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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 inKiss1KO 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 inKiss1-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 STATEMENTOvulation 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.

Background: Tsukamurellaspp. are obligate aerobic, gram-positive, non-motile, and slightly acid-fast bacilli belonging to the Actinomycetes family. Theyshare many characteristics with Nocardia, Rhodococcus, Gordonia, and the rapidly growing Mycobacterium species. Therefore, standard testing may misidentify Tsukamurella spp. as another species. Accurate and rapid diagnosis is critical for proper infection management, but identification of this bacterium is difficult in the standard laboratory setting. Case presentation: A bloodstream infection caused by a gram-positive bacterium and related to a central venous catheter was identified in an immunocompromised 2-year-old girl. Tsukamurella tyrosinosolvens was identified by modified secA1 sequencing. Antibiotic treatment and removal of the central venous catheter resolved the infection. Inappropriate management of the catheter during an overnight stay outside of the hospital was considered as a possible source of infection. Conclusions: SecA1 sequencing may be a useful diagnostic tool in the identification of T. tyrosinosolvens. Providing proper central venous catheter care instructions to patients, their families, and medical staff is important for infection prevention.

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.

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.

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 gonadotropin-releasing hormone (GnRH)/luteinizing hormone (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 3rd ventricle blocked the suppression of LH pulses and part of gluconeogenesis induced by intravenous (iv) 2DG administration in ovariectomized rats treated with a negative feedback level of estradiol-17β (OVX + low E2). The iv 2DG administration significantly increased the number of Penk (enkephalin gene)-positive cells co-expressing 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 co-expressed 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 co-expressed 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.

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

Endogenous opioid peptides have attracted attention as critical neuropeptides in the central mechanism regulating female reproduction ever since the discovery that arcuate dynorphin neurons that coexpress kisspeptin and neurokinin B (NKB), which are also known as kisspeptin/neurokinin B/dynorphin (KNDy) neurons, play a role as a master regulator of pulsatile gonadotropin-releasing hormone (GnRH) release in mammals. In this study, we first focus on the role of dynorphin released by KNDy neurons in the GnRH pulse generation. Second, we provide a historical overview of studies on endogenous opioid peptides. Third, we discuss how endogenous opioid peptides modulate tonic GnRH/gonadotropin release in female mammals as a mediator of inhibitory internal and external cues, such as ovarian steroids, nutritional status, or stress, on reproduction. Then, we discuss the role of endogenous opioid peptides in GnRH surge generation in female mammals.

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.

Objective: The present study aimed to survey seasonal changes in reproductive performance of local cows receiving artificial insemination (AI) in the Pursat province of Cambodia, a tropical country, to investigate if ambient conditions affect the reproductive performance of cows as to better understand the major problems regarding cattle production.Methods: The number of cows receiving AI, resultant number of calving, and calving rate were analyzed for those receiving the first AI from 2016 to 2017. The year was divided into three seasons: cool/dry (from November to February), hot/dry (from March to June), and wet (from July to October), based on the maximal temperature and rainfall in Pursat, to analyze the relationship between ambient conditions and the reproductive performance of cows. Body condition scores (BCS) and feeding schemes were also analyzed in these seasons.Results: The number of cows receiving AI was significantly higher in the cool/dry season than the wet season. The number of calving and calving rate were significantly higher in cows receiving AI in the cool/dry season compared with the hot/dry and wet seasons. The cows showed higher BCSs in the cool/dry season compared to the hot/dry and wet seasons probably due to the seasonal changes in the feeding schemes: these cows grazed on wild grasses in the cool/dry season but fed with a limited amount of grasses and straw in the hot/dry and wet seasons.Conclusion: The present study suggests that the low number of cows receiving AI, low number of calving, and low calving rate could be mainly due to poor body condition as a result of the poor feeding schemes during the hot/dry and wet seasons. The improvement of body condition by the refinement of feeding schemes may contribute to an increase in the reproductive performance in cows during the hot/dry and wet seasons in Cambodia.

