Raphe glucose-sensing serotonergic neurons stimulate KNDy neurons to enhance LH pulses via 5HT2CR: rat and goat studies.

Dysfunction of central serotonergic neurons is known to cause depressive disorders in humans, who often show reproductive and/or glucose metabolism disorders. This study examined whether dorsal raphe (DR) serotonergic neurons sense high glucose availability to upregulate reproductive function via activating hypothalamic arcuate (ARC) kisspeptin neurons (= KNDy neurons), a dominant stimulator of gonadotropin-releasing hormone (GnRH)/gonadotropin pulses, using female rats and goats. RNA-seq and histological analysis revealed that stimulatory serotonin-2C receptor (5HT2CR) was mainly expressed in the KNDy neurons in female rats. The serotonergic reuptake inhibitor administration into the mediobasal hypothalamus (MBH), including the ARC, significantly blocked glucoprivic suppression of luteinizing hormone (LH) pulses and hyperglycemia induced by intravenous 2-deoxy-D-glucose (2DG) administration in female rats. A local infusion of glucose into the DR significantly increased in vivo serotonin release in the MBH and partly restored LH pulses and hyperglycemia in the 2DG-treated female rats. Furthermore, central administration of serotonin or a 5HT2CR agonist immediately evoked GnRH pulse generator activity, and central 5HT2CR antagonism blocked the serotonin-induced facilitation of GnRH pulse generator activity in ovariectomized goats. These results suggest that DR serotonergic neurons sense high glucose availability to reduce gluconeogenesis and upregulate reproductive function by activating GnRH/LH pulse generator activity in mammals.

Neonatal Aromatase Inhibition Blocked Defeminization of AVPV Kiss1 Neurons and LH Surge-Generating System in Male Rats.

The neuroendocrine system that controls the preovulatory surge of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH), which triggers ovulation in female mammals, is sexually differentiated in rodents. A transient increase in circulating testosterone levels in male rats within a few hours of birth is primarily responsible for the defeminization of anteroventral periventricular nucleus (AVPV) kisspeptin neurons, which are critical regulators of the GnRH/LH surge. The present study aimed to determine whether neonatal estradiol-17ß (E2) converted from testosterone by aromatase primarily causes the defeminization of AVPV kisspeptin neurons and the surge of GnRH/LH in male rodents. The results of the present study showed that the neonatal administration of letrozole (LET), a nonsteroidal aromatase inhibitor, within 2 hours of birth rescued AVPV Kiss1 expression and the LH surge in adult male rats, while the neonatal administration of testosterone propionate (TP) irreversibly attenuated AVPV Kiss1 expression and the LH surge in adult female rats. Furthermore, the neonatal LET-treated Kiss1-Cre-activated tdTomato reporter males exhibited a comparable number of AVPV Kiss1-Cre-activated tdTomato-expressing cells to that of vehicle-treated female rats, while neonatal TP-treated females showed fewer AVPV Kiss1-Cre-activated tdTomato-expressing cells than vehicle-treated females. Moreover, neonatal TP administration significantly decreased the number of arcuate Kiss1-expressing and Kiss1-Cre-activated tdTomato-positive cells and suppressed LH pulses in adult gonadectomized female rats; however, neonatal LET administration failed to affect them. These results suggest that E2 converted from neonatal testosterone is primarily responsible for the defeminization of AVPV kisspeptin neurons and the subsequent GnRH/LH surge generation in male rats.

Conditional Oprk1-dependent Kiss1 deletion in kisspeptin neurons caused estrogen-dependent LH pulse disruption and LH surge attenuation in female rats.

The gonadotropin-releasing hormone (GnRH) pulse and surge are considered to be generated by arcuate kisspeptin/neurokinin B/dynorphin A (KNDy) neurons and anteroventral periventricular nucleus (AVPV) kisspeptin neurons, respectively, in female rodents. The majority of KNDy and AVPV kisspeptin neurons express κ-opioid receptors (KORs, encoded by Oprk1) in female rodents. Thus, this study aimed to investigate the effect of a conditional Oprk1-dependent Kiss1 deletion in kisspeptin neurons on the luteinizing hormone (LH) pulse/surge and fertility using Kiss1-floxed/Oprk1-Cre rats, in which Kiss1 was deleted in cells expressing or once expressed the Oprk1/Cre. The Kiss1-floxed/Oprk1-Cre female rats, with Kiss1 deleted in a majority of KNDy neurons, showed normal puberty while having a one-day longer estrous cycle and fewer pups than Kiss1-floxed controls. Notably, ovariectomized (OVX) Kiss1-floxed/Oprk1-Cre rats showed profound disruption of LH pulses in the presence of a diestrous level of estrogen but showed apparent LH pulses without estrogen treatment. Furthermore, Kiss1-floxed/Oprk1-Cre rats, with Kiss1 deleted in approximately half of AVPV kisspeptin neurons, showed a lower peak of the estrogen-induced LH surge than controls. These results suggest that arcuate and AVPV kisspeptin neurons expressing or having expressed Oprk1 have a role in maintaining normal GnRH pulse and surge generation, the normal length of the estrous cycle, and the normal offspring number in female rats.

