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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.

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.

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.

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.

Fusion failure of the Mullerian ducts is thought to occur congenitally in cattle. We aimed to elucidate the contribution of incomplete fusion of the Mullerian ducts to reproductive difficulties in dairy cattle. We observed the vaginas of Holstein cattle to classify the anomalies into mild and severe types, based on severity of incomplete fusion, and recorded information about the cattle at the time of artificial insemination (AI) or embryo transfer. Of the 1054 Holstein cattle examined, 22 (2.09%) individuals showed incomplete fusion of the Mullerian ducts. Among them, 17 (77.3%) had mild type and 5 (22.7%) had severe type incomplete fusion. We analyzed the changes in the prevalence of these anomalies in previous studies and the present study. The prevalence of incomplete fusion of the Mullerian ducts varied from 0% to 6.98% by dairy breed or region. Linear regression analysis showed that the change in the prevalence over time was not statistically significant, with a regression coefficient of −0.04% per year (r2 = 0.27; P = 0.07). The effect of incomplete fusion of the Mullerian ducts on reproductive performance was evaluated by univariate analysis: first service pregnancy rate, number of services, and days from first service to pregnancy were significantly affected in the heifers with the severe types. We next analyzed the effect of incomplete fusion of the Mullerian ducts on conception, using logistic regression analysis. Mild and severe types of incomplete fusion of the Mullerian ducts were selected as explanatory variables, along with heat stress, parity, the number of previous services, AI after ovulation, and sex-sorted semen. The severe types (OR = 0.24, P = 0.03), but not the mild types (OR = 1.01, P = 0.98), were significantly associated with conception. In the present study, we divided the incomplete fusion of the Mullerian ducts by severity and demonstrated that the severe types had a significant effect on poor conception in Holstein cattle. Since the adjusted odds of conception of the severe types of incomplete fusion of the Mullerian ducts were approximately 4 times lower than those of the normal cattle, it is important to determine severe incomplete fusion of the Mullerian ducts prior to service.

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.

Kisspeptin, identified as a natural ligand of GPR54 in 2001, is now considered as a master regulator of puberty and subsequent reproductive functions in mammals. Our previous studies using Kiss1 knockout (KO) rats clearly demonstrated the indispensable role of kisspeptin in gonadotropin-releasing hormone (GnRH)/gonadotropin secretion. In addition, behavioral analyses of Kiss1 KO rats revealed an organizational effect of kisspeptin on neural circuits controlling sexual behaviors. Our studies using transgenic mice carrying a region-specific Kiss1 enhancer-driven reporter gene provided a clue as to the mechanism by which estrogen regulates Kiss1 expression in hypothalamic kisspeptin neurons. Analyses of Kiss1 expression and gonadotropin secretion during the pubertal transition shed light on the mechanism triggering GnRH/gonadotropin secretion at the onset of puberty in rats. Here, we summarize data obtained from the aforementioned studies and revisit the physiological roles of kisspeptin in the mechanism underlying reproductive functions in mammals.

The mammalian olfactory system employs sophisticated mechanisms to detect and recognize an extensive range of smells. In rodents, the olfactory epithelium (OE), situated within the nasal cavity, mainly comprises four defined endoturbinates and several ectoturbinates. Olfactory receptors (ORs) belong to a large family, comprising over 1,000 genes in rodents, which are expressed in olfactory sensory neurons in the OE that detect odor molecules. The rodent OE is divided into four topographically distinct zones, defined by individual OR distribution. However, although the structural complexity and the zonal organization of mammalian OE may contribute to successfully interpreting olfactory information, it remains poorly understood. In this study, we investigated the nasal cavity structure and zonal organization of the OE in goats. Morphological observations revealed that the goat nasal cavity possessed well-developed endoturbinates and ectoturbinates and had a structure similar to that of rodents and sheep, previously reported in other studies. In situ hybridization was used to analyze the expression pattern of ORs, NADPH:quinone oxidoreductase 1, and olfactory cell adhesion molecules as markers of zonal organization in the goat OE. Based on the expression patterns of these genes, we concluded that the goat OE was divided into four zones. The well-developed structure of the nasal cavity and distribution of each OR in the OE were similar to those found in rodents, suggesting that these features were highly conserved between mammals and may have fundamental roles in discriminating among numerous odor molecules in the environment.

The present study aims to examine the effect of tropical temperatures on autonomic nervous activity in Cambodian dairy cattle by analyzing heart rate variability (HRV). Holter-type electrocardiograms were recorded in adult crossbred cows (Cambodian native × Holstein) either in a sheltered area or under direct sunlight. Rectal temperatures and heart rates increased in animals under direct sunlight as compared to those in the shelter. The power spectral analysis of HRV revealed that three out of the five cows studied underwent a decrease in parasympathetic nervous activity under direct sunlight with the remaining two cows showing no apparent change. The HRV analysis would prove to be a useful tool to reveal information about heat tolerance in dairy cows.

Cellular senescence, defined as an irreversible cell cycle arrest, exacerbates the tissue microenvironment. Our previous study demonstrated that mouse uterine senescent cells were physiologically increased according to gestational days and that their abnormal accumulation was linked to the onset of preterm delivery. We hypothesized that there is a mechanism for removal of senescent cells after parturition to maintain uterine function. In the current study, we noted abundant uterine senescent cells and their gradual disappearance in wild-type postpartum mice. F4/80+ macrophages were present specifically around the area rich in senescent cells. Depletion of macrophages in the postpartum mice using anti-F4/80 antibody enlarged the area of senescent cells in the uterus. We also found excessive uterine senescent cells and decreased second pregnancy success rate in a preterm birth model using uterine p53-deleted mice. Furthermore, a decrease in F4/80+ cells and an increase in CD11b+ cells with a senescence-associated inflammatory microenvironment were observed in the p53-deleted uterus, suggesting that uterine p53 deficiency affects distribution of the macrophage subpopulation, interferes with senescence clearance, and promotes senescence-induced inflammation. These findings indicate that the macrophage is a key player in the clearance of uterine senescent cells to maintain postpartum uterine function.

