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In mammals, secretion of tonic (pulsatile) gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) is often suppressed during lactation. Suppression of GnRH/LH pulses in lactating dams is assumed to be caused by suckling stimuli and a chronic negative energy balance due to milk production. The present study aimed to investigate whether the central enkephalin-δ opioid receptor (DOR) signaling mediated the suppression of LH secretion by acute suckling stimuli and/or chronic negative energy balance due to milk production in rats during late lactation when dams were under a heavy energy demand. On postpartum day 16, the number of Penk (enkephalin mRNA)-expressing cells in the arcuate nucleus was significantly higher in lactating rats than in non-lactating control rats. Pulsatile LH secretion was suppressed in rats with chronic suckling or acute 1-h suckling stimuli 6 h after pup removal on day 16 of lactation. Central DOR antagonism significantly increased the mean LH concentrations and the baseline of LH pulses in rats with chronic suckling but not with acute suckling stimuli on day 16 of lactation. Besides, central κ opioid receptor (KOR) antagonism increased the amplitude of LH pulses in rats with the acute suckling stimuli on day 16 of lactation. These results suggest that central DOR signaling mediates the suppression of LH secretion caused by a negative energy balance in rats receiving chronic suckling during late lactation. On the other hand, central KOR signaling likely mediates acute suckling stimuli-induced suppression of LH secretion in rats during late lactation.

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

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

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.

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.

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.

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.

Protein–protein interactions (PPIs) are recognized as important targets in drug discovery. The characteristics of molecules that inhibit PPIs differ from those of small-molecule compounds. We developed a novel chemical library database system (DLiP) to design PPI inhibitors. A total of 32,647 PPI-related compounds are registered in the DLiP. It contains 15,214 newly synthesized compounds, with molecular weight ranging from 450 to 650, and 17,433 active and inactive compounds registered by extracting and integrating known compound data related to 105 PPI targets from public databases and published literature. Our analysis revealed that the compounds in this database contain unique chemical structures and have physicochemical properties suitable for binding to the protein–protein interface. In addition, advanced functions have been integrated with the web interface, which allows users to search for potential PPI inhibitor compounds based on types of protein–protein interfaces, filter results by drug-likeness indicators important for PPI targeting such as rule-of-4, and display known active and inactive compounds for each PPI target. The DLiP aids the search for new candidate molecules for PPI drug discovery and is available online (https://skb-insilico.com/dlip).

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.

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.

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.

Accumulating evidence suggests that KNDy neurons located in the hypothalamic arcuate nucleus (ARC), which are reported to express kisspeptin, neurokinin B, and dynorphin A, are indispensable for the gonadotropin-releasing hormone (GnRH) pulse generation that results in rhythmic GnRH secretion. The aims of the present study were to investigate the effects of peripheral administration of the neurokinin 3 receptor (NK3R/TACR3, a receptor for neurokinin B) antagonist, SB223412, on GnRH pulse-generating activity and pulsatile luteinizing hormone (LH) secretion in ovariectomized Shiba goats treated with luteal phase levels of estrogen. The NK3R antagonist was infused intravenously for 4 h {0.16 or 1.6 mg/(kg body weight [BW]·4 h)} during which multiple unit activity (MUA) in the ARC was recorded, an electrophysiological technique commonly employed to monitor GnRH pulse generator activity. In a separate experiment, the NK3R antagonist (40 or 200 mg/[kg BW·day]) was administered orally for 7 days to determine whether the NK3R antagonist could modulate pulsatile LH secretion when administered via the oral route. Intravenous infusion of the NK3R antagonist significantly increased the interval of episodic bursts of MUA compared with that of the controls. Oral administration of the antagonist for 7 days also significantly prolonged the interpulse interval of LH pulses. The results of this study demonstrate that peripheral administration of an NK3R antagonist suppresses pulsatile LH secretion by acting on the GnRH pulse generator, suggesting that NK3R antagonist administration could be used to modulate reproductive functions in ruminants.

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.

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.

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.

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

The discovery of the role of kisspeptin neurons in the regulation of mammalian reproduction in 2003 was one of the biggest breakthroughs in reproductive endocrinology within the last few decades. Research during the past two decades since the discovery of kisspeptin has been unveiling the mechanism of how the hypothalamic kisspeptin neurons control reproductive functions through regulation of gonadotropin-releasing hormone (GnRH) secretion. This article aims to overview kisspeptin research, including the most recent studies from ours and other research groups, and to discuss the possibility of new strategies to control reproductive functions in farm animals. In the first section, we introduce the critical role of kisspeptin neurons in puberty onset and reproductive functions in mammals, including the regulation of two modes of GnRH/gonadotropin secretion, namely pulsatile and surge modes. The next section focuses more on the mechanism of how the kisspeptin neurons in the arcuate nucleus in the hypothalamus precisely controls GnRH pulse using other two neuropeptides, neurokinin B and dynorphin A. The article also discusses the mechanism suppressing reproductive function during lactation and other stress conditions through inhibition of kisspeptin neurons and consequent GnRH/gonadotropin secretion, to provide insights on the possibility of new strategies to control reproductive performance in domestic farm animals.

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.

Hindbrain ependymocytes are postulated to have a glucose-sensing role in regulating gonadal functions. Previous studies have suggested that malnutrition-induced suppression of gonadotropin secretion is mediated by noradrenergic inputs from the A2 region in the solitary tract nucleus to the paraventricular nucleus (PVN), and by corticotropin-releasing hormone (CRH) release in the hypothalamus. However, no morphological evidence to indicate the neural pathway from the hindbrain ependymocytes to hypothalamic kisspeptin neurons, a center for reproductive function in mammals, currently exists. The present study aimed to examine the existence of a neuronal pathway from the hindbrain ependymocytes to kisspeptin neurons in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV). To determine this, wheat-germ agglutinin (WGA), a trans-synaptic tracer, was injected into the fourth ventricle (4V) in heterozygous Kiss1-tandem dimer Tomato (tdTomato) rats, where kisspeptin neurons were visualized by tdTomato fluorescence. 48 h after the WGA injection, brain sections were taken from the forebrain, midbrain and hindbrain and subjected to double immunohistochemistry for WGA and dopamine β-hydroxylase (DBH) or CRH. WGA immunoreactivities were found in vimentin-immunopositive ependymocytes of the 4V and the central canal (CC), but not in the third ventricle. The WGA immunoreactivities were detected in some tdTomato-expressing cells in the ARC and AVPV, DBH-immunopositive cells in the A1–A7 noradrenergic nuclei, and CRH-immunopositive cells in the PVN. These results suggest that the hindbrain ependymocytes have neuronal connections with the kisspeptin neurons, most probably via hindbrain noradrenergic and CRH neurons to relay low energetic signals for regulation of reproduction.

Abstract Purpose Hypothalamic kisspeptin neurons are considered to play a critical role in regulating mammalian reproduction and integrating humoral and neuronal inputs that control gonadotropin‐releasing hormone (GnRH)/gonadotropin release. The present study aimed to investigate the upstream regulator candidates for kisspeptin neurons. Methods Visualized kisspeptin neurons that were taken from the arcuate nucleus (ARC) of Kiss1‐tdTomato rats were subjected to next‐generation sequencing (NGS) analysis. In situ hybridization (ISH) for the calcitonin receptor gene (Calcr) was performed throughout the whole forebrain of ovariectomized wild‐type female rats that had been implanted with a negative feedback level of estrogen, because the Calcr expression was evident in the ARC kisspeptin neurons from the NGS analysis. Then, a double ISH was performed for the Calcr and kisspeptin gene (Kiss1) in the brain regions, containing either the anteroventral periventricular nucleus (AVPV) or ARC of the female rats. Results The NGS analysis revealed that the Calcr was highly expressed in the ARC kisspeptin neurons. It was found that the Calcr was co‐expressed in 12% and 22% of the Kiss1‐expressing cells in the ARC and AVPV, respectively. Conclusion The present study suggests that calcitonin receptor signaling could be involved in the regulation of reproductive function through the direct control of the ARC and/or AVPV kisspeptin neurons, and then GnRH/gonadotropin release.

Pubertal onset is thought to be timed by an increase in pulsatile gonadotropin-releasing hormone (GnRH)/gonadotropin secretion in mammals. The underlying mechanism of pubertal onset in mammals is still an open question. Evidence accumulated in the last 15 years suggests that kisspeptin/neurokinin B/dynorphin A (KNDy) neurons in the hypothalamic arcuate nucleus play a key role in pubertal onset by triggering pulsatile GnRH/gonadotropin secretin in mammals. Specifically, KNDy neurons are now considered a part of GnRH pulse generator, in which neurokinin B facilitates and dynorphin A inhibits, the synchronized discharge of KNDy neurons in autocrine and/or paracrine manners. Kisspeptin serves as a potent secretagogue of GnRH secretion and thus its release is fundamental to pubertal increase in GnRH/gonadotropin secretion in mammals. Proposed mechanisms inhibiting Kiss1 (kisspeptin gene) expression during childhood to juvenile varies from species to species: we envisage that negative feedback action of estrogen plays a key role in the inhibition of Kiss1 expression in KNDy neurons in rodents and sheep, whereas estrogen-independent inhibition of kisspeptin secretion by γ-amino butyric acid or neuropeptide Y are suggested to be responsible for the pre-pubertal suppression of GnRH/gonadotropin secretion in primates. Taken together, the timing of pubertal onset is postulated to be controlled by upstream regulators for kisspeptin biosynthesis and secretion in mammals.

