Reproductive Hormone-Dependent and -Independent Contributions to Developmental Changes in Kisspeptin in GnRH-Deficient Hypogonadal Mice

Kisspeptin is a potent activator of GnRH-induced gonadotropin secretion and is a proposed central regulator of pubertal onset. In mice, there is a neuroanatomical separation of two discrete kisspeptin neuronal populations, which are sexually dimorphic and are believed to make distinct contributions to reproductive physiology. Within these kisspeptin neuron populations, Kiss1 expression is directly regulated by sex hormones, thereby confounding the roles of sex differences and early activational events that drive the establishment of kisspeptin neurons. In order to better understand sex steroid hormone-dependent and -independent effects on the maturation of kisspeptin neurons, hypogonadal (hpg) mice deficient in GnRH and its downstream effectors were used to determine changes in the developmental kisspeptin expression. In hpg mice, sex differences in Kiss1 mRNA levels and kisspeptin immunoreactivity, typically present at 30 days of age, were absent in the anteroventral periventricular nucleus (AVPV). Although immunoreactive kisspeptin increased from 10 to 30 days of age to levels intermediate between wild type (WT) females and males, corresponding increases in Kiss1 mRNA were not detected. In contrast, the hpg arcuate nucleus (ARC) demonstrated a 10-fold increase in Kiss1 mRNA between 10 and 30 days in both females and males, suggesting that the ARC is a significant center for sex steroid-independent pubertal kisspeptin expression. Interestingly, the normal positive feedback response of AVPV kisspeptin neurons to estrogen observed in WT mice was lost in hpg females, suggesting that exposure to reproductive hormones during development may contribute to the establishment of the ovulatory gonadotropin surge mechanism. Overall, these studies suggest that the onset of pubertal kisspeptin expression is not dependent on reproductive hormones, but that gonadal sex steroids critically shape the hypothalamic kisspeptin neuronal subpopulations to make distinct contributions to the activation and control of the reproductive hormone cascade at the time of puberty.     

Comparative analysis of kisspeptin-immunoreactivity reveals genuine differences in the hypothalamic Kiss1 systems between rats and mice

Kiss1 mRNA and its corresponding peptide products, kisspeptins, are expressed in two restricted brain areas of rodents, the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC). The concentration of mature kisspeptins may not directly correlate with Kiss1 mRNA levels, because mRNA translation and/or posttranslational modification, degradation, transportation and release of kisspeptins could be regulated independently of gene expression, and there may thus be differences in kisspeptin expression even in species with similar Kiss1 mRNA profiles. We measured and compared kisspeptin-immunoreactivity in both nuclei and both sexes of rats and mice and quantified kisspeptin-immunoreactive nerve fibers. We also determined Kiss1 mRNA levels and measured kisspeptin-immunoreactivity in colchicine pretreated rats. Overall, we find higher levels of kisspeptin-immunoreactivity in the mouse compared to the rat, independently of brain region and gender. In the female mouse AVPV high numbers of kisspeptin-immunoreactive neurons were present, while in the rat, the female AVPV displays a similar number of kisspeptin-immunoreactive neurons compared to the level of Kiss1 mRNA expressing cells, only after axonal transport inhibition. Interestingly, the density of kisspeptin innervation in the anterior periventricular area was higher in female compared to male in both species. Species differences in the ARC were evident, with the mouse ARC containing dense fibers, while the rat ARC contains clearly discernable cells. In addition, we show a marked sex difference in the ARC, with higher kisspeptin levels in females. These findings show that the translation of Kiss1 mRNA and/or the degradation/transportation/release of kisspeptins are different in mice and rats.     