KNDy neurons are named for their co-expression of three neuropeptides, kisspeptin, neurokinin B and dynorphin A (Dyn A). These cells, located in the hypothalamic arcuate nucleus (ARC), are assumed to be the generator of GnRH/LH pulses that control follicular growth, spermatogenesis and steroidogenesis. It is essential to clarify the mechanisms that regulate GnRH pulse generator to understand the reproductive functions in mammals. However, the inhibitory mechanism of GnRH pulse generator by Dyn A is unclear including the location of κ-opioid receptors (KOR; the receptor for Dyn A) in the hypothalamus. To elucidate the mechanism, we analyzed the localization and projections of KOR neurons in rats. We used a lentiviral vector that contains GFP and wheat germ agglutinin (WGA) connected with picornaviral 2A peptide, which are driven by KOR promoter, to visualize KOR neurons. This lentiviral vector was administrated into the region close to the arcuate nucleus (ARC) and ventromedial hypothalamic nucleus (VMH) in female rats. Two weeks after the injection, the rats were sacrificed to analyze the localization and projections of KOR neurons by in situ hybridization (ISH) and immunohistochemistry (IHC). GFP was strongly expressed in cells in the VMH. Double staining of KOR ISH and GFP IHC revealed that all GFP-positive cells were Oprk1 (KOR gene) positive in the VMH, demonstrating that we succeeded to obtain animals that specifically express GFP in KOR neurons. Double staining of GFP and kisspeptin IHC detected the axons of kisspeptin neurons near the cell body of GFP-positive neurons. Likewise, axons of GFP-positive neurons were observed near the Kiss1-positive cells by double staining of GFP IHC and Kiss1 (kisspeptin gene) ISH. In addition, we found axons expressing WGA in the ARC by IHC. Taken together, we found the localization of KOR neurons in the VMH and projections of KOR neurons to KNDy neurons and those of KNDy neurons to KOR neurons as well. These results suggest that KOR neurons in the VMH, which receive Dyn A signal from KNDy neurons, directly inhibit the activity of KNDy neurons. WGA-positive axons in the ARC imply the existence of unknown neurons mediating signals of KOR neurons to KNDy neurons. Our rats fluorescently labeled with KOR neurons will be useful to analyze detailed characteristics of KOR neurons, which will make major progress in elucidating the inhibitory mechanism of reproductive function.

Accumulating evidence suggests that kisspeptin/Gpr54 signaling is indispensable for GnRH/gonadotropin secretion and consequent reproductive functions in mammals. Conventional Kiss1 knockout mice and rats are reported to be infertile. To date, however, no report investigating the effect of inducible central Kiss1 knockout on pulsatile gonadotropin release is available. Here we report in vivo analysis of inducible conditional Kiss1 knockout male mice generated by infection with adeno-associated virus (AAV) vectors driving Cre recombinase (AAV-Cre) in the brain of Kiss1-floxed male mice, in which exon 3 of the Kiss1gene were floxed with LoxP sites. Adult Kiss1-floxed male mice were injected with AAV-Cre or AAV vectors driving GFP (AAV-GFP) bilaterally into the hypothalamic arcuate nucleus (ARC). Four weeks after the AAV-Cre injection, the male mice showed profound decrease in the number of ARC Kiss1-expressing cells and 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 animals infected with AAV-GFP showed apparent LH pulses and Kiss1 expression in the ARC both at 4 or 8 weeks after the AAV-GFP injection. These results suggest that ARC Kiss1-expressing cells are responsible for pulsatile LH secretion in male mice and the possibility of compensation mechanism to restore GnRH/LH pulse generation in central Kiss1 knockout mice. This work was supported in part by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research 18H03973 (to H.T.).

The neurokinin B (NKB)-neurokinin-3 receptor (NK3R) signaling positively regulates the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus. The NK3R-selective antagonists may suppress the reproductive functions of mammals. For development of novel NK3R antagonists with reduced environmental toxicity, a structure-activity relationship study of an NK3R antagonist, talnetant, was carried out. Among several talnetant derivatives with labile functional groups in the natural environment, 3-mercaptoquinoline 2f exhibited a comparable biological activity to that of the parent talnetant. Additionally, compound 2f was converted into the disulfide 3f or isothiazolone 8 by air-oxidation, both of which showed no binding affinity to NK3R.

Elucidating the physiological mechanisms that control reproduction is an obvious strategy for improving the fertility of cattle and developing new agents to control reproductive functions. The present study aimed to identify kisspeptin neurons in the bovine hypothalamus, clarifying that a central mechanism is also present in the cattle brain, as kisspeptin is known to play an important role in the stimulation of gonadotropin-releasing hormone (GnRH)/gonadotropin secretion in other mammals. To characterize kisspeptin neurons in the bovine hypothalamus, the co-localizations of kisspeptin and neurokinin B (NKB) or kisspeptin and dynorphin A (Dyn) were examined. Hypothalamic tissue was collected from Japanese Black or Japanese Black × Holstein crossbred cows during the follicular and luteal phases. Brain sections, including the arcuate nucleus (ARC) and the preoptic area (POA), were dual immunostained with kisspeptin and either NKB or Dyn. In the ARC, both NKB and Dyn were co-localized in kisspeptin neurons during both the follicular and luteal phases, demonstrating the presence of kisspeptin/NKB/Dyn-containing neurons, referred to as KNDy neurons, in cows. In the POA, no co-localization of kisspeptin with either NKB or Dyn was detected. Kisspeptin expression in the follicular phase was higher than that in the luteal phase, suggesting that kisspeptin expression in the POA is positively controlled by estrogen in cows. The kisspeptin neuronal populations in the ARC and POA likely play important roles in regulating the GnRH pulse and surge, respectively, in cows.