Sex difference in developmental changes in visualized Kiss1 neurons in newly generated Kiss1-Cre rats.

Hypothalamic kisspeptin neurons are master regulators of mammalian reproduction via direct stimulation of gonadotropin-releasing hormone and consequent gonadotropin release. Here, we generated novel Kiss1 (kisspeptin gene)-Cre rats and investigated the developmental changes and sex differences in visualized Kiss1 neurons of Kiss1-Cre-activated tdTomato reporter rats. First, we validated Kiss1-Cre rats by generating Kiss1-expressing cell-specific Kiss1 knockout (Kiss1-KpKO) rats, which were obtained by crossing the current Kiss1-Cre rats with Kiss1-floxed rats. The resulting male Kiss1-KpKO rats lacked Kiss1 expression in the brain and exhibited hypogonadotropic hypogonadism, similar to the hypogonadal phenotype of global Kiss1 KO rats. Histological analysis of Kiss1 neurons in Kiss1-Cre-activated tdTomato reporter rats revealed that tdTomato signals in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) were not affected by estrogen, and that tdTomato signals in the ARC, AVPV, and medial amygdala (MeA) were sexually dimorphic. Notably, neonatal AVPV tdTomato signals were detected only in males, but a larger number of tdTomato-expressing cells were detected in the AVPV and ARC, and a smaller number of cells in the MeA was detected in females than in males at postpuberty. These findings suggest that Kiss1-visualized rats can be used to examine the effect of estrogen feedback mechanisms on Kiss1 expression in the AVPV and ARC. Moreover, the Kiss1-Cre and Kiss1-visualized rats could be valuable tools for further detailed analyses of sexual differentiation in the brain and the physiological role of kisspeptin neurons across the brain in rats.

Enkephalin-δ opioid receptor signaling partly mediates suppression of LH release during early lactation in rats.

Gonadal function is often suppressed during lactation in mammals including rodents, ruminants, and primates. This suppression is thought to be mostly due to the inhibition of the tonic (pulsatile) release of gonadotropin-releasing hormone (GnRH) and consequent gonadotropin. Accumulating evidence suggests that kisspeptin neurons in the arcuate nucleus (ARC) play a critical role in the regulation of pulsatile GnRH/gonadotropin release, and kisspeptin mRNA (Kiss1) and/or kisspeptin expression in the ARC are strongly suppressed by the suckling stimuli in lactating rats. This study aimed to examine whether the central enkephalin-δ-opioid receptor (DOR) signaling mediates the suckling-induced suppression of luteinizing hormone (LH) release in lactating rats. Central administration of a selective DOR antagonist increased the mean plasma LH levels and baseline of LH pulses in ovariectomized lactating mother rats compared to vehicle-injected control dams on day 8 of lactation without affecting the number of Kiss1-expressing cells and the intensity of Kiss1 mRNA signals in the ARC. Furthermore, the suckling stimuli significantly increased the number of enkephalin mRNA (Penk)-expressing cells and the intensity of Penk mRNA signals in the ARC compared to non-lactating control rats. Collectively, these results suggest that central DOR signaling, at least in part, mediates the suppression of LH release induced by suckling stimuli in lactating rats via indirect and/or direct inhibition of ARC kisspeptin neurons.

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.

Hindbrain Adenosine 5-Triphosphate (ATP)-Purinergic Signaling Triggers LH Surge and Ovulation via Activation of AVPV Kisspeptin Neurons in Rats.