Sex steroid control of hypothalamic Kiss1 expression in sheep and rodents: comparative aspects

In recent years, the Kiss1 gene has been cast into the reproductive spotlight. In the short period since the discovered link between kisspeptins, the encoded peptides of Kiss1, and fertility, these peptides are now known to be critical for the neuroendocrine control of reproduction. Kisspeptin producing cells in the hypothalamus are poised to become the 'missing link' in the sex steroid feedback control of GnRH secretion. These cells contain all the necessary components to relay information of the sex steroid environment to GnRH neurons, which possess the kisspeptin receptor, GPR54. Sex steroids regulate Kiss1 mRNA, and kisspeptin expression in the hypothalamus, in a manner consistent with both negative and positive feedback control of GnRH. The precise nature of sex steroid effects, in particular those of estrogen, on Kiss1 expression have been extensively studied in the female rodent and ewe. In the arcuate nucleus (ARC) of both species, kisspeptin cells appear to forward signals pertinent to negative feedback regulation of GnRH, although in the ewe it appears this population of Kiss1 cell is also responsible for positive feedback regulation of GnRH at the time of the preovulatory GnRH/LH surge. In rodents, these positive feedback signals appear to be mediated by kisspeptin cells exclusively within the anteroventral periventricular nucleus (AVPV). There are no Kiss1 cells in the ovine AVPV, but there is a population in the preoptic area. The role these preoptic area cells play in the sex steroid feedback regulation of GnRH secretion, if any, is yet to be revealed.     

Sexual differentiation and the Kiss1 system: hormonal and developmental considerations

The nervous system (both central and peripheral) is anatomically and physiologically differentiated between the sexes, ranging from gender-based differences in the cerebral cortex to motoneuron number in the spinal cord. Although genetic factors may play a role in the development of some sexually differentiated traits, most identified sex differences in the brain and behavior are produced under the influence of perinatal sex steroid signaling. In many species, the ability to display an estrogen-induced luteinizing hormone (LH) surge is sexually differentiated, yet the specific neural population(s) that allows females but not males to display such estrogen-mediated "positive feedback" has remained elusive. Recently, the Kiss1/kisspeptin system has been implicated in generating the sexually dimorphic circuitry underlying the LH surge. Specifically, Kiss1 gene expression and kisspeptin protein levels in the anteroventral periventricular (AVPV) nucleus of the hypothalamus are sexually differentiated, with females displaying higher levels than males, even under identical hormonal conditions as adults. These findings, in conjunction with accumulating evidence implicating kisspeptins as potent secretagogues of gonadotropin-releasing hormone (GnRH), suggest that the sex-specific display of the LH surge (positive feedback) reflects sexual differentiation of AVPV Kiss1 neurons. In addition, developmental kisspeptin signaling via its receptor GPR54 appears to be critical in males for the proper sexual differentiation of a variety of sexually dimorphic traits, ranging from complex social behavior to specific forebrain and spinal cord neuronal populations. This review discusses the recent data, and their implications, regarding the bi-directional relationship between the Kiss1 system and the process of sexual differentiation.     

Kisspeptins in reproductive biology: consensus knowledge and recent developments

Kisspeptins, a family of neuropeptides encoded by the Kiss1 gene that are mainly expressed in discrete neuronal populations of the hypothalamus, have recently emerged as essential upstream regulatory elements of GnRH (gonadotropin-releasing hormone) neurons and, thereby, potent elicitors of gonadotropin secretion. Indeed, kisspeptins are now recognized as important regulators of key aspects of the maturation and function of the reproductive axis, including the sexual differentiation of the brain, the timing of puberty, the adult regulation of gonadotropin secretion by gonadal hormones, and the control of fertility by metabolic and environmental (e.g., photoperiod) cues. Appreciation of these fundamental biological features has led to the contention that kisspeptins are indispensable elements of the reproductive brain whose relevance goes beyond their crucial physiological roles and may pose potential pathophysiological and therapeutic interest. In spite of such a consensus, recent developments in the field have helped to expand, and somewhat challenged, our current understanding of the neuroendocrine and molecular mechanisms whereby some of the effects of kisspeptins are conducted. This review aims to provide a synoptic and balanced account of the consensus knowledge and recent findings in the field of kisspeptin physiology, which we predict will be crucial in shaping the progress of our understanding of the roles played by this family of neuropeptides in reproductive biology.     