Kisspeptin, encoded by KISS1/Kiss1 gene, is now considered a master regulator of reproductive functions in mammals owing to its involvement in the direct activation of gonadotropin-releasing hormone (GnRH) neurons after binding to its cognate receptor, GPR54. Ever since the discovery of kisspeptin, intensive studies on hypothalamic expression of KISS1/Kiss1 and on physiological roles of hypothalamic kisspeptin neurons have provided clues as to how the brain controls sexual maturation at the onset of puberty and subsequent reproductive performance in mammals. Additionally, emerging evidence indicates the potential involvement of extra-hypothalamic kisspeptin in reproductive functions. Here, we summarize data regarding kisspeptin inside and outside the hypothalamus and revisit the physiological roles of central and peripheral kisspeptins in the reproductive functions of mammals.

Sulfhydryl oxidation is part of the sperm maturation process essential for the acquisition of sperm fertilization competency and its structural stabilization; however, the specific sulfhydryl oxidases that fulfill these roles have yet to be identified. In this study, we investigate the potential involvement of one atypical thiol oxidase family called quiescin Q6/sulfhydryl oxidase (QSOX) using the mouse epididymis as our model system. With multidisciplinary approaches, we show that QSOX isoform 1 and 2 exhibit complementary distribution throughout the epididymal duct, but that each variant possesses distinct subcellular localization within the epididymal principal cells. While QSOX2 was exclusively present in the Golgi apparatus of the caput and corpus epididymis, QSOX1c, the most profusely express QSOX1 variant, was abundantly present in the cauda luminal fluids. Moreover, immunohistochemistry studies together with proteomic identification in isolated epididymosomes provided evidence substantiating the release of QSOX2, but not QSOX1c, via an apocrine secretory pathway. Furthermore, we demonstrate for the first time, distinct association of QSOX1c and QSOX2 with the sperm acrosome and implantation fossa, during different stages of their epididymal maturation. In conclusion, our study provides the first comprehensive comparisons between QSOX1 and QSOX2 in the mouse epididymis, revealing their distinct epididymal distribution, cellular localization, mechanisms of secretion and sperm membrane association. Together, these data suggest that QSOX1 and QSOX2 have discrete biological functions in male germ cell development.Summary SentenceMouse epididymal sulfhydryl oxidases exhibit complementary tissue distribution with disparate cellular localization, the distinct secretory mechanism and sperm membrane association suggesting their participation in different sperm maturation processes.

The mammalian olfactory system employs sophisticated mechanisms to detect and recognize an extensive range of smells. In rodents, the olfactory epithelium (OE), situated within the nasal cavity, mainly comprises four defined endoturbinates and several ectoturbinates. Olfactory receptors (ORs) belong to a large family, comprising over 1,000 genes in rodents, which are expressed in olfactory sensory neurons in the OE that detect odor molecules. The rodent OE is divided into four topographically distinct zones, defined by individual OR distribution. However, although the structural complexity and the zonal organization of mammalian OE may contribute to successfully interpreting olfactory information, it remains poorly understood. In this study, we investigated the nasal cavity structure and zonal organization of the OE in goats. Morphological observations revealed that the goat nasal cavity possessed well-developed endoturbinates and ectoturbinates and had a structure similar to that of rodents and sheep, previously reported in other studies. In situ hybridization was used to analyze the expression pattern of ORs, NADPH:quinone oxidoreductase 1, and olfactory cell adhesion molecules as markers of zonal organization in the goat OE. Based on the expression patterns of these genes, we concluded that the goat OE was divided into four zones. The well-developed structure of the nasal cavity and distribution of each OR in the OE were similar to those found in rodents, suggesting that these features were highly conserved between mammals and may have fundamental roles in discriminating among numerous odor molecules in the environment.

We quantified the transpiration cooling effect by comparing a living climber greenwall (CGW) against a real but non-transpiring one after confirming the cessation of transpiration and similar vegetation coverages of both of them. We severed stems of plants in one of the CGWs to stop transpiration. By monitoring the changes in transpiration rate, leaf temperature, and percentage coverages of plants with and without severance of stems, we tried to confirm the cessation of transpiration and similar vegetation coverages. In parallel with it, we measured the indoor thermal environments in the well-covered houses of the two treatments, as well as a non-covered house, and calculated the transpiration cooling effect. The transpiration rates of plants with stem severance dropped to less than 1.0 mmol m −2 s −1 within 35 min after severance. Almost all leaf temperatures in the severed treatment were similar to the dry reference leaf temperatures. In contrast, those in the non-severed treatment remained the same as before severance. In addition, the percentage points of percentage coverages between the living CGW and the real but non-transpiring one was reduced to just 1.5% two days after severance. Thus, we quantified the transpiration cooling effect of CGWs using the indoor thermal environmental data two days after severance. The average transpiration cooling effect calculated from the indoor wall surface temperatures (=severed plant-covered house − intact plant-covered house) was 0.23 °C, although the average shading cooling effect (=non-covered house − severed plant-covered house) was 8.55 °C under global solar radiation on a vertical south surface between 400 and 600 W m −2 . In addition, those calculated from room temperatures under the same environmental conditions were 0.15 and 4.00 °C, respectively. Therefore, we concluded that the transpiration cooling effect of CGWs, which were separately set from the walls, on the indoor thermal environment of houses was much smaller than the shading cooling effect. Using these methodologies, we recommend further investigation of the appropriate distance between CGWs and walls to increase the transpiration cooling effect.