Context Daily growth hormone (GH) injections can be burdensome for patients and carers. Somapacitan is a long-acting, reversible albumin-binding GH derivative in development for once-weekly administration in patients with growth hormone deficiency (GHD). Objective The objective of this study is to evaluate the efficacy, safety, and tolerability of once-weekly somapacitan vs once-daily GH. Design REAL 3 is a multicenter, randomized, controlled, double-blind (somapacitan doses), phase 2 study with a 26-week main and 26-week extension phase (NCT02616562). Setting This study took place at 29 sites in 11 countries. Patients Fifty-nine GH treatment-naive prepubertal children with GHD were randomly assigned; 58 completed the trial. Interventions Interventions comprised 3 somapacitan doses (0.04 [n = 16], 0.08 [n = 15], or 0.16 mg/kg/wk [n = 14]) and daily GH (0.034 mg/kg/d [n = 14]), administered subcutaneously. Main Outcome Measures The primary end point was height velocity (HV) at week 26. Secondary efficacy end points included HV SD score (SDS) and insulin-like growth factor-I (IGF-I) SDS. Results At week 26, mean (SD) annualized HV for the somapacitan groups was 8.0 (2.0), 10.9 (1.9), and 12.9 (3.5) cm/year, respectively, vs 11.4 (3.3) cm/year for daily GH; estimated treatment difference (somapacitan 0.16 mg/kg/week—daily GH): 1.7 [95% CI –0.2 to 3.6] cm/year. HV was sustained at week 52, and significantly greater with somapacitan 0.16 mg/kg/week vs daily GH. Mean (SD) change from baseline in HV SDS at week 52 was 4.72 (2.79), 6.14 (3.36), and 8.60 (3.15) for the somapacitan groups, respectively, vs 7.41 (4.08) for daily GH. Model-derived mean (SD) IGF-I SDS for the somapacitan groups was −1.62 (0.86), −1.09 (0.78), and 0.31 (1.06), respectively, vs −0.40 (1.50) observed for daily GH. Safety and tolerability were consistent with the profile of daily GH. Conclusions In children with GHD, once-weekly somapacitan 0.16 mg/kg/week provided the closest efficacy match with similar safety and tolerability to daily GH after 26 and 52 weeks of treatment. A short visual summary of our work is available (1).

Accumulating evidence suggests that kisspeptin-GPR54 signaling is indispensable for gonadotropin-releasing hormone (GnRH)/gonadotropin secretion and consequent reproductive functions in mammals. Conventional Kiss1 knockout (KO) mice and rats are reported to be infertile. To date, however, no study has investigated the effect of inducible central Kiss1 KO/knockdown on pulsatile gonadotropin release in male mammals. Here we report an in vivo analysis of inducible conditional Kiss1 knockdown male mice. The mice were generated by a bilateral injections of either adeno-associated virus (AAV) vectors driving Cre recombinase (AAV-Cre) or AAV vectors driving GFP (AAV-GFP, control) into the hypothalamic arcuate nucleus (ARC) of Kiss1-floxed male mice, in which exon 3 of the Kiss1 gene were floxed with loxP sites. Four weeks after the AAV-Cre injection, the mice showed a profound decrease in the both number of ARC Kiss1-expressing cells and the luteinizing hormone (LH) pulse frequency. Interestingly, pulsatile LH secretion was apparent 8 weeks after the AAV-Cre injection despite the suppression of ARC Kiss1 expression. The control Kiss1-floxed mice infected with AAV-GFP showed apparent LH pulses and Kiss1 expression in the ARC at both 4 and 8 weeks after the AAV-GFP injection. These results with an inducible conditional Kiss1 knockdown in the ARC of male mice suggest that ARC kisspeptin neurons are responsible for pulsatile LH secretion in male mice, and indicate the possibility of a compensatory mechanism that restores GnRH/LH pulse generation.

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.

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.

