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.

Capturing temperature changes on the ocular surface along with estrus and ovulation using infrared thermography in Japanese Black cows.

Pre-ovulatory follicles are cooler than the neighboring reproductive organs in cows. Thus, measuring the temperature of reproductive organs could be a useful method for predicting estrus and ovulation in cows, and the establishment of a non-invasive technique is required. In this study, we used infrared thermography (IRT) to measure ocular surface temperature as a potential surrogate for reproductive organ temperature. Five Japanese Black cows with synchronized estrus were subjected to temperature measurements in five regions of the ocular surface, including the nasal conjunctiva, nasal limbus, center cornea, temporal limbus, and temporal conjunctiva, twice a day (0800 h and 1600 h) during the experimental period. The temperatures in the five regions significantly declined in cows from estrus to ovulation. To the best of our knowledge, this study is the first to use IRT to show a temperature decrease in the ocular surface along with estrus to ovulation in Japanese Black cows.

Direct evidence that KNDy neurons maintain gonadotropin pulses and folliculogenesis as the GnRH pulse generator.

The gonadotropin-releasing hormone (GnRH) pulse is fundamental for mammalian reproduction: GnRH pulse regimens are needed as therapies for infertile women as continuous GnRH treatment paradoxically inhibits gonadotropin release. Circumstantial evidence suggests that the hypothalamic arcuate KNDy neurons expressing kisspeptin (encoded by Kiss1), neurokinin B (encoded by Tac3), and dynorphin A serve as a GnRH pulse generator; however, no direct evidence is currently available. Here, we show that rescuing >20% KNDy neurons by transfecting Kiss1 inside arcuate Tac3 neurons, but not outside of these neurons, recovered folliculogenesis and luteinizing hormone (LH) pulses, an indicator of GnRH pulses, in female global Kiss1 knockout (KO) rats and that >90% conditional arcuate Kiss1 KO in newly generated Kiss1-floxed rats completely suppressed LH pulses. These results first provide direct evidence that KNDy neurons are the GnRH pulse generator, and at least 20% of KNDy neurons are sufficient to maintain folliculogenesis via generating GnRH/gonadotropin pulses.

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.

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.

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.

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

In brief: 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. Abstract: 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 interleukin1ß 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.

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.

Kobayashi Award 2019: The neuroendocrine regulation of the mammalian reproduction.

Mammalian reproductive function is a complex system of many players orchestrated by the hypothalamus-pituitary-gonadal (HPG) axis. The hypothalamic gonadotropin-releasing hormone (GnRH) and the consequent pituitary gonadotropin release show two modes of secretory patterns, namely the surge and pulse modes. The surge mode is triggered by the positive feedback action of estrogen secreted from the mature ovarian follicle to induce ovulation in females of most mammalian species. The pulse mode of GnRH release is required for stimulating tonic gonadotropin secretion to drive folliculogenesis, spermatogenesis and steroidogenesis and is negatively fine-tuned by the sex steroids. Accumulating evidence suggests that hypothalamic kisspeptin neurons are the master regulator for animal reproduction to govern the HPG axis. Specifically, kisspeptin neurons located in the anterior hypothalamus, such as the anteroventral periventricular nucleus (AVPV) in rodents and preoptic nucleus (POA) in ruminants, primates and others, and the neurons located in the arcuate nucleus (ARC) in posterior hypothalamus in most mammals are considered to play a key role in generating the surge and pulse modes of GnRH release, respectively. The present article focuses on the role of AVPV (or POA) kisspeptin neurons as a center for GnRH surge generation and of the ARC kisspeptin neurons as a center for GnRH pulse generation to mediate estrogen positive and negative feedback mechanisms, respectively, and discusses how the estrogen epigenetically regulates kisspeptin gene expression in these two populations of neurons. This article also provides the mechanism how malnutrition and lactation suppress GnRH/gonadotropin pulses through an inhibition of the ARC kisspeptin neurons. Further, the article discusses the programming effect of estrogen on kisspeptin neurons in the developmental brain to uncover the mechanism underlying the sex difference in GnRH/gonadotropin release as well as an irreversible infertility induced by supra-physiological estrogen exposure in rodent models.

