Abstract. Metastin/kisspeptin, the KiSS-1 gene product, has been identified as an endogenous ligand of GPR54 that reportedly regulates GnRH/LH surges and estrous cyclicity in female rats. The aim of the present study was to determine if metastin/kisspeptin neurons are a target of estrogen positive feedback to induce GnRH/LH surges. We demonstrated that preoptic area (POA) infusion of the antirat metastin/kisspeptin monoclonal antibody blocked the estrogen-induced LH surge, indicating that endogenous metastin/kisspeptin released around the POA mediates the estrogen positive feedback effect on GnRH/LH release. Metastin/kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) may be responsible for mediating the feedback effect because the percentage of c-Fosexpressing KiSS-1 mRNA-positive cells to total KiSS-1 mRNA-positive cells was significantly higher in the afternoon than in the morning in the anteroventral periventricular nucleus (AVPV) of high estradiol (E2)-treated females. The percentage of c-Fos-expressing metastin/kisspeptin neurons was not different between the afternoon and morning in the arcuate nucleus (ARC). Most of the KiSS-1 mRNA expressing cells contain ERα immunoreactivity in the AVPV and ARC. In addition, AVPV KiSS-1 mRNA expressions were highest in the proestrous afternoon and lowest in the diestrus 1 in females and were increased by estrogen treatment in ovariectomized animals. On the other hand, the ARC KiSS-1 mRNA expressions were highest at diestrus 2 and lowest at proestrous afternoon and were increased by ovariectomy and decreased by high estrogen treatment. Males lacking the surge mode of GnRH/LH release showed no obvious cluster of metastin/kisspeptin-immunoreactive neurons in the AVPV when compared with high E2-treated females, which showed a much greater density of these neurons. Taken together, the present study demonstrates that the AVPV metastin/kisspeptin neurons are a target of estrogen positive feedback to induce GnRH/LH surges in female rats.

Ovulation is caused by a sequence of neuroendocrine events: GnRH and LH surges that are induced by positive feedback action of estrogen secreted by the mature ovarian follicles. The central mechanism of positive feedback action of estrogen on GnRH/LH secretion, however, is not fully understood yet. The present study examined whether metastin, the product of metastasis suppressor gene KiSS-1, is a central neuropeptide regulating GnRH/LH surge and then estrous cyclicity in the female rat. Metastin had a profound stimulation on LH secretion by acting on the preoptic area (POA), where most GnRH neurons projecting to the median eminence are located, because injection of metastin into the third ventricle or POA increased plasma LH concentrations in estrogen-primed ovariectomized rats. Metastin neurons were immunohistochemically found in the arcuate nucleus (ARC) to be colocalized with estrogen receptors with some fibers in the preoptic area (POA) in close apposition with GnRH neuronal cell bodies or fibers. Quantitative RT-PCR has revealed that KiSS-1 and GPR54 mRNAs were expressed in the ARC and POA, respectively. The blockade of local metastin action in the POA with a specific monoclonal antibody to rat metastin completely abolished proestrous LH surge and inhibited estrous cyclicity. Metastin-immunoreactive cell bodies in the ARC showed a marked increase and c-Fos expression in the early proestrus afternoon compared with the day of diestrus. Thus, metastin released in the POA is involved in inducing the preovulatory LH surge and regulating estrous cyclicity.

Pulsatile release of gonadotrophin-releasing hormone (GnRH) is indispensable to maintain normal gonadotrophin secretion. The pulsatile secretion of GnRH is associated with synchronised electrical activity in the mediobasal hypothalamus (i.e. multiple unit activity; MUA), which is considered to reflect the rhythmic oscillations in the activity of the neuronal network that drives pulsatile GnRH secretion. However, the cellular source of this ultradian rhythm in GnRH activity is unknown. Direct input from kisspeptin neurones in the arcuate nucleus (ARC) to GnRH cell bodies in the medial preoptic area or their terminals in the median eminence could be the intrinsic source for driving the GnRH pulse generator. To determine whether kisspeptin signalling could be responsible for producing pulsatile GnRH secretion, we studied goats, measured plasma levels of luteinising hormone (LH) and recorded MUA in the posterior ARC, where the majority of kisspeptin neuronal cell bodies are located. Rhythmic volleys of MUA were found to be accompanied by LH pulses with regular intervals in the ARC, where kisspeptin neuronal cell bodies were found. Exogenous administration of kisspeptin stimulated a sustained increase in LH secretion, without influencing MUA, suggesting that the GnRH pulse generator, as reflected by MUA, originated from outside of the network of GnRH neurones, and could plausibly reflect the pacemaker activity of kisspeptin neurones, whose projections reach the median eminence where GnRH fibres project. These observations suggest that the kisspeptin neurones in the ARC may be the intrinsic source of the GnRH pulse generator.