The present study aimed to evaluate whether novel conditional kisspeptin neuron-specific Kiss1 knockout (KO) mice utilizing the Cre-loxP system could recapitulate the infertility of global Kiss1 KO models, thereby providing further evidence for the fundamental role of hypothalamic kisspeptin neurons in regulating mammalian reproduction. We generated Kiss1-floxed mice and hypothalamic kisspeptin neuron-specific Cre-expressing transgenic mice and then crossed these two lines. The conditional Kiss1 KO mice showed pubertal failure along with a suppression of gonadotropin secretion and ovarian atrophy. These results indicate that newly-created hypothalamic Kiss1 KO mice obtained by the Cre-loxP system recapitulated the infertility of global Kiss1 KO models, suggesting that hypothalamic kisspeptin, but not peripheral kisspeptin, is critical for reproduction. Importantly, these Kiss1-floxed mice are now available and will be a valuable tool for detailed analyses of roles of each population of kisspeptin neurons in the brain and peripheral kisspeptin-producing cells by the spatiotemporal-specific manipulation of Cre expression.

The reproductive performance of cattle can be suppressed by heat stress. Reproductive organ temperature, especially ovarian temperature, may affect follicle development and ovulation. The establishment of a technique for long-term measurement of ovarian temperature could prove useful in understanding the mechanisms underlying the temperature-dependent changes in follicular development and subsequent ovulation in cows. Here we report a novel method facilitating long-term and continuous recording of ovarian parenchymal temperature in cows. The method revealed that the ovarian temperature in the luteal phase was constantly maintained lower than the vaginal temperature, and that the diurnal temperature variation in the ovary was significantly greater than that in the vagina, suggesting that the ovaries may require a lower temperature than other organs to maintain their functions. This novel method could be used for the further understanding of ovarian functions during estrous cycles in cows.

Accumulating evidence suggests that kisspeptin neurons in the arcuate nucleus (ARC), which coexpress neurokinin B and dynorphin, are involved in gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) pulse generation, while the anteroventral periventricular nucleus (AVPV) kisspeptin neurons are responsible for GnRH/LH surge generation. The present study aims to examine whether GnRH(1-5), a GnRH metabolite, regulates LH release via kisspeptin neurons. GnRH(1-5) was intracerebroventricularly injected to ovariectomized and estrogen-treated Wistar-Imamichi female rats. Immediately after the central GnRH(1-5) administration at 2 nmol, plasma LH concentration increased, resulting in significantly higher levels of the area under the curve and baseline of plasma LH concentrations compared to vehicle-injected controls. On the other hand, in Kiss1 knockout rats, GnRH(1-5) administration failed to affect LH secretion, suggesting that the facilitatory effect of GnRH(1-5) on LH release is mediated by kisspeptin neurons. Double in situ hybridization (ISH) for Kiss1 and Gpr101, a GnRH(1-5) receptor gene, revealed that few Kiss1-expressing cells coexpress Gpr101 in both ARC and AVPV. On the other hand, double ISH for Gpr101 and Slc17a6, a glutamatergic marker gene, revealed that 29.2% of ARC Gpr101-expressing cells coexpress Slc17a6. Further, most of the AVPV and ARC Kiss1-expressing cells coexpress Grin1, a gene encoding a subunit of NMDA receptor. Taken together, these results suggest that the GnRH(1-5)-GPR101 signaling facilitates LH release via indirect activation of kisspeptin neurons and that glutamatergic neurons may mediate the signaling. This provides a new aspect of kisspeptin- and GnRH-neuronal communication with the presence of stimulation from GnRH to kisspeptin neurons in female 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

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 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.

Additional file 1.

When people evaluate explanations in uncertain situations, the latent scope bias occurs. It refers to the tendency to perceive explanations that do not include unobservable events as plausible. Previous studies have proposed the inferred evidence account, which states that the bias is caused by underestimating the occurrence probability of unobservable events. Additionally, this account assumes that humans use Bayesian probability reasoning in evaluating such explanations. However, previous studies on this bias have not examined the Bayesian probabilistic reasoning component. This study measured subjective probabilities of explanations and modeled the reasoning process. As a result, it was found that latent scope bias is caused by Bayesian probabilistic reasoning, compared to the inference using psychological utility. The results also suggest that there are considerable individual differences in the occurrence of latent scope bias. These results support the inferred evidence account. Future studies are required to investigate the factors causing such individual differences.