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.

Establishment of embryo transfer in the musk shrew (Suncus murinus).

The present study established techniques to induce pseudopregnancy, in vitro oocyte cultures from pronuclear to 2- to 4-cell stages, and embryo transfer in musk shrews, a reflex ovulator. Offspring were subsequently obtained by transferring in vivo-developed or in vitro-cultured embryos. Female musk shrews received human chronic gonadotropin (hCG), with or without mating stimuli, from vasectomized males to produce pseudopregnant recipients. Embryos at the 2- to 4-cell stage were collected 44-48 h after mating. Another set of embryos was collected 26-27 h after mating and then cultured for 20 h from the pronuclear to 2- to 4-cell stages. Subsequently, embryos were transferred into the oviducts of pseudopregnant recipients 24 or 48 h after the induction of pseudopregnancy. Offsprings were successfully obtained from recipients that received hCG 24 h before embryo transfer, regardless of mating stimuli. These techniques may be valuable for producing transgenic musk shrews.

Kiss1-dependent and independent release of luteinizing hormone and testosterone in perinatal male rats.

Prenatal and postnatal biphasic increases in plasma testosterone levels derived from perinatal testes are considered critical for defeminizing/masculinizing the brain mechanism that regulates sexual behavior in male rats. Hypothalamic kisspeptin neurons are indispensable for stimulating GnRH and downstream gonadotropin, as well as the consequent testicular testosterone production/release in adult male rats. However, it is unclear whether kisspeptin is responsible for the increase in plasma testosterone levels in perinatal male rats. The present study aimed to investigate the role of Kiss1/kisspeptin in generating perinatal plasma LH and the consequent testosterone increase in male rats by comparing the plasma testosterone and LH profiles of wild-type (Kiss1+/+) and Kiss1 knockout (Kiss1-/-) male rats. A biphasic pattern of plasma testosterone levels, with peaks in the prenatal and postnatal periods, was found in both Kiss1+/+ and Kiss1-/- male rats. Postnatal plasma testosterone and LH levels were significantly lower in Kiss1-/- male rats than in Kiss1+/+ male rats, whereas the levels in the prenatal embryonic period were comparable between the genotypes. Exogenous kisspeptin challenge significantly increased plasma testosterone and LH levels and the number of c-Fos-immunoreactive GnRH neurons in neonatal Kiss1-/- and Kiss1+/+ male rats. Kiss1 and Gpr54 (kisspeptin receptor gene) were found in the testes of neonatal rats, but kisspeptin treatment failed to stimulate testosterone release in the cultured testes of both genotypes. These findings suggest that postnatal, but not prenatal, testosterone increase in male rats is mainly induced by central kisspeptin-dependent stimulation of GnRH and consequent LH release.

Intronic Enhancer Is Essential for Nr5a1 Expression in the Pituitary Gonadotrope and for Postnatal Development of Male Reproductive Organs in a Mouse Model.

Nuclear receptor subfamily 5 group A member 1 (NR5A1) is expressed in the pituitary gonadotrope and regulates their differentiation. Although several regulatory regions were implicated in Nr5a1 gene expression in the pituitary gland, none of these regions have been verified using mouse models. Furthermore, the molecular functions of NR5A1 in the pituitary gonadotrope have not been fully elucidated. In the present study, we generated mice lacking the pituitary enhancer located in the 6th intron of the Nr5a1 gene. These mice showed pituitary gland-specific disappearance of NR5A1, confirming the functional importance of the enhancer. Enhancer-deleted male mice demonstrated no defects at fetal stages. Meanwhile, androgen production decreased markedly in adult, and postnatal development of reproductive organs, such as the seminal vesicle, prostate, and penis was severely impaired. We further performed transcriptomic analyses of the whole pituitary gland of the enhancer-deleted mice and controls, as well as gonadotropes isolated from Ad4BP-BAC-EGFP mice. These analyses identified several genes showing gonadotrope-specific, NR5A1-dependent expressions, such as Spp1, Tgfbr3l, Grem1, and Nr0b2. These factors are thought to function downstream of NR5A1 and play important roles in reproductive organ development through regulation of pituitary gonadotrope functions.