Ovulation disorders are a serious problem for humans and livestock. In female rodents, kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) are responsible for generating a luteinizing hormone (LH) surge and consequent ovulation. Here, we report that adenosine 5-triphosphate (ATP), a purinergic receptor ligand, is a possible neurotransmitter that stimulates AVPV kisspeptin neurons to induce an LH surge and consequent ovulation in rodents. Administration of an ATP receptor antagonist (PPADS) into the AVPV blocked the LH surge in ovariectomized (OVX) rats treated with a proestrous level of estrogen (OVX + high E2) and significantly reduced the ovulation rate in proestrous ovary-intact rats. AVPV ATP administration induced a surge-like LH increase in OVX + high E2 rats in the morning. Importantly, AVPV ATP administration could not induce the LH increase in Kiss1 KO rats. Furthermore, ATP significantly increased intracellular Ca2+ levels in immortalized kisspeptin neuronal cell line, and coadministration of PPADS blocked the ATP-induced Ca2+ increase. Histologic analysis revealed that the proestrous level of estrogen significantly increased the number of P2X2 receptor (an ATP receptor)-immunopositive AVPV kisspeptin neurons visualized by tdTomato in Kiss1-tdTomato rats. The proestrous level of estrogen significantly increased varicosity-like vesicular nucleotide transporter (a purinergic marker)-immunopositive fibers projecting to the vicinity of AVPV kisspeptin neurons. Furthermore, we found that some hindbrain vesicular nucleotide transporter-positive neurons projected to the AVPV and expressed estrogen receptor α, and the neurons were activated by the high E2 treatment. These results suggest that hindbrain ATP-purinergic signaling triggers ovulation via activation of AVPV kisspeptin neurons.SIGNIFICANCE STATEMENT Ovulation disorders, which cause infertility and low pregnancy rates, are a serious problem for humans and livestock. The present study provides evidence that adenosine 5-triphosphate, acting as a neurotransmitter in the brain, stimulates kisspeptin neurons in the anteroventral periventricular nucleus, known as the gonadotropin-releasing hormone surge generator, via purinergic receptors to induce the gonadotropin-releasing hormone/luteinizing hormone surge and ovulation in rats. In addition, histologic analyses indicate that adenosine 5-triphosphate is likely to be originated from the purinergic neurons in the A1 and A2 of the hindbrain. These findings may contribute to new therapeutic controls for hypothalamic ovulation disorders in humans and livestock.

Enkephalin-δ Opioid Receptor Signaling Mediates Glucoprivic Suppression of LH Pulse and Gluconeogenesis in Female Rats.

Energy availability is an important regulator of reproductive function at various reproductive phases in mammals. Glucoprivation induced by 2-deoxy-D-glucose (2DG), an inhibitor of glucose utilization, as an experimental model of malnutrition suppresses the pulsatile release of GnRH/LH and induces gluconeogenesis. The present study was performed with the aim of examining whether enkephalin-δ-opioid receptor (DOR) signaling mediates the suppression of pulsatile GnRH/LH release and gluconeogenesis during malnutrition. The administration of naltrindole hydrochloride (NTI), a selective DOR antagonist, into the third ventricle blocked the suppression of LH pulses and part of gluconeogenesis induced by IV 2DG administration in ovariectomized rats treated with a negative feedback level of estradiol-17â€…ß (OVX + low E2). The IV 2DG administration significantly increased the number of Penk (enkephalin gene)-positive cells coexpressing fos (neuronal activation marker gene) in the paraventricular nucleus (PVN), but not in the arcuate nucleus (ARC) in OVX + low E2 rats. Furthermore, double in situ hybridization for Penk/Pdyn (dynorphin gene) in the PVN revealed that approximately 35% of the PVN Penk-expressing cells coexpressed Pdyn. Double in situ hybridization for Penk/Crh (corticotropin-releasing hormone gene) in the PVN and Penk/Kiss1 (kisspeptin gene) in the ARC revealed that few Penk-expressing cells coexpressed Crh and Kiss1. Taken together, these results suggest that central enkephalin-DOR signaling mediates the suppression of pulsatile LH release during malnutrition. Moreover, the current study suggests that central enkephalin-DOR signaling is also involved in gluconeogenesis during malnutrition in female rats.

Dynorphin-κ-opioid receptor signaling, but not µ-opioid receptor signaling, partly mediates the suppression of luteinizing hormone release during late lactation in rats.