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.

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.

Background:Primary cilia are sensory organelles crucial for organ development. The pivotal structure of the primary cilia is a microtubule that is generated via tubulin polymerization reaction that occurs in the basal body. It remains to be elucidated how molecules with distinct physicochemical properties contribute to the formation of the primary cilia., Results:Here we show that brain expressed X-linked 1 (Bex1) plays an essential role in tubulin polymerization and primary cilia formation. The Bex1 protein shows the physicochemical property of being an intrinsically disordered protein (IDP). Bex1 shows cell density-dependent accumulation as a condensate either in nucleoli at a low cell density or at the apical cell surface at a high cell density. The apical Bex1 localizes to the basal body. Bex1 knockout mice present ciliopathy phenotypes and exhibit ciliary defects in the retina and striatum. Bex1 recombinant protein shows binding capacity to guanosine triphosphate (GTP) and forms the condensate that facilitates tubulin polymerization in the reconstituted system., Conclusions:Our data reveals that Bex1 plays an essential role for the primary cilia formation through providing the reaction field for the tubulin polymerization.

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.

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.

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.

Lowered glucose availability, sensed by the hindbrain, has been suggested to enhance gluconeogenesis and food intake as well as suppress reproductive function. In fact, our previous histological and in vitro studies suggest that hindbrain ependymal cells function as a glucose sensor. The present study aimed to clarify the hindbrain glucose sensor-hypothalamic neural pathway activated in response to hindbrain glucoprivation to mediate counterregulatory physiological responses. Administration of 2-deoxy-D-glucose (2DG), an inhibitor of glucose utilization, into the fourth ventricle (4V) of male rats for 0.5 hour induced messenger RNA (mRNA) expression of c-fos, a marker for cellular activation, in ependymal cells in the 4V, but not in the lateral ventricle, the third ventricle or the central canal without a significant change in blood glucose and testosterone levels. Administration of 2DG into the 4V for 1 hour significantly increased blood glucose levels, food intake, and decreased blood testosterone levels. Simultaneously, the expression of c-Fos protein was detected in the 4V ependymal cells; dopamine β-hydroxylase-immunoreactive cells in the C1, C2, and A6 regions; neuropeptide Y (NPY) mRNA-positive cells in the C2; corticotropin-releasing hormone (CRH) mRNA-positive cells in the hypothalamic paraventricular nucleus (PVN); and NPY mRNA-positive cells in the arcuate nucleus (ARC). Taken together, these results suggest that lowered glucose availability, sensed by 4V ependymal cells, activates hindbrain catecholaminergic and/or NPY neurons followed by CRH neurons in the PVN and NPY neurons in the ARC, thereby leading to counterregulatory responses, such as an enhancement of gluconeogenesis, increased food intake, and suppression of sex steroid secretion.

Ovulation is an essential phenomenon for reproduction in mammalian females along with follicular growth. It is well established that gonadal function is controlled by the neuroendocrine system called the hypothalamus-pituitary-gonadal (HPG) axis. Gonadotropin-releasing hormone (GnRH) neurons, localized in the hypothalamus, had been considered to be the head in governing the HPG axis for a long time until the discovery of kisspeptin. In females, induction of ovulation and folliculogenesis has been linked to a surge mode and pulse mode of GnRH releases, respectively. The mechanisms of how the two modes of GnRH are differently regulated had long remained elusive. The discovery of kisspeptin neurons, distributed in two hypothalamic nuclei, such as the arcuate nucleus in the caudal hypothalamus and preoptic area or the anteroventral periventricular nucleus in the rostral hypothalamic regions, and analyses of the detailed functions of kisspeptin neurons have led marked progress on the understanding of different mechanisms regulating GnRH surges (ovulation) and GnRH pulses (folliculogenesis). The present review will focus on the role of kisspeptin neurons as the GnRH surge generator, including the sexual differentiation of the surge generation system and factors that regulate the surge generator. Comparative aspects between mammalian species are especially focused on.