Ependymocytes are one of the energy-sensing cells that regulate animal reproduction through their responsiveness to changes in extracellular glucose levels and the expression of pancreatic-type glucokinase and glucose transporter 2, which play a critical role in sensing blood glucose levels in pancreatic β-cells. Molecular mechanisms underlying glucose sensing in the ependymocytes remain poorly understood. The AMP-activated protein kinase (AMPK), a serine/threonine kinase highly conserved in all eukaryotic cells, has been suggested to be an intracellular fuel gauge that detects cellular energy status. The present study aims to clarify the role AMPK of the lower brainstem ependymocytes has in sensing glucose levels to regulate reproductive functions. First, we will show that administration of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, an AMPK activator, into the 4th ventricle suppressed pulsatile LH release in female rats. Second, we will demonstrate the presence of AMPK catalytic subunit immunoreactivities in the rat lower brainstem ependymocytes. Third, transgenic mice were generated to visualize the ependymocytes with Venus, a green fluorescent protein, expressed under the control of the mouse vimentin promoter for further in vitro study. The Venus-labeled ependymocytes taken from the lower brainstem of transgenic mice revealed that AMPK activation by 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, an AMPK activator, increased in vitro intracellular calcium concentrations. Taken together, malnutrition-induced AMPK activation of ependymocytes of the lower brainstem might be involved in suppression of GnRH/LH release and then gonadal activities.

Kisspeptin, encoded by the Kiss1 gene, has attracted attention as a key candidate neuropeptide in controlling puberty and reproduction via regulation of gonadotrophin-releasing hormone (GnRH) secretion in mammals. Pioneer studies with Kiss1 or its cognate receptor Gpr54 knockout (KO) mice showed the indispensable role of kisspeptin-GPR54 signalling in the control of animal reproduction, although detailed analyses of gonadotrophin secretion, especially pulsatile and surge-mode of luteinising hormone (LH) secretion, were limited. Thus, in the present study, we have generated Kiss1 KO rats aiming to evaluate a key role of kisspeptin in governing reproduction via pulse and surge modes of GnRH/LH secretion. Kiss1 KO male and female rats showed a complete suppression of pulsatile LH secretion, which is responsible for folliculogenesis and spermatogenesis, and an absence of puberty and atrophic gonads. Kiss1 KO female rats showed no spontaneous LH/follicle-stimulating hormone surge and an oestrogen-induced LH surge, suggesting that the GnRH surge generation system, which is responsible for ovulation, does not function without kisspeptin. Furthermore, challenge of major stimulatory neurotransmitters, such as monosodium glutamate, NMDA and norepinephrine, failed to stimulate LH secretion in Kiss1 KO rats, albeit they stimulated LH release in wild-type controls. Taken together, the results of the present study confirm that kisspeptin plays an indispensable role in generating two modes (pulse and surge) of GnRH/gonadotrophin secretion to regulate puberty onset and normal reproductive performance. In addition, the present study suggests that kisspeptin neurones play a critical role as a hub integrating major stimulatory neural inputs to GnRH neurones, using newly established Kiss1 KO rats, which serve as a useful model for detailed analysis of hormonal profiles.

Pulsatile secretion of GnRH plays a pivotal role in follicular development via stimulating tonic gonadotropin secretion in mammals. Kisspeptin neurons, located in the arcuate nucleus (ARC), are considered to be an intrinsic source of the GnRH pulse generator. The present study aimed to determine ARC-specific enhancer(s) of the Kiss1 gene by an in vivo reporter assay. Three green fluorescent protein (GFP) reporter constructs (long, medium length, and short) were generated by insertion of GFP cDNA at the Kiss1 locus. Transgenic female mice bearing the long and medium-length constructs showed apparent GFP signals in kisspeptin-immunoreactive cells in both the ARC and anteroventral periventricular nucleus, in which another population of kisspeptin neurons are located. On the other hand, transgenic mice bearing 5′-truncated short construct showed few GFP signals in the ARC kisspeptin-immunoreactive cells, whereas they showed colocalization of GFP- and kisspeptin-immunoreactivities in the anteroventral periventricular nucleus. In addition, chromatin immunoprecipitation and chromosome conformation capture assays revealed recruitment of unoccupied estrogen receptor-α in the 5′-upstream region and intricate chromatin loop formation between the 5′-upstream and promoter regions of Kiss1 locus in the ARC. Taken together, the present results indicate that 5′-upstream region of Kiss1 locus plays a critical role in Kiss1 gene expression in an ARC-specific manner and that the recruitment of estrogen receptor-α and formation of a chromatin loop between the Kiss1 promoter and the 5′ enhancer region may be required for the induction of ARC-specific Kiss1 gene expression. These results suggest that the 5′-upstream region of Kiss1 locus functions as an enhancer for ARC Kiss1 gene expression in mice.