Follicular development and ovulation are profoundly suppressed during lactation. This suppression is suggested to be due to the suckling-induced inhibition of the kisspeptin gene (the master regulator of reproduction) in the arcuate nucleus (ARC) and subsequent inhibition of pulsatile gonadotropin-releasing hormone (GnRH)/gonadotropin release. The present study examined whether hypothalamic κ-opioid receptor (KOR) or µ-opioid receptor (MOR) signaling mediates the suppression of luteinizing hormone (LH) release induced by suckling stimulus during late lactation in rats. Central administration of a selective KOR antagonist blocked the suppression of LH release on Day 16 of lactation; however, central administration of a selective MOR antagonist failed to block the suppression. The suckling stimulus significantly increased the number of fos (a marker for neural activation)-positive Pdyn (dynorphin gene)-expressing cells in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) but not in the ARC. Taken together, these results suggest that central KOR signaling, but not MOR signaling, at least partly, mediates the suppression of LH release induced by suckling stimulus during late lactation, and PVN and SON Dyn neurons may be involved in the suppression in rats.

Possibility of Multiple Drug-Drug Interactions in Patients Treated with Statins: Analysis of Data from the Japanese Adverse Drug Event Report (JADER) Database and Verification by Animal Experiments.

Adverse drug events due to drug-drug interactions can be prevented by avoiding concomitant use of causative drugs; therefore, it is important to understand drug combinations that cause drug-drug interactions. Although many attempts to identify drug-drug interactions from real-world databases such as spontaneous reporting systems have been performed, little is known about drug-drug interactions caused by three or more drugs in polypharmacy, i.e., multiple drug-drug interactions. Therefore, we attempted to detect multiple drug-drug interactions using decision tree analysis using the Japanese Adverse Drug Event Report (JADER) database, a Japanese spontaneous reporting system. First, we used decision tree analysis to detect drug combinations that increase the risk of rhabdomyolysis in cases registered in the JADER database that used six statins. Next, the risk of three or more drug combinations that significantly increased the risk of rhabdomyolysis was validated with in vivo experiments in rats. The analysis identified a multiple drug-drug interaction signal only for pitavastatin. The reporting rate of rhabdomyolysis for pitavastatin in the JADER database was 0.09, and it increased to 0.16 in combination with allopurinol. Furthermore, the rate was even higher (0.40) in combination with valsartan. Additionally, necrosis of leg muscles was observed in some rats simultaneously treated with these three drugs, and their creatine kinase and myoglobin levels were elevated. The combination of pitavastatin, allopurinol, and valsartan should be treated with caution as a multiple drug-drug interaction. Since multiple drug-drug interactions were detected with decision tree analysis and the increased risk was verified in animal experiments, decision tree analysis is considered to be an effective method for detecting multiple drug-drug interactions.

Peptide-to-Small Molecule: A Pharmacophore-Guided Small Molecule Lead Generation Strategy from High-Affinity Macrocyclic Peptides.

Recent technological innovations have led to the development of methods for the rapid identification of high-affinity macrocyclic peptides for a wide range of targets; however, it is still challenging to achieve the desired activity and membrane permeability at the same time. Here, we propose a novel small molecule lead discovery strategy, ″Peptide-to-Small Molecule″, which is a combination of rapid identification of high-affinity macrocyclic peptides via peptide display screening followed by pharmacophore-guided de novo design of small molecules, and demonstrate the applicability using nicotinamide N-methyltransferase (NNMT) as a target. Affinity selection by peptide display technology identified macrocyclic peptide 1 that exhibited good enzymatic inhibitory activity but no cell-based activity. Thereafter, a peptide pharmacophore-guided de novo design and further structure-based optimization resulted in highly potent and cell-active small molecule 14 (cell-free IC50 = 0.0011 µM, cell-based IC50 = 0.40 µM), indicating that this strategy could be a new option for drug discovery.

Screening check test to confirm the relative reactivity and applicability of 2,4-dinitrophenylhydrazine impregnated-filters for formaldehyde on other compounds.

OBJECTIVES: A simple check test method was designed to confirm whether a 2,4-dinitrophenylhydrazine (DNPH) filter for formaldehyde can be used to measure other compounds. METHODS: Sample mixtures containing the same concentrations of formaldehyde, acetaldehyde, and acetone were spiked to the DNPH-filter, extracted, and then measured using high performance liquid chromatography with photodiode array detector (HPLC-PDA). The amounts of DNPH-derivatives versus the amounts of spiked samples were then plotted. RESULTS: When the amount of DNPH << the total amount of spiked samples, the amount of DNPH-derivatives was formaldehyde > acetaldehyde >> acetone. This order corresponded to the relative rate constants for the reaction. Therefore, this study confirmed that acetone was not collected at the formaldehyde sampling rate. CONCLUSIONS: This check test easily measured the reaction rate order and can be used as a simple test to determine whether other samples can be measured by the analytical methods used for the specified sample.