Pulsatile gonadotropin-releasing hormone (GnRH) secretion is indispensable for reproduction in mammals. Kisspeptin neurons in the hypothalamic arcuate nucleus (ARC), referred to as KNDy neurons because of the coexpression of neurokinin B and dynorphin A, are considered as components of the GnRH pulse generator that produces rhythmic GnRH secretion. The present study aimed to investigate if peripheral administration of PF-4455242, a κ-opioid receptor (KOR, a dynorphin A receptor) antagonist, facilitates pulsatile luteinizing hormone (LH) secretion and GnRH pulse generator activity in estrogen-treated ovariectomized Shiba goats to determine the possibility of using KOR antagonists to artificially control ovarian activities. PF-4455242 was intravenously infused for 4 h (1 or 10 μmol/kg body weight/4 h) or as a single subcutaneous injection (1 or 10 μmol/kg body weight). In a separate experiment, the same KOR antagonist (10 μmol/kg body weight/4 h) was intravenously infused during the recording of multiple unit activity (MUA) in the ARC that reflects the activity of the GnRH pulse generator to test the effects of KOR antagonist administration on GnRH pulse generator activity. Intravenous infusion and single subcutaneous injection of the KOR antagonist significantly increased the frequency of LH pulses compared with controls. Intravenous infusion of KOR antagonist also significantly increased the frequency of episodic bursts in the MUA. The present study demonstrates that peripherally administered KOR antagonist stimulates pulsatile LH secretion by acting on the GnRH pulse generator, and peripheral administration of PF-4455242 can be used to facilitate pulsatile LH secretion, which in turn facilitates ovarian activities in farm animals.

Aberrant exposure to estrogen-like compounds during the critical developmental period may cause improper hypothalamic programming, thus resulting in reproductive dysfunction in adulthood in male mammals. Kisspeptin-neurokinin B-dynorphin A (KNDy) neurons in the arcuate nucleus (ARC) have been suggested to govern tonic GnRH/gonadotropin release to control reproduction in male mammals. In this study, we report that chronic exposure to supraphysiological levels of estrogen during the neonatal period caused an irreversible suppression of KNDy genes in the ARC, resulting in reproductive dysfunction in male rats. Daily estradiol benzoate (EB) administration from days 0 to 10 postpartum caused smaller seminiferous tubules, abnormal spermatogenesis, and a decrease in plasma testosterone in adult male rats. The neonatal EB treatment profoundly suppressed LH pulse and ARC KNDy gene expression at adulthood, but it failed to affect the number of GnRH gene-expressing cells in male rats. The EB treatment failed to affect gene expression of other neuropeptides, such as GHRH, proopiomelanocortin, and agouti-related protein in the ARC, suggesting that ARC KNDy neurons would be a specific target of neonatal estrogen to cause male reproductive dysfunction. Because LH secretory responses to kisspeptin challenge and GnRH expression were spared in male rats with the EB treatment, LH pulse suppression is most probably due to ARC KNDy deficiency. Taken together, the current study indicates that chronic exposure to estrogenic chemicals in the developing brain causes a defect of ARC KNDy neurons, resulting in an inhibition of pulsatile GnRH/LH release and the failure of spermatogenesis and steroidogenesis.

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.

Follicular development and ovulation are profoundly suppressed during lactation in mammals. This suppression is suggested to be mainly due to the suckling-induced inhibition of kisspeptin gene (Kiss1) expression in the arcuate nucleus (ARC) and consequent inhibition of pulsatile GnRH/LH release. We examined whether central somatostatin (SST) signaling mediates the suckling-induced suppression of pulsatile LH secretion. SST has been reported to be expressed in the posterior intralaminar thalamic nucleus (PIL), where the suckling stimulus is postulated to be relayed to the hypothalamus during lactation. SST inhibitory receptors (SSTRs) are abundantly expressed in the ARC, where kisspeptin/neurokinin B/dynorphin A (KNDy) neurons are located. Histological and quantitative studies revealed that the suckling stimulus increased the number of SST-expressing cells in the PIL, and Sstr2 expression in the ARC. Furthermore, a central injection of an SSTR2 antagonist caused a significant increase in pulsatile LH release in lactating rats. Double labeling of Sstr2 and the neurokinin B gene, as a marker for ARC KNDy neurons, showed Sstr2 expression was abundantly detected in the ARC, but few KNDy neurons coexpressed Sstr2 in lactating rats. Taken together, these findings suggest the suckling-induced activation of SST-SSTR2 signaling mediates, at least in part, the suppression of pulsatile LH secretion during lactation in rats, probably via the indirect effects of SST on KNDy neurons. These results provide a new aspect on the role of central SST-SSTR signaling in understanding the mechanism underlying lactational anestrus.