Administration of leptin during undernutrition improves reproductive function, but whether this occurs at the level of the brain, pituitary, or gonads is not yet clear. The present study tested the hypothesis that one important mechanism is the control of pulsatile gonadotropin-releasing hormone (GnRH) secretion. Our approach was to determine if leptin could prevent the marked suppression of pulsatile luteinizing hormone (LH) secretion which occurs during fasting. Leptin (3 µg/g i.p.; three times/48 h) or vehicle was administered during a 48-hour fast in adult ovariectomized and estrogen-treated ovariectomized rats (n = 5–7/group). LH was measured in blood samples collected every 6 min for 2 h before and after fasting. In vehicle-treated animals, plasma insulin and leptin levels decreased after fasting. As expected, the LH pulse frequency also decreased markedly. When circulating leptin remained artificially elevated during fasting, the suppression of LH pulse frequency did not occur. Leptin treatment maintained a high LH pulse frequency in the presence or absence of estrogen. The finding that leptin modulates LH pulse frequency indicates that this fat-derived hormone conveys information about nutrition to mechanisms which regulate pulsatile gonadotropin-releasing hormone secretion. Because this occurs in the absence of estrogen, the mechanism does not necessarily involve modulation of negative feedback.

Recently, a novel physiologically active peptide, kisspeptin (metastin), has been reported to facilitate sexual maturation and ovulation by directly stimulating GnRH neurons in several mammalian species. Despite its importance in the neuroendocrine regulation of reproduction, kisspeptin neurons have only been studied in mammals, and there has been no report on the kisspeptin or kisspeptin neuronal systems in nonmammalian vertebrates. We used medaka for the initial identification of the KiSS-1 gene and the anatomical distribution of KiSS-1 mRNA expressing neurons (KiSS-1 neurons) in the brain of nonmammalian species. In situ hybridization for the medaka KiSS-1 gene cloned here proved that two kisspeptin neuronal populations are localized in the hypothalamic nuclei, the nucleus posterioris periventricularis and the nucleus ventral tuberis (NVT). Furthermore, NVT KiSS-1 neurons were sexually dimorphic in number (male neurons >> female neurons) under the breeding conditions. We also found that the number of KiSS-1 neurons in the NVT but not that in the nucleus posterioris periventricularis was positively regulated by ovarian estrogens. The fact that there were clear differences in the number of NVT KiSS-1 neurons between the fish under the breeding and nonbreeding conditions strongly suggests that the steroid-sensitive changes in the KiSS-1 mRNA expression in the NVT occur physiologically, according to the changes in the reproductive state. From the present results, we conclude that the medaka KiSS-1 neuronal system is involved in the central regulation of reproductive functions, and, given many experimental advantages, the medaka brain may serve as a good model system to study its physiology.