Fish Protein Promotes Skeletal Muscle Hypertrophy via the Akt/mTOR Signaling Pathways.

Skeletal muscle is the largest organ in the body and has a broad range of plasticity, undergoing atrophy in response to aging or disease and hypertrophy in response to nutritional supplements or exercise. Loss of skeletal muscle mass and force increases the risk of falls, impairs mobility, and leads to reduced quality of life. In a previous study, we demonstrated that taking in Alaska pollock protein (APP) for only 7 d increased the gastrocnemius muscle mass in rats. This study was conducted to identify hypertrophic myofibers and analyze how hypertrophy occurs within them. Twenty male rats were randomly divided into two groups and administered a diet of casein or APP for 7 d. The expression of each myosin heavy chain (MyHC) isoform in a cross-sectional area was then measured. MyHC IIb and IIx isoforms exhibited hypertrophic features in the gastrocnemius muscles of the APP-fed rats. Furthermore, comprehensive proteomic analyses were conducted to identify changes in protein expression due to muscle hypertrophy. Our results, evaluated by pathway analyses, indicated that the activity of the growth factor signaling pathway was significantly impacted by APP consumption. Moreover, APP could promote protein synthesis by activating the protein kinase B/mechanistic target of the rapamycin signaling pathway, which is also promoted by exercise.

Central somatostatin-somatostatin receptor 2 signaling mediates lactational suppression of luteinizing hormone release via the inhibition of glutamatergic interneurons during late lactation in rats.

Reproductive function is suppressed during lactation owing to the suckling-induced suppression of the kisspeptin gene (Kiss1) expression in the arcuate nucleus (ARC) and subsequent suppression of luteinizing hormone (LH) release. Our previous study revealed that somatostatin (SST) neurons mediate suckling-induced suppression of LH release via SST receptor 2 (SSTR2) in ovariectomized lactating rats during early lactation. This study examined whether central SST-SSTR2 signaling mediates the inhibition of ARC Kiss1 expression and LH release in lactating rats during late lactation and whether the inhibition of glutamatergic neurons, stimulators of LH release, is involved in the suppression of LH release mediated by central SST-SSTR2 signaling in lactating rats. A central injection of the SSTR2 antagonist CYN154806 (CYN) significantly increased ARC Kiss1 expression in lactating rats on day 16 of lactation. Dual in situ hybridization revealed that few ARC Kiss1-positive cells co-expressed Sstr2, and some of the ARC Slc17a6 (a glutamatergic neuronal marker)-positive cells co-expressed Sstr2. Furthermore, almost all ARC Kiss1-positive cells co-expressed Grin1, a subunit of N-methyl-D-aspartate (NMDA) receptors. The numbers of Slc17a6/Sstr2 double-labeled and Slc17a6 single-labeled cells were significantly lower in lactating dams than in non-lactating rats whose pups had been removed after parturition. A central injection of an NMDA antagonist reversed the CYN-induced increase in LH release in lactating rats. Overall, these results suggest that central SST-SSTR2 signaling, at least partly, mediates the suppression of ARC Kiss1 expression and LH release by inhibiting ARC glutamatergic interneurons in lactating rats.

Generation of Tfap2c-T2A-tdTomato knock-in reporter rats via adeno-associated virus-mediated efficient gene targeting.

Gene editing in mammalian zygotes enables us to generate genetically modified animals rapidly and efficiently. In this study, we compare multiple gene targeting strategies in rat zygotes by generating a novel knock-in reporter rat line to visualize the expression pattern of transcription factor AP-2 gamma (Tfap2c). The targeting vector is designed to replace the stop codon of Tfap2c with T2A-tdTomato sequence. We show that the combination of electroporation-mediated transduction of CRISPR/Cas9 components with adeno-associated virus-mediated transduction of the targeting vector is the most efficient in generating the targeted rat line. The Tfap2c-T2A-tdTomato fluorescence reflects the endogenous expression pattern of Tfap2c in preimplantation embryo, germline, placenta, and forebrain during rat embryo development. The reporter line generated here will be a reliable resource for identifying and purifying Tfap2c expressing cells in rats, and the gene targeting strategy we used can be widely applied for generating desired animals.

Kisspeptin neurons as a key player bridging the endocrine system and sexual behavior in mammals.