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.

Significance Reports suggest that at least 25% of women suffering from reproductive disorders are due to hypothalamic dysfunction. Pulsatile gonadotropin-releasing hormone (GnRH) regimens are used for infertile women. Thus, elucidation of the GnRH/gonadotropin pulse generator is warranted to improve therapeutic approaches for these disorders. Normal pulsatile gonadotropin release and folliculogenesis were rescued by transfecting the kisspeptin gene ( Kiss1 ) into the hypothalamic neurokinin B (NKB) neurons in global Kiss1 knockout infertile female rats but not by transfecting outside the NKB neurons. Our results demonstrate that kisspeptin/NKB/dynorphin A (KNDy) neurons serve as the GnRH pulse generator, and >20% KNDy neurons are enough to maintain GnRH/gonadotropin pulses and folliculogenesis in female rats. The findings provide a potential therapeutic aspect for hypothalamic reproductive disorders.

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.

Malnutrition suppresses reproductive functions in mammals, which is considered to be mostly due to the inhibition of pulsatile gonadotropin-releasing hormone (GnRH)/gonadotropin secretion. Accumulating evidence suggests that kisspeptin neurons in the arcuate nucleus (ARC) play a critical role in the regulation of pulsatile GnRH/gonadotropin release. The present study aimed to examine if the hypothalamic dynorphin A (Dyn) neurons mediate the suppression of GnRH/luteinizing hormone (LH) pulses during malnutrition. Ovariectomized rats treated with a negative feedback level of estradiol-17β-treated (OVX+E2) were administered with intravenous (iv) or fourth cerebroventricle (4V) 2-deoxy-D-glucose (2DG), an inhibitor of glucose utilization, to serve as a malnutrition model. Central administration of a Dyn receptor antagonist blocked the iv- or 4V-2DG-induced suppression of LH pulses in OVX+E2 rats. The 4V 2DG administration significantly increased the number of Pdyn (Dyn gene)-positive cells co-expressing fos in the paraventricular nucleus (PVN), but not in the ARC and supraoptic nucleus (SON), and the iv 2DG treatment significantly increased the number of fos and Pdyn-co-expressing cells in the PVN and SON, but decreased it in the ARC. The E2 treatment significantly increased Pdyn expression in the PVN, but not in the ARC and SON. Double in situ hybridization for Kiss1 (kisspeptin gene) and Oprk1 (Dyn receptor gene) revealed that around 60% of ARC Kiss1-expressing cells co-expressed Oprk1. These results suggest that the PVN Dyn neurons, at least in part, mediate LH pulse suppression induced by the hindbrain or peripheral glucoprivation, and Dyn neurons may directly suppress the ARC kisspeptin neurons in female rats.