Pulsatile secretion of gonadotrophin-releasing hormone (GnRH)/luteinising hormone is indispensable for the onset of puberty and reproductive activities at adulthood in mammalian species. A cohort of neurones expressing three neuropeptides, namely kisspeptin, encoded by the Kiss1 gene, neurokinin B (NKB) and dynorphin A, localised in the hypothalamic arcuate nucleus (ARC), so-called KNDy neurones, comprises a putative intrinsic source of the GnRH pulse generator. Synchronous activity among KNDy neurones is considered to be required for pulsatile GnRH secretion. It has been reported that gap junctions play a key role in synchronising electrical activity in the central nervous system. Thus, we hypothesised that gap junctions are involved in the synchronised activities of KNDy neurones, which is induced by NKB-NK3R signalling. We determined the role of NKB-NK3R signalling in Ca2+ oscillation (an indicator of neuronal activities) of KNDy neurones and its synchronisation mechanism among KNDy neurones. Senktide, a selective agonist for NK3R, increased the frequency of Ca2+ oscillations in cultured Kiss1-GFP cells collected from the mediobasal hypothalamus of the foetal Kiss1-green fluorescent protein (GFP) mice. The senktide-induced Ca2+ oscillations were synchronised in the Kiss1-GFP and neighbouring glial cells. Confocal microscopy analysis of these cells, which have shown synchronised Ca2+ oscillations, revealed close contacts between Kiss1-GFP cells, as well as between Kiss1-GFP cells and glial cells. Dye coupling experiments suggest cell-to-cell communication through gap junctions between Kiss1-GFP cells and neighbouring glial cells. Connexin-26 and -37 mRNA were found in isolated ARC Kiss1 cells taken from adult female Kiss1-GFP transgenic mice. Furthermore, 18β-glycyrrhetinic acids and mefloquine, which are gap junction inhibitors, attenuated senktide-induced Ca2+ oscillations in Kiss1-GFP cells. Taken together, these results suggest that NKB-NK3R signalling enhances synchronised activities among neighbouring KNDy neurones, and that both neurone-neurone and neurone-glia communications via gap junctions possibly contribute to synchronised activities among KNDy neurones.

Guinea pigs are important zoo animals and have been recommended for animal-assisted activities or therapy, however there are problems concerning testosterone inducing aggressive or sexual behaviors in male guinea pigs. Testicular testosterone secretion is regulated by pulsatile gonadotropin releasing hormone (GnRH)/luteinizing hormone (LH) release in mammals. The mechanism generating GnRH/LH pulses is thought to be governed by kisspeptin neurons, which coexpress neurokinin B (NKB) and dynorphin A (Dyn), in the arcuate nucleus (ARC). Kisspeptin neurons in the ARC are frequently referred to as KNDy neurons. The purpose of this study was to examine whether the antagonization of NKB-neurokinin-3 receptor (NK3R) signaling can manipulate testosterone secretion in male guinea pigs. A single subcutaneous administration or 7 days of oral administration of an NK3R-selective antagonist, SB223412 (50 mg/body), significantly decreased plasma testosterone levels in male guinea pigs. In vitro binding assays confirmed that SB223412 has a high affinity to guinea pig NK3R. These results suggest that SB223412 could be used as an orally-available compound to suppress testosterone levels in male guinea pigs. Double labeling in situ hybridization of kisspeptin and either NKB or Dyn showed that kisspeptin-expressing neurons contained NKB (77.9%) or Dyn (62.3%) in the ARC, suggesting the presence of KNDy neurons in the ARC of guinea pigs. In conclusion, the present study shows that SB223412 could be a candidate compound to suppress testosterone secretion in male guinea pigs for controlling sexual and aggressive behaviors in the species.

The oestrogen-induced luteinising hormone (LH) surge is evident in male primates, including humans, whereas male rodents never show the LH surge, even when treated with a preovulatory level of oestrogen. This suggests that the central mechanism governing reproductive hormones in primates is different from that in rodents. The present study aimed to investigate whether male Japanese monkeys conserve a brain mechanism mediating the oestrogen-induced LH surge via activation of kisspeptin neurones. Adult male and female Japanese monkeys were gonadectomised and then were treated with oestradiol-17β for 2 weeks followed by a bolus injection of oestradiol benzoate. Both male and female monkeys showed an oestrogen-induced LH surge. In gonadectomised monkeys sacrificed just before the anticipated time of the LH surge, oestrogen treatment significantly increased the number of KISS1-expressing cells in the preoptic area (POA) and enhanced the expression of c-fos in POA KISS1-positive cells of males and females. The oestrogen treatment failed to induce c-fos expression in the arcuate nucleus (ARC) kisspeptin neurones in both sexes just prior to LH surge onset. Thus, kisspeptin neurones in the POA but not in the ARC might be involved in the positive-feedback action of oestrogen that induces LH surge in male Japanese monkeys, as well as female monkeys. The present results indicate that oestrogen-induced activation of POA kisspeptin neurones may contribute to the LH surge generation in both sexes. The conservation of the LH surge generating system found in adult male primates, unlike rodents, could be a result of the capability of oestrogen to induce POA kisspeptin expression and activation.

This study aimed to evaluate a technique to cool livestock buildings by covering it with kudzu vine ( Pueraria lobata ). Specifically, we first examined whether one-year-old nursery stocks of kudzu that were planted in spring can cover livestock buildings with standard size by summer in Japan. Then, we quantified the cooling effects of changes of kudzu vine covering on the room temperatures of fabricated livestock buildings under field conditions. Our results showed that the vines reached the roof of standard-sized livestock buildings by summer. The estimated average percentage coverages and the sorted room temperature reductions (RTRs) showed positively significant relationships under the global solar radiation on a horizontal surface greater than 0.2 kW m −2 . The maximum sorted RTR was 3.44 °C, when the estimated average percentage coverage was 43.9%. Thus, we conclude that the technique to cool livestock buildings using one-year-old nursery stocks of kudzu vine is applicable to livestock buildings.