Reproductive behaviors are sexually differentiated: for example, male rodents show mounting behavior, while females in estrus show lordosis behavior as sex-specific sexual behaviors. Kisspeptin neurons govern reproductive function via direct stimulation of gonadotropin-releasing hormone (GnRH) and subsequent gonadotropin release for gonadal steroidogenesis in mammals. First, we discuss the role of hypothalamic kisspeptin neurons as an indispensable regulator of sexual behavior by stimulating the synthesis of gonadal steroids, which exert "activational effects" on the behavior in adulthood. Second, we discuss the central role of kisspeptin neurons that are directly involved in neural circuits controlling sexual behavior in adulthood. We then focused on the role of perinatal hypothalamic kisspeptin neurons in the induction of perinatal testosterone secretion for its "organizational effects" on masculinization/defeminization of the male brain in rodents during a critical period. We subsequently concluded that kisspeptin neurons are key players in bridging the endocrine system and sexual behavior in mammals.

Cellular and molecular mechanisms regulating the KNDy neuronal activities to generate and modulate GnRH pulse in mammals.

Accumulating findings during the past decades have demonstrated that the hypothalamic arcuate kisspeptin neurons are supposed to be responsible for pulsatile release of gonadotropin-releasing hormone (GnRH) to regulate gametogenesis and steroidogenesis in mammals. The arcuate kisspeptin neurons express neurokinin B (NKB) and dynorphin A (Dyn), thus, the neurons are also referred to as KNDy neurons. In the present article, we mainly focus on the cellular and molecular mechanisms underlying GnRH pulse generation, that is focused on the action of NKB and Dyn and an interaction between KNDy neurons and astrocytes to control GnRH pulse generation. Then, we also discuss the factors that modulate the activity of KNDy neurons and consequent pulsatile GnRH/LH release in mammals.

Role of KNDy Neurons Expressing Kisspeptin, Neurokinin B, and Dynorphin A as a GnRH Pulse Generator Controlling Mammalian Reproduction.

Increasing evidence accumulated during the past two decades has demonstrated that the then-novel kisspeptin, which was discovered in 2001, the known neuropeptides neurokinin B and dynorphin A, which were discovered in 1983 and 1979, respectively, and their G-protein-coupled receptors, serve as key molecules that control reproduction in mammals. The present review provides a brief historical background and a summary of our recent understanding of the roles of hypothalamic neurons expressing kisspeptin, neurokinin B, and dynorphin A, referred to as KNDy neurons, in the central mechanism underlying gonadotropin-releasing hormone (GnRH) pulse generation and subsequent tonic gonadotropin release that controls mammalian reproduction.

Kisspeptin Neurons and Estrogen-Estrogen Receptor α Signaling: Unraveling the Mystery of Steroid Feedback System Regulating Mammalian Reproduction.

Estrogen produced by ovarian follicles plays a key role in the central mechanisms controlling reproduction via regulation of gonadotropin-releasing hormone (GnRH) release by its negative and positive feedback actions in female mammals. It has been well accepted that estrogen receptor α (ERα) mediates both estrogen feedback actions, but precise targets had remained as a mystery for decades. Ever since the discovery of kisspeptin neurons as afferent ERα-expressing neurons to govern GnRH neurons, the mechanisms mediating estrogen feedback are gradually being unraveled. The present article overviews the role of kisspeptin neurons in the arcuate nucleus (ARC), which are considered to drive pulsatile GnRH/gonadotropin release and folliculogenesis, in mediating the estrogen negative feedback action, and the role of kisspeptin neurons located in the anteroventral periventricular nucleus-periventricular nucleus (AVPV-PeN), which are thought to drive GnRH/luteinizing hormone (LH) surge and consequent ovulation, in mediating the estrogen positive feedback action. This implication has been confirmed by the studies showing that estrogen-bound ERα down- and up-regulates kisspeptin gene (Kiss1) expression in the ARC and AVPV-PeN kisspeptin neurons, respectively. The article also provides the molecular and epigenetic mechanisms regulating Kiss1 expression in kisspeptin neurons by estrogen. Further, afferent ERα-expressing neurons that may regulate kisspeptin release are discussed.

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 ß-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-17ß (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 ß-endorphin-MOR signaling mediates the suppression of pulsatile LH release and that the ß-endorphin may indirectly suppress the arcuate kisspeptin neurons, a master regulator for GnRH/LH pulses during malnutrition. Furthermore, the current study suggests that central ß-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.