Restoration of spermatogenesis and fertility is a major issue to be solved in male mammals with hypogonadotropic hypogonadism. Kiss1 knockout (KO) male mice are postulated to be a suitable animal model to investigate if hormonal replacement rescues spermatogenesis in mammals with this severe reproductive hormone deficiency, because KO mice replicate the hypothalamic disorder causing hypogonadism. The present study investigated whether testosterone supplementation was able to restore spermatogenesis and in vitro fertilization ability in Kiss1 KO mice. To this end, spermatogenesis, in vitro fertilization ability of Kiss1 KO sperm, and preimplantation development of wild-type embryos inseminated with Kiss1 KO sperm, were examined. The newly generated Kiss1 KO male mice showed infertility with cryptorchidism. Subcutaneous testosterone supplementation for 6 weeks restored plasma and intratesticular testosterone levels, elicited testicular descent, and induced complete spermatogenesis from spermatocytes to elongated spermatids in the testis, resulting in an increase in epididymal sperm number in testosterone-supplemented Kiss1 KO male mice. Epididymal sperm derived from the testosterone-supplemented Kiss1 KO mice showed normal in vitro fertilization ability, and the fertilized eggs showed normal preimplantation development, while the males failed to impregnate females. These results suggest that the failure of spermatogenesis in Kiss1 KO mice is mainly due to a lack of testosterone production, and that Kiss1 KO sperm are capable of fertilizing eggs if the animals receive the appropriate testosterone supplementation without local kisspeptin signaling in the testis and epididymis. Thus, testosterone supplementation would restore spermatogenesis of male mammals showing hypogonadotropic hypogonadism with genetic inactivation of the KISS1/Kiss1 gene.

The present study aimed to evaluate whether novel conditional kisspeptin neuron-specific Kiss1 knockout (KO) mice utilizing the Cre-loxP system could recapitulate the infertility of global Kiss1 KO models, thereby providing further evidence for the fundamental role of hypothalamic kisspeptin neurons in regulating mammalian reproduction. We generated Kiss1-floxed mice and hypothalamic kisspeptin neuron-specific Cre-expressing transgenic mice and then crossed these two lines. The conditional Kiss1 KO mice showed pubertal failure along with a suppression of gonadotropin secretion and ovarian atrophy. These results indicate that newly-created hypothalamic Kiss1 KO mice obtained by the Cre-loxP system recapitulated the infertility of global Kiss1 KO models, suggesting that hypothalamic kisspeptin, but not peripheral kisspeptin, is critical for reproduction. Importantly, these Kiss1-floxed mice are now available and will be a valuable tool for detailed analyses of roles of each population of kisspeptin neurons in the brain and peripheral kisspeptin-producing cells by the spatiotemporal-specific manipulation of Cre expression.

The reproductive performance of cattle can be suppressed by heat stress. Reproductive organ temperature, especially ovarian temperature, may affect follicle development and ovulation. The establishment of a technique for long-term measurement of ovarian temperature could prove useful in understanding the mechanisms underlying the temperature-dependent changes in follicular development and subsequent ovulation in cows. Here we report a novel method facilitating long-term and continuous recording of ovarian parenchymal temperature in cows. The method revealed that the ovarian temperature in the luteal phase was constantly maintained lower than the vaginal temperature, and that the diurnal temperature variation in the ovary was significantly greater than that in the vagina, suggesting that the ovaries may require a lower temperature than other organs to maintain their functions. This novel method could be used for the further understanding of ovarian functions during estrous cycles in cows.

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.

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.

Pulsatile gonadotropin-releasing hormone (GnRH) secretion is essential for regulating reproductive functions in mammals. GnRH pulses are governed by a neural mechanism that is termed the GnRH pulse generator. In the present study, we investigated the role of central calcitonin receptor (CTR) signaling in the regulation of the GnRH pulse generator activity in ovariectomized goats by administering amylin, an endogenous ligand for CTR, into the lateral ventricle. GnRH pulse generator activity was measured using multiple unit activity (MUA) recordings in the mediobasal hypothalamus. We analyzed changes in the interval of characteristic increases in MUA (MUA volleys). The MUA volley interval shortened immediately after amylin administration, followed by prolonged intervals. Double in situ hybridization for KISS1 (kisspeptin gene) and CALCR (CTR gene) revealed that low expression levels of CALCR were found in the arcuate kisspeptin neurons, which is suggested as the main population of neurons, involved in GnRH pulse generator activity. These results suggest that central amylin-CTR signaling has a biphasic role in the regulation of GnRH pulse generator activity by acting on cells other than the arcuate kisspeptin neurons in goats.