Kisspeptins are neuropeptides that induce the secretion of gonadotropin-releasing hormone via the activation of the cognate receptor, G-protein coupled receptor 54 (GPR54). The kisspeptin-GPR54 axis is associated with the onset of puberty and the maintenance of the reproductive system. In this study, several fluorescent probes have been designed and synthesized for rat GPR54 through the modification of the N-terminus of rat kisspeptins to allow for the visualization of the expression and localization of kisspeptin receptor(s) in living cells and native tissues. The tetramethylrhodamine (TMR) and rhodamine green (RG)-labeled kisspeptins exhibited good binding and agonistic activities towards GPR54, and the results of the application studies demonstrated that these fluorescent probes could be used effectively for the detection of GPR54 receptors in flow cytometry and confocal microscopy experiments.

Hypothalamo-pituitary-gonadal axis has been recognized as a functional unit orchestrating reproductive performance in mammals. The mechanism governing puberty onset, however, is still one of the great mysteries of biology. The present article reviews previous studies over the last century, which gradually elucidated the brain mechanism controlling puberty onset in mammals along with the discovery of gonadotrophins and the gonadotrophin-releasing hormone and the establishment of the concept for the hypothalamo-pituitary-gonadal axis. The discovery of kisspeptin and subsequent progress of neuroendocrinology in the last decade has resulted in much greater understanding of the way the brain governs puberty onset in mammals.

Puberty is associated with an increase in gonadotropin secretion as a result of an increase in gonadotropin-releasing hormone (GnRH) secretion. Kisspeptin is considered to play a key role in puberty onset in many mammalian species, including rodents, ruminants and primates. The present study aimed to determine if changes in hypothalamic expression of the KISS1 gene, encoding kisspeptin, are associated with the onset of puberty in pigs. The animals (n=4 in each group) were perfused with 4% paraformaldehyde at 0, 1, 2, 3 and 4 months old, as prepubertal stages, and at 5 months old, as the peripubertal stage, following each blood sampling. KISS1 gene expressions in coronal sections of brains were visualized by in situ hybridization. Plasma luteinizing hormone (LH) was measured by radioimmunoassay. KISS1 mRNA signals were observed in the arcuate nucleus (ARC) at all ages examined without any significant difference in the number of KISS1-expressing cells, indicating that the KISS1 gene is constantly expressed in the ARC throughout pubertal development in pigs. The plasma LH concentration was the highest in 0-month-old piglets and significantly decreased in the 1- and 2 month-old groups (P

Green facades have long been used to reduce the overheating of buildings by excessive solar gain, but little research attention has been paid to the plant traits that reduce the surface temperature of urban structures. This study aimed to identify the key traits contributing to the cooling effects of green facades by comparing five vine plant species. The vines were trained to climb a plastic net that was attached on the freestanding walls under outdoor conditions. One of the walls was not covered with plants to serve as control. We measured the longest vine length, total number of leaves, percentage coverage, leaf transpiration rate, leaf solar transmittance, wall surface temperatures, global solar radiation on a vertical south surface (GSR) and ambient temperature. We identified the percentage coverage as the key trait determining the overall cooling effect: a statistically positive relationship (P

Female rats show a gonadotrophin-releasing hormone (GnRH)/luteinising hormone (LH) surge in the presence of a preovulatory level of oestrogen, whereas males do not because of brain defeminisation during the developmental period by perinatal oestrogen converted from androgen. The present study aimed to identify the site(s) of oestrogen action and the critical period for defeminising the mechanism regulating the GnRH/LH surge. Animals given perinatal treatments, such as steroidal manipulations, brain local implantation of oestradiol (E(2) ) or administration of an NMDA antagonist, were examined for their ability to show an E(2) -induced LH surge at adulthood. Lordosis behaviour was examined to compare the mechanisms defeminising the GnRH/LH surge and sexual behaviour. A single s.c. oestradiol-benzoate administration on either the day before birth (E21), the day of birth (D0) or day 5 (D5) postpartum completely abolished the E(2) -induced LH surge at adulthood in female rats, although the same treatment did not inhibit lordosis. Perinatal castration on E21 or D0 partially rescued the E2-induced LH surge in genetically male rats, whereas castration from E21 to D5 totally rescued lordosis. Neonatal E(2) implantation in the anterior hypothalamus including the anteroventral periventricular nucleus (AVPV)/preoptic area (POA) abolished the E(2) -induced LH surge in female rats, whereas E(2) implantation in the mid and posterior hypothalamic regions had no inhibitory effect on the LH surge. Lordosis was not affected by neonatal E(2) implantation in any hypothalamic regions. In male rats, neonatal NMDA antagonist treatment rescued lordosis but not the LH surge. Taken together, these results suggest that an anterior hypothalamic region such as the AVPV/POA region is a perinatal site of oestrogen action where the GnRH/LH regulating system is defeminised to abolish the oestrogen-induced surge. The mechanism for defeminisation of the GnRH/LH surge system might be different from that of sexual behaviour, in terms of the site(s) of oestrogen action and critical period, as well as the neurotransmitter system involved.

Kisspeptin is a key molecule that stimulates gonadotropin secretion via release of gonadotropin-releasing hormone (GnRH). In the present study, our aim was to investigate whether kisspeptin has stimulatory effects on follicular development via GnRH/gonadotropin secretion in cows. Japanese Black beef cows were intravenously injected with full-length bovine kisspeptin [Kp-53 (0.2 or 2 nmol/kg)] or vehicle 5 days after they exhibited standing estrus (Day 0). In cows injected with Kp-53 at 2 nmol/kg, the follicular sizes of the first dominant follicles increased on Day 6 and thereafter. Ovulation of the first dominant follicle occurred in 1 out of 4 cows treated with Kp-53 at 2 nmol/kg. Injection of Kp-53 at 2 nmol/kg increased the concentration of plasma luteinizing hormone (LH) but not follicle-stimulating hormone, over a 4-h period following injection in all cows. The present study suggests that administration of full-length kisspeptin causes LH secretion, which is sustained for a few hours, and it is capable of stimulating follicular development and/or ovulation.

Neurokinin B (NKB), encoded by TAC3, is thought to be an important accelerator of pulsatile gonadotropin-releasing hormone release. This study aimed to clarify the transcriptional regulatory mechanism of goat TAC3. First, we determined the full-length mRNA sequence of goat TAC3 from the hypothalamus to be 820 b, including a 381 b coding region, with the putative transcription start site located 143-b upstream of the start codon. The deduced amino acid sequence of NKB, which is produced from preproNKB, was completely conserved among goat, cattle, and human. Next, we cloned 5’-upstream region of goat TAC3 up to 3400 b from the translation initiation site, and this region was highly homologous with cattle TAC3 (89%). We used this goat TAC3 5’-upstream region to perform luciferase assays. We created a luciferase reporter vector containing DNA constructs from –2706, –1837, –834, –335, or –197 to +166 bp (the putative transcription start site was designated as +1) of goat TAC3 and these were transiently transfected into mouse hypothalamus-derived N7 cells and human neuroblastoma-derived SK-N-AS cells. The luciferase activity gradually increased with the deletion of the 5’-upstream region, suggesting that the transcriptional suppressive region is located between –2706 and –336 bp and that the core promoter exists downstream of –197 bp. Estradiol treatment did not lead to significant suppression of luciferase activity of any constructs, suggesting the existence of other factor(s) that regulate goat TAC3 transcription.

Olfactory stimuli play an important role in regulating reproductive functions in mammals. The present study investigated the effect of olfactory signals derived from male rats on kisspeptin neuronal activity and luteinizing hormone (LH) secretion in female rats. Wistar-Imamichi strain female rats were ovariectomised (OVX) and implanted with preovulatory levels of oestradiol-17β (E2). OVX+E2 rats were sacrificed 1 hour after exposure to either: clean bedding, female-soiled bedding or male-soiled bedding. Dual staining for Kiss1 mRNA in situ hybridization and c-Fos immunohistochemistry revealed that the numbers of Kiss1-expressing cells and c-Fos-immunopositive Kiss1-expressing cells in the anteroventral periventricular nucleus (AVPV) were significantly higher in OVX+E2 rats exposed to male-soiled bedding than those of the other groups. No significant difference was found in the number of c-Fos-immunopositive Kiss1-expressing cells in the arcuate nucleus and c-Fos-immunopositive Gnrh1-expressing cells between the groups. The number of c-Fos-immunopositive cells was also significantly higher in the limbic system consisting of several nuclei, such as the bed nucleus of the stria terminalis, the cortical amygdala and the medial amygdala, in OVX+E2 rats exposed to male-soiled bedding than the other groups. OVX+E2 rats exposed to male-soiled bedding showed apparent LH surges, and the peak of the LH surge and area under the curve of LH concentrations in the OVX+E2 group were significantly higher than those of the other two groups. These results suggest that olfactory signals derived from male rats activate AVPV kisspeptin neurones, likely via the limbic system, resulting in enhancement of the peak of the LH surge in female rats. Taken together, the present study suggests that AVPV kisspeptin neurones are a target of olfactory signals to modulate LH release in female rats. This article is protected by copyright. All rights reserved.

Exposure to estrogen during the developmental period causes reproductive dysfunction in mammals, because the developing brain is highly sensitive to estrogens. In the present study, we report that long-term exposure to supraphysiological doses of estrogen during the neonatal critical period causes irreversible suppression of Kiss1/kisspeptin expression in the arcuate nucleus (ARC) via estrogen receptor-alpha (ERα) and ERβ, resulting in reproductive dysfunction in female rats. Daily estradiol-benzoate (EB) administration from days 0 to 10 postpartum caused persistent vaginal diestrus in female rats. The female rats showed profound suppression of pulsatile luteinizing hormone (LH) release and ARC Kiss1/kisspeptin expression even after ovariectomy at adulthood. In contrast, female rats treated with a single EB injection at day 5 postpartum exhibited persistent vaginal estrus and showed comparable LH pulses and numbers of ARC Kiss1-expressing cells to vehicle-treated controls after ovariectomy at adulthood. Because the LH secretory response to exogenous kisspeptin was spared in female rats with neonatal long-term estrogen exposure, the LH pulse suppression was most probably due to ARC kisspeptin deficiency. Furthermore, neonatal estrogen might act through both ERα and ERβ, because EB exposure significantly reduced the number of ARC Kiss1-expressing cells in wild-type mice but not in ERα or ERβ knockout mice. Taken together, long-term exposure to supraphysiological doses of estrogen in the developing brain might cause defects in ARC kisspeptin neurons via ERα and ERβ, resulting in inhibition of pulsatile LH release and lack of estrous cyclicity.