Inotropic action of the puberty hormone kisspeptin in rat, mouse and human: cardiovascular distribution and characteristics of the kisspeptin receptor

Kisspeptins, the ligands of the kisspeptin receptor known for its roles in reproduction and cancer, are also vasoconstrictor peptides in atherosclerosis-prone human aorta and coronary artery. The aim of this study was to further investigate the cardiovascular localisation and function of the kisspeptins and their receptor in human compared to rat and mouse heart. Immunohistochemistry and radioligand binding techniques were employed to investigate kisspeptin receptor localisation, density and pharmacological characteristics in cardiac tissues from all three species. Radioimmunoassay was used to detect kisspeptin peptide levels in human normal heart and to identify any pathological changes in myocardium from patients transplanted for cardiomyopathy or ischaemic heart disease. The cardiac function of kisspeptin receptor was studied in isolated human, rat and mouse paced atria, with a role for the receptor confirmed using mice with targeted disruption of Kiss1r. The data demonstrated that kisspeptin receptor-like immunoreactivity localised to endothelial and smooth muscle cells of intramyocardial blood vessels and to myocytes in human and rodent tissue. [¹²⁵I]KP-14 bound saturably, with subnanomolar affinity to human and rodent myocardium (K_D = 0.12 nM, human; K_D = 0.44 nM, rat). Positive inotropic effects of kisspeptin were observed in rat, human and mouse. No response was observed in mice with targeted disruption of Kiss1r. In human heart a decrease in cardiac kisspeptin level was detected in ischaemic heart disease. Kisspeptin and its receptor are expressed in the human, rat and mouse heart and kisspeptins possess potent positive inotropic activity. The cardiovascular actions of the kisspeptins may contribute to the role of these peptides in pregnancy but the consequences of receptor activation must be considered if kisspeptin receptor agonists are developed for use in the treatment of reproductive disorders or cancer.     

Time-related effects of various LED light spectra on reproductive hormones in the brain of the goldfish Carassius auratus

In this study, we examined differences among the gonadotropin-inhibitory hormone (GnIH), kisspeptin 1 (Kiss1), Kiss 1 receptor (G-protein-coupled receptor 54; GPR54), melatonin receptor 1 (MT1), and melatonin levels in brain cells of goldfish Carassius auratus exposed to white fluorescent light and three light-emitting diode (LED) wavelength and melatonin treatments in the culture medium. In the green and blue LED treatment groups, GnIH and MT1 mRNA expression levels were significantly lower than in the other groups; conversely, levels significantly increased in the melatonin treatment groups. Additionally, expression levels of Kiss1 and its receptor, GPR54, in the white fluorescent and red LED light groups were significantly lower than the other groups, but levels also significantly decreased in the melatonin treatment groups. These results suggest that white fluorescent and red wavelengths downregulate the production of neurohormones in the brains of C. auratus and thus may inhibit sexual maturation in goldfish.     

Kisspeptins and the control of gonadotropin secretion in male and female rodents

Kisspeptins, the products of KiSS-1 gene acting via G protein-coupled receptor 54 (GPR54), have recently emerged as fundamental gatekeepers of gonadal function by virtue of their ability to stimulate gonadotropin secretion. Indeed, since the original disclosure of the reproductive facet of the KiSS-1/GPR54 system, an ever-growing number of studies have substantiated the extraordinary potency of kisspeptins to elicit gonadotropin secretion in different mammalian species, under different physiologic and experimental conditions, and through different routes of administration. In this context, studies conducted in laboratory rodents have been enormously instrumental to characterize: (i) the primary mechanisms of action of kisspeptins in the control of gonadotropin secretion; (ii) the pharmacological consequences of acute vs. continuous activation of GPR54; (iii) the roles of specific populations of kisspeptin-producing neurons at the hypothalamus in mediating the feedback effects of sex steroids; (v) the function of kisspeptins in the generation of the pre-ovulatory surge of gonadotropins; and (iv) the influence of sex steroids on GnRH/gonadotropin responsiveness to kisspeptins. While some of those aspects of kisspeptin function will be covered elsewhere in this Special Issue, we summarize herein the most salient data, obtained in laboratory rodents, that have helped to define the physiologic roles and putative pharmacological implications of kisspeptins in the control of male and female gonadotropic axis.     