Objectives: In 2014, immediately prior to the revision of Article 25-2 of the Pharmacists’ Act, we conducted a survey on pharmacists’ and patients’ perceptions of pharmacists’ roles. A role discrepancy between the two was identified. The objective was to examine changes in role perceptions and awareness of pharmacists as medication specialists following revision to the Pharmacists’ Act. Methods: The survey was conducted using an Internet-based questionnaire. A total of 469 patients and 354 pharmacists responded to 12 questions about the perceived roles of pharmacists. Results: Analysis revealed that the only evaluation that changed as a result of revisions was pharmacists’ role as “family or regular pharmacist,” with scores dropping by about half. As in 2014, the high rating rate for pharmacists surpassed the high rating of patients for all other items. The greatest discrepancy in role perception was observed for the same three items (“Understanding the effects of the drugs the patients are taking,” “Understanding the health changes caused by the drugs dispensed to the patients,” and “Consciously protecting patients from the adverse effects of drugs”) as 2014. Conclusion: A major role discrepancy continues to exist between patients and pharmacists, and it is necessary for pharmacists to take on a more advanced role in patient care. Results suggest that pharmacists must monitor changes in patients’ lifestyles and provide clear explanations for patients to rate them highly as medication specialists.

Distinct classes of SOX10 mutations result in peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, Waardenburg syndrome, and Hirschsprung disease, collectively known as PCWH. Meanwhile, SOX10 haploinsufficiency caused by allelic loss-of-function mutations leads to a milder non-neurological disorder, Waardenburg–Hirschsprung disease. The cellular pathogenesis of more complex PCWH phenotypes in vivo has not been thoroughly understood. To determine the pathogenesis of PCWH, we have established a transgenic mouse model. A known PCWH-causing SOX10 mutation, c.1400del12, was introduced into mouse Sox10-expressing cells by means of bacterial artificial chromosome (BAC) transgenesis. By crossing the multiple transgenic lines, we examined the effects produced by various copy numbers of the mutant transgene. Within the nervous systems, transgenic mice revealed a delay in the incorporation of Schwann cells in the sciatic nerve and the terminal differentiation of oligodendrocytes in the spinal cord. Transgenic mice also showed defects in melanocytes presenting as neurosensory deafness and abnormal skin pigmentation, and a loss of the enteric nervous system. Phenotypes in each lineage were more severe in mice carrying higher copy numbers, suggesting a gene dosage effect for mutant SOX10. By uncoupling the effects of gain-of-function and haploinsufficiency in vivo, we have demonstrated that the effect of a PCWH-causing SOX10 mutation is solely pathogenic in each SOX10-expressing cellular lineage in a dosage-dependent manner. In both the peripheral and central nervous systems, the primary consequence of SOX10 mutations is hypomyelination. The complex neurological phenotypes in PCWH patients likely result from a combination of haploinsufficiency and additive dominant effect.

Smoking Cessation Treatment (SCT) is a policy that has to be promoted for health economics, and expectations for the success of treatments with varenicline (VAR) are large. However, the Food and Drug Administration (FDA) have issued a warning on VAR-induced depression and suicide. In the present study, utilizing the FDA Adverse Event Reporting System (FAERS), we searched for antidepressants (ADs) used during SCT that cause fewer suicide-related events (SRE) (Study 1). We also investigated whether VAR concomitantly administered with ADs increases the risk of SRE (Study 2). In addition, we investigated whether the use of VAR alone is a latent risk factor of SRE. The backgrounds of cases with and without SRE were matched using the Propensity Score. In Study 1, the highest integrated Reporting Odds Ratio (iROR) was noted in concomitantly administered mirtazapine (iROR 6.98; 95% Confidence Interval (CI) 1.57-30.99), while the lowest ratio was noted in concomitantly administered amitriptyline (iROR 0.59; iROR95%CI 0.23-1.50). Study 2 clarified that SCT increases the risk of SRE in AD-treated cases (iROR 8.02; iROR95%CI 5.47-11.76; not significance). Of ADs concomitantly used during SCT with VAR, amitriptyline and mirtazapine showed the lowest and highest risks, respectively (Study 1). It was clarified that concomitant use of VAR in the treatment of depression with ADs increased the risk of SRE (Study 2). The results of Studies 1 and 2 suggested that the use of VAR alone is a latent risk factor inducing suicide.

Minoru Tomita, Naoko Inoue, Tadahiko TsuruShinagawa LASIK Center, Tokyo, JapanPurpose: To report on keratoectasia after laser in situ keratomileusis (LASIK) in two eyes of two patients who had no obvious preoperative risk factors, and treatment of keratoectasia by intrastromal corneal ring segments.Method: Clinical interventional case report.Results: Two patients underwent LASIK in both eyes at Shinagawa LASIK center, Tokyo. Preoperative standard ophthalmological examinations demonstrated no specific risk factors of keratoectasia. However, each eye of the two patients developed keratoectasia as defined by corneal topography. Treatment for keratoectasia was performed by inserting intrastromal corneal ring segments (ICRS) in the affected eyes. In one eye, KeraRing (MEDIPHACOS, Ltd, Brazil) was inserted, and Intacs SK (Addition Technology Inc, USA) was inserted in the other.Conclusion: Even in eyes where no risk factors for keratoectasia were found following thorough preoperative examinations, keratoectasia could develop after LASIK. ICRS could be effective for the treatment of keratoectasia after LASIK.Keywords: keratoectasia, LASIK, intrastromal corneal ring segments