Testosterone regulation on quiescin sulfhydryl oxidase 2 synthesis in the epididymis.

The epididymis is an androgen-responsive organ, whose structure and functions are modulated by the coordination between androgen and epididymal cues. Highly regulated molecular interaction within the epididymis is required to support viable sperm development necessary for subsequent fertilization. In the present study, we extended our earlier findings on a promising epididymal protein, quiescin sulfhydryl oxidase 2 (QSOX2), and demonstrated a positive correlation between testosterone and QSOX2 protein synthesis through the use of loss- and restore-of-function animal models. Moreover, based on transcriptomic analyses and 2D culture system, we determined that an additional polarized effect of glutamate is indispensable for the regulatory action of testosterone on QSOX2 synthesis. In conclusion, we propose noncanonical testosterone signaling supports epididymal QSOX2 protein synthesis, providing a novel perspective on the regulation of sperm maturation within the epididymis.

Estrogen increases KISS1 expression in newly generated immortalized KISS1-expressing cell line derived from goat preoptic area.

Kisspeptin neurons located in the hypothalamic preoptic area (POA) are suggested to be responsible for the induction of the gonadotropin-releasing hormone (GnRH) surge and the following luteinizing hormone (LH) surge to regulate female mammals' ovulation. Accumulating evidence demonstrates that the preovulatory level of estrogen activates the POA kisspeptin neurons (estrogen positive feedback), which in turn induces a GnRH/LH surge. This study aimed to derive a cell line from goat POA kisspeptin neurons as an in vitro model to analyze the estrogen positive feedback mechanism in ruminants. Neuron-derived cell clones obtained by the immortalization of POA tissue from a female Shiba goat fetus were analyzed for the expression of kisspeptin (KISS1) and estrogen receptor α (ESR1) genes using quantitative real-time reverse transcription-polymerase chain reaction and three cell clones were selected as POA kisspeptin neuron cell line candidates. One cell line (GP64) out of the three clones showed significant increase in the KISS1 level by incubation with estradiol for 24 h, indicating that the GP64 cells mimic endogenous goat POA kisspeptin neurons. The GP64 cells showed immunoreactivities for kisspeptin and estrogen receptor α and retained a stable growth rate throughout three passages. Further, intracellular calcium levels in the GP64 cells were increased by the KCl challenge, indicating their neurosecretory ability. In conclusion, we generated a new KISS1-expressing cell line derived from goat POA. The current GP64 cell line could be a useful model to elucidate the estrogen positive feedback mechanism responsible for the GnRH/LH surge generation in ruminants.

Reduction of arcuate kappa-opioid receptor-expressing cells increased luteinizing hormone pulse frequency in female rats.

The brain mechanism responsible for the pulsatile secretion of gonadotropin-releasing hormone (GnRH) is important for maintaining reproductive function in mammals. Accumulating evidence suggests that kisspeptin/neurokinin B/dynorphin A (KNDy) neurons in the hypothalamic arcuate nucleus (ARC) play a critical role in the regulation of pulsatile GnRH and subsequent gonadotropin secretion. Dynorphin A (Dyn) and its receptor, kappa-opioid receptor (KOR, encoded by Oprk1), have been shown to be involved in the suppression of pulsatile GnRH/luteinizing hormone (LH) release. On the other hand, it is still unclear whether the inhibitory Dyn signaling affects KNDy neurons or KOR-expressing non-KNDy cells in the ARC or other brain regions. We therefore aimed to clarify the role of ARC-specific Dyn-KOR signaling in the regulation of pulsatile GnRH/LH release by the ARC specific cell deletion of KOR-expressing cells using Dyn-conjugated-saporin (Dyn-SAP). Estrogen-primed ovariectomized female rats were administered Dyn-SAP to the ARC. In situ hybridization of Oprk1 showed that ARC Dyn-SAP administration significantly decreased the number of Oprk1-expressing cells in the ARC, but not in the ventromedial hypothalamic nucleus and paraventricular nucleus. The frequency of LH pulses significantly increased in animals bearing the ARC Dyn-SAP administration. The number of Kiss1-expressing cells in the ARC was not affected by ARC Dyn-SAP treatment. Dyn-KOR signaling within the ARC seems to mediate the suppression of the frequency of pulsatile GnRH/LH release, and ARC non-KNDy KOR neurons may be involved in the mechanism modulating GnRH/LH pulse generation.

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.

Retinoblastoma binding protein 7 is involved in Kiss1 mRNA upregulation in rodents.

Kisspeptin, encoded by Kiss1, is essential for reproduction in mammals. Kiss1 expression is regulated by estrogen via histone acetylation in the Kiss1 promotor region. Thus, elucidation of histone modification factor(s) involved in the regulation of Kiss1 expression is required to gain further understanding of the mechanisms of its control. The RNA-seq analysis of isolated kisspeptin neurons, obtained from the arcuate nucleus (ARC) of female rats, revealed that Rbbp7, encoding retinoblastoma binding protein 7 (RBBP7), a member of histone modification and chromatin remodeling complexes, is highly expressed in the ARC kisspeptin neurons. Thus, the present study aimed to investigate whether RBBP7 is involved in Kiss1 expression. Histological analysis using in situ hybridization (ISH) revealed that Rbbp7 expression was located in several hypothalamic nuclei, including the ARC and the anteroventral periventricular nucleus (AVPV), where kisspeptin neurons are located. Double ISH for Rbbp7 and Kiss1 showed that a majority of kisspeptin neurons (more than 85%) expressed Rbbp7 mRNA in both the ARC and the AVPV of female rats. Further, Rbbp7 mRNA knockdown significantly decreased in vitro expression of Kiss1 in a mouse immortalized kisspeptin neuronal cell line (mHypoA-55). Estrogen treatment significantly decreased and increased Kiss1 mRNA levels in the ARC and AVPV of ovariectomized female rats, respectively, but failed to affect Rbbp7 mRNA levels in both the nuclei. Taken together, these findings suggest that RBBP7 is involved in the upregulation of Kiss1 expression in kisspeptin neurons of rodents in an estrogen-independent manner.

Mating-induced increase in Kiss1 mRNA expression in the anteroventral periventricular nucleus prior to an increase in LH and testosterone release in male rats.

Kisspeptin has an indispensable role in gonadotropin-releasing hormone/gonadotropin secretion in mammals. In rodents, kisspeptin neurons are located in distinct brain regions, namely the anteroventral periventricular nucleus-periventricular nucleus continuum (AVPV/PeN), arcuate nucleus (ARC), and medial amygdala (MeA). Among them, the physiological role of AVPV/PeN kisspeptin neurons in males has not been clarified yet. The present study aims to investigate the acute effects of the olfactory and/or mating stimulus with a female rat on hypothalamic and MeA Kiss1 mRNA expression, plasma luteinizing hormone (LH) and testosterone levels in male rats. Intact male rats were exposed to the following stimuli: exposure to clean bedding; exposure to female-soiled bedding as a female-olfactory stimulus; exposure to female-soiled bedding and mating stimulus with a female rat. The mating stimulus significantly increased the number of the AVPV/PeN Kiss1 mRNA-expressing cells in males within 5 minutes after the exposure, and significantly increased LH and testosterone levels, followed by an increase in male sexual behavior. Whereas, the males exposed to female-soiled bedding showed a moderate increase in LH levels and no significant change in testosterone levels and the number of the AVPV/PeN Kiss1 mRNA-expressing cells. Importantly, none of the stimuli affected the number of Kiss1 mRNA-expressing cells in the ARC and MeA. These results suggest that the mating-induced increase in AVPV/PeN Kiss1 mRNA expression may be, at least partly, involved in stimulating LH and testosterone release, and might consequently ensure male mating behavior. This study would be the first report suggesting that the AVPV/PeN kisspeptin neurons in males may play a physiological role in ensuring male reproductive performance.