Formation of Kiss1R/GPER Heterocomplexes Negatively Regulates Kiss1R-mediated Signalling through Limiting Receptor Cell Surface Expression

Kisspeptin receptor (Kiss1R) is an important receptor that plays central regulatory roles in reproduction by regulating hormone release in the hypothalamus. We hypothesize that the formation of heterocomplexes between Kiss1R and other hypothalamus G protein-coupled receptors (GPCRs) affects their cellular signaling. Through screening of potential interactions between Kiss1R and hypothalamus GPCRs, we identified G protein-coupled estrogen receptor (GPER) as one interaction partner of Kiss1R. Based on the recognised function of kisspeptin and estrogen in regulating the reproductive system, we investigated the Kiss1R/GPER heterocomplex in more detail and revealed that complex formation significantly reduced Kiss1R-mediated signaling. GPER did not directly antagonize Kiss1R conformational changes upon ligand binding, but it rather reduced the cell surface expression of Kiss1R. These results therefore demonstrate a regulatory mechanism of hypothalamic hormone receptors via receptor cooperation in the reproductive system and modulation of receptor sensitivity.     

Differential and gonad stage-dependent roles of kisspeptin1 and kisspeptin2 in reproduction in the modern teleosts, morone species

Kisspeptin is an important regulator of reproduction in many vertebrates. The involvement of the two kisspeptins, Kiss1 and Kiss2, and their receptors, Gpr54-1 and Gpr54-2, in controlling reproduction was studied in the brains of the modern teleosts, striped and hybrid basses. In situ hybridization and laser capture microdissection followed by quantitative RT (QRT)-PCR detected coexpression of kiss1 and kiss2 in the hypothalamic nucleus of the lateral recess. Neurons expressing gpr54-1 and gpr54-2 were detected in several brain regions. In the preoptic area, gpr54-2 was colocalized in GnRH1 neurons while gpr54-1 was expressed in cells attached to GnRH1 fibers, indicating two different modes of GnRH1 regulation. The expression of all four genes was measured in the brains of males and females at different life stages using QRT-PCR. The levels of kiss1 and gpr54-1 mRNA, the latter being expressed in minute levels, were consistently lower than those of kiss2 and gpr54-2. While neither gene's expression increased at prepuberty, all were dramatically elevated in mature females. The levels of kiss2 mRNA increased also in mature males. Kiss1 peptide was less potent than Kiss2 in elevating plasma luteinizing hormone levels and in up-regulating gnrh1 and gpr54-2 expression in prepubertal hybrid bass in vivo. In contrast, during recrudescence, Kiss1 was more potent than Kiss2 in inducing luteinizing hormone release, and Kiss2 down-regulated gnrh1 and gpr54-2 expression. This is the first report in fish to demonstrate the alternating actions and the importance of both neuropeptides for reproduction. The organization of the kisspeptin system suggests a transitional evolutionary state between early to late evolving vertebrates.     

Preweaning over- and underfeeding alters onset of puberty in the rat without affecting kisspeptin

The perinatal nutritional environment can permanently influence body weight, potentially leading to changes in puberty onset and reproductive function. We hypothesized that perinatal under- or overfeeding would alter puberty onset and influence concentrations of a neuropeptide crucial for successful puberty, kisspeptin. We manipulated Wistar rat litter sizes to derive small (SL), control (CL), and large (LL) litters containing 4, 12, and 20 rat pups respectively. This manipulation results in an overweight phenotype in SL rats and a lean phenotype in LL that persists throughout life. To investigate whether successful puberty onset is affected by neonatal under- or overfeeding, we examined indices of growth and development, including the onset of puberty, as well as the central expression of Kiss1 mRNA in these pups. Male LL rats reached puberty later than those from CL. These males also had reduced plasma testosterone and elevated 17beta-estradiol concentrations at puberty. The age at puberty onset was not affected in SL males despite accelerated growth. In females, puberty onset was not significantly delayed by having a lean phenotype, and steroid hormones were not affected. The age at onset was, however, younger in the SL females. Kiss1 mRNA in the hypothalamus was not affected by neonatal nutrition either at puberty or 7 days later. Our findings show early life underfeeding in males and overfeeding in females significantly affects puberty onset, altering steroid hormone concentrations in males, but this is not related to changes in hypothalamic kisspeptin.     

Kisspeptins Modulate the Biology of Multiple Populations of Gonadotropin-Releasing Hormone Neurons during Embryogenