals treated with saline. We conclude from this study that impaired GnRH or gonadotrope cell function is not the primary cause of reduced LH secretion in this model of metabolic stress. Since kisspeptin neurons in the arcuate nucleus likely form the GnRH/LH pulse generator, we next determined whether arcuate kisspeptin cells were inhibited during IIH. For this purpose, we used a mouse model expressing GFP in kisspeptin cells (Kiss1-Cre-GFP). Mice were fasted and injected with insulin or saline, as above, and fixed neural tissue was collected for assessment of c-Fos immunoreactivity in arcuate Kiss1 cells. Two hours following saline injection ~20% of Kiss1 neurons contained c-Fos. In contrast, only 6.6 ± 0.5% of Kiss1 neurons contained c-Fos in animals treated with insulin. Thus, reduced gonadotropin secretion in IIH may be mediated by an inhibition of the ARC kisspeptin cells. Together these data support the hypothesis that inhibition of LH pulses by metabolic stress is mediated by neural factors including arcuate kisspeptin cells. Future work will investigate kisspeptin cell afferents that convey the inhibitory signal initiated by metabolic stressors.

Mapping neuronal inputs to Kiss1 neurons in the arcuate nucleus of the mouse.

The normal function of the mammalian reproductive axis is strongly influenced by physiological, metabolic and environmental factors. Kisspeptin neuropeptides, encoded by the Kiss1 gene, are potent regulators of the mammalian reproductive axis by stimulating gonadodropin releasing hormone secretion from the hypothalamus. To understand how the reproductive axis is modulated by higher order neuronal inputs we have mapped the afferent circuits into arcuate (ARC) Kiss1 neurons. We used a transgenic mouse that expresses the CRE recombinase in Kiss1 neurons for conditional viral tracing with genetically modified viruses. CRE-mediated activation of these viruses in Kiss1 neurons allows the virus to move transynaptically to label neurons with primary or secondary afferent inputs into the Kiss1 neurons. Several regions of the brain showed synaptic connectivity to arcuate Kiss1 neurons including proopiomelanocortin neurons in the ARC itself, kisspeptin neurons in the anteroventral periventricular nucleus, vasopressin neurons in the supraoptic and suprachiasmatic nuclei, thyrotropin releasing neurons in the paraventricular nucleus and unidentified neurons in other regions including the subfornical organ, amygdala, interpeduncular nucleus, ventral premammilary nucleus, basal nucleus of stria terminalis and the visual, somatosensory and piriform regions of the cortex. These data provide an insight into how the activity of Kiss1 neurons may be regulated by metabolic signals and provide a detailed neuroanatomical map for future functional studies.

Ad libitum feeding triggers puberty onset associated with increases in arcuate Kiss1 and Pdyn expression in growth-retarded rats.

Increasing evidence shows that puberty onset is largely dependent on body weight rather than chronological age. To investigate the mechanism involved in the energetic control of puberty onset, the present study examined effects of chronic food restriction during the prepubertal period and the resumption of ad libitum feeding for 24 and 48 h on estrous cyclicity, Kiss1 (kisspeptin gene), Tac3 (neurokinin B gene) and Pdyn (dynorphin A gene) expression in the hypothalamus, luteinizing hormone (LH) secretion and follicular development in female rats. When animals weighed 75 g, they were subjected to a restricted feeding to retard growth to 70–80 g by 49 days of age. Then, animals were subjected to ad libitum feeding or remained food-restricted. The growth-retarded rats did not show puberty onset associated with suppression of both Kiss1 and Pdyn expression in the arcuate nucleus (ARC). 24-h ad libitum feeding increased tonic LH secretion and the number of Graafian and non-Graafian tertiary follicles with an increase in the numbers of ARC Kiss1- and Pdyn-expressing cells. 48-h ad libitum feeding induced the vaginal proestrus and a surge-like LH increase with an increase in Kiss1-expressing cells in the anteroventral periventricular nucleus (AVPV). These results suggest that the negative energy balance causes pubertal failure with suppression of ARC Kiss1 and Pdyn expression and then subsequent gonadotropin secretion and ovarian function, while the positive energetic cues trigger puberty onset via an increase in ARC Kiss1 and Pdyn expression and thus gonadotropin secretion and follicular development in female rats.