In mammals, maturation of the gonadal axis at puberty appears to be timed by an increase in tonic gonadotropin release from the anterior lobe of pituitary gland, where the synthesis and release of gonadotropins, such as the follicle-stimulating hormone and luteinizing hormone, are stimulated by the gonadotropin-releasing hormone (GnRH) released from the hypothalamus. The concept of hypothalamic regulation of the gonadal axis dates back to the middle of last century, and our understanding for the GnRH/gonadotropin-releasing system has become abundantly clearer from studies in the last few decades or so. To date, the most plausible interpretation is that KNDy neurons, which produce kisspeptin, neurokinin B, and dynorphin A, serve as a master regulator of tonic GnRH/gonadotropin release, which governs the pubertal maturation of gonadal activity, i.e., gametogenesis and steroidogenesis. Estrogen derived from the immature ovary appears to play a critical role in prepubertal restraint of kisspeptin biosynthesis and hence GnRH/gonadotropin release in rodents and sheep. Decreased sensitivity to estrogen negative feedback during the pubertal transition is likely a key event for pubertal maturation of the hypothalamic mechanism regulating GnRH and then gonadal activity. It is noteworthy that steroid dependency differs from species to species. In primates, steroid-independent inhibition is mainly underlying the prepubertal restraint of GnRH/gonadotropin-releasing system. In all mammalian species, the timing of puberty onset is dependent on body weight rather than chronological age. Thus, hypothalamic mechanisms underlying the relationship between nutritional statuses and relieving the prepubertal restraint of kisspeptin biosynthesis are still unanswered questions.

Follicular development and ovulation are strongly suppressed during lactation in mammals via a profound suppression of gonadotrophin secretion. The present study aimed to examine the role of oestrogen feedback action in suppressing luteinising hormone (LH) secretion and hypothalamic kisspeptin expression during the latter half of lactation. Plasma LH concentrations kept at low levels throughout the lactating period in intact and oestrogen-replaced ovariectomised (OVX) lactating rats, whereas plasma LH concentrations gradually elevated from day 10 postpartum in lactating OVX rats. OVX lactating rats showed frequent LH pulses at late lactation, although the LH pulses were significantly inhibited by an oestrogen replacement, which is much less effective on LH release in nonlactating rats. Oestrogen replacement in lactating OVX rats significantly reduced the number of Kiss1 mRNA-expressing cells in the arcuate nucleus (ARC) at late lactation, although the same oestrogen treatment did not affect the number of Kiss1-expressing cells in nonlactating controls. Exogenous kisspeptin challenge (0.2 nmol) into the third cerebroventricle significantly increased LH secretion in lactating OVX, lactating OVX + subcutaneous 17β-oestradiol and intact lactating rats at day 16 postpartum. These results suggest that LH pulse suppression during late lactation could be a result of the enhanced oestrogen-dependent suppression of ARC kisspeptin expression.

kisspeptins that are encoded by kiss1 gene are now considered the key regulator of reproduction from a number of studies in mammals. In most vertebrates, a paralogue of kiss1, called kiss2, is also present, and the functional significance of kisspeptins is not known precisely. In the present study, we have cloned kiss2 from a perciform teleost, the red seabream Pagrus major. The amino acid sequence deduced from the red seabream kiss2 contained a highly conserved 10-amino-acid residue, Kiss2(10) or kp-10. A kiss1-like transcript was also identified, but it appears to be non-functional due to the presence of a "premature" stop codon. Neurons that express kiss2 mRNA were distributed in the dorsal (NRLd) and ventral (NRLv) parts of nucleus recessi lateralis in the hypothalamus. In some fish a few kiss2-expressing neurons were detected in the preoptic area and nucleus ventralis tuberis. The number of kiss2-expressing neurons in the NRLd was larger during the first spawning season in both males and females compared with fish in the post-spawning periods. In males the number of kiss2 neurons in the NRLd of maturing fish was also larger than those in the post-spawning periods. In males the number of kiss2 neurons in the NRLv showed a similar pattern of changes to that of NRLd, while significant changes were not detected for females. The numbers of gonadotropin-releasing hormone 1 (GnRH1)-immunoreactive neurons in the preoptic area showed a similar pattern of change as those of kiss2 cells of the NRLd in both males and females (and also the NRLv in males). These results are in good agreement with the hypothesis that kiss2 neurons are involved in pubertal processes via regulatory influences on GnRH1 neurons in red seabream.

The present study investigated whether kisspeptin–G protein-coupled receptor 54 (GPR54) signaling plays a role in mediating mating-induced ovulation in the musk shrew ( Suncus murinus ), a reflex ovulator. For this purpose, we cloned suncus Kiss1 and Gpr54 cDNA from the hypothalamus and found that suncus kisspeptin (sKp) consists of 29 amino acid residues (sKp-29). Injection of exogenous sKp-29 mimicked the mating stimulus to induce follicular maturation and ovulation. Administration of several kisspeptins and GPR54 agonists also induced presumed ovulation in a dose-dependent manner, and Gpr54 mRNA was distributed in the hypothalamus, showing that kisspeptins induce ovulation through binding to GPR54. The sKp-29–induced ovulation was blocked completely by pretreatment with a gonadotropin-releasing hormone (GnRH) antagonist, suggesting that kisspeptin activates GnRH neurons to induce ovulation in the musk shrew. In addition, in situ hybridization revealed that Kiss1 -expressing cells are located in the medial preoptic area (POA) and arcuate nucleus in the musk shrew hypothalamus. The number of Kiss1 -expressing cells in the POA or arcuate nucleus was up-regulated or down-regulated by estradiol, suggesting that kisspeptin neurons in these regions were the targets of the estrogen feedback action. Finally, mating stimulus largely induced c-Fos expression in Kiss1 -positive cells in the POA, indicating that the mating stimulus activates POA kisspeptin neurons to induce ovulation. Taken together, these results indicate that kisspeptin–GPR54 signaling plays a role in the induction of ovulation in the musk shrew, a reflex ovulator, as it does in spontaneous ovulators.