The evolution of brain infarction after transient focal cerebral ischemia was studied in mice using multiparametric imaging techniques. One-hour focal cerebral ischemia was induced by occluding the middle cerebral artery using the intraluminal filament technique. Cerebral protein synthesis (CPS) and the regional tissue content of adenosine triphosphate (ATP) were measured after recirculation times from 0 hours to 3 days. The observed changes were correlated with the expression of the mRNAs of hsp-70, c-fos, and junB, as well as the distribution of DNA double-strand breaks, visualized by TUNEL. At the end of 1 hour of ischemia, protein synthesis was suppressed in a larger tissue volume than ATP in accordance with the biochemical differentiation between core and penumbra. Hsp70 mRNA was selectively expressed in the cortical penumbra, whereas c-fos and junB mRNAs were increased both in the lateral part of the penumbra and in the ipsilateral cingulate cortex with normal metabolism. During reperfusion after withdrawal of the intraluminal filament, suppression of CPS persisted except in the most peripheral parts of the middle cerebral artery territory, in which it recovered between 6 hours and 3 days. ATP, in contrast, returned to normal levels within 1 hour but secondarily deteriorated from 3 hours on until, between 1 and 3 days, the ATP-depleted area merged with that of suppressed protein synthesis leading to delayed brain infarction. Hsp70 mRNA, but not c-fos and junB, was strongly expressed during reperfusion, peaking at 3 hours after reperfusion. TUNEL-positive cells were detected from 3 hours on, mainly in areas with secondary ATP depletion. These results stress the importance of an early recovery of CPS for the prevention of ischemic injury and suggest that TUNEL is an unspecific response of delayed brain infarction.

The brain mechanism regulating gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release is sexually differentiated in rodents. Kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) have been suggested to be sexually dimorphic and involved in the GnRH/LH surge generation. The present study aimed to determine the significance of neonatal testicular androgen to defeminize AVPV kisspeptin expression and the GnRH/LH surge-generating system. To this end, we tested whether neonatal castration feminizes AVPV kisspeptin neurons and the LH surge-generating system in male rats and whether neonatal estradiol benzoate (EB) treatment suppresses the kisspeptin expression and the LH surge in female rats. Immunohistochemistry, in situ hybridization, and quantitative real-time RT-PCR were performed to investigate kisspeptin and Kiss1 mRNA expressions. Male rats were castrated immediately after birth, and females were treated with EB on postnatal Day 5. Neonatal castration caused an increase in AVPV kisspeptin expression at peptide and mRNA levels in the genetically male rats, and the animals showed surge-like LH release in the presence of the preovulatory level of estradiol (E2) at adulthood. On the other hand, neonatal EB treatment decreased the number of AVPV kisspeptin neurons and caused an absence of E2-induced LH surge in female rats. Semiquantitative RT-PCR analysis showed that neonatal steroidal manipulation affects Kiss1 expression but does not significantly affect gene expressions of neuropeptides (neurotensin and galanin) and enzymes or transporter for neurotransmitters (gamma-aminobutyric acid, glutamate, and dopamine) in the AVPV, suggesting that the manipulation specifically affects Kiss1 expressions. Taken together, our present results provide physiological evidence that neonatal testicular androgen causes the reduction of AVPV kisspeptin expression and failure of LH surge in genetically male rats. Thus, it is plausible that perinatal testicular androgen causes defeminization of the AVPV kisspeptin system, resulting in the loss of the surge system in male rats.

Kisspeptin, a peptide encoded by the Kiss1 gene, has been considered as a potential candidate for a factor triggering the onset of puberty, and its expression in the hypothalamus was found to increase during peripubertal period in rodent models. The present study aimed to clarify the oestrogenic regulation of peripubertal changes in Kiss1 mRNA expression in the anteroventral periventricular nucleus (AVPV) and hypothalamic arcuate nucleus (ARC), and to determine which population of kisspeptin neurones shows a change in kisspeptin expression parallel to that in luteinising hormone (LH) pulses at the peripubertal period. Quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry revealed an apparent increase in the ARC Kiss1 mRNA expression and kisspeptin immunoreactivity around the time of vaginal opening in intact female rats. The AVPV Kiss1 mRNA levels also increased at day 26, but decreased at day 31, and then increased at day 36/41. In ovariectomised (OVX) rats, ARC Kiss1 mRNA expression did not show peripubertal changes and was kept at a high level throughout peripubertal periods. Apparent LH pulses were found in these prepubertal OVX rats. Oestradiol replacement suppressed ARC Kiss1 mRNA expression in OVX prepubertal rats, but not in adults. Similarly, LH pulses were suppressed by oestradiol in the prepubertal period (days 21 and 26), but regular pulses were found in adulthood. The present study suggests that a pubertal increase of Kiss1/kisspeptin expression both in the ARC and AVPV is involved in the onset of puberty. These results also suggest that both LH pulses and ARC Kiss1 expression are more negatively regulated by oestrogen in prepubertal female rats compared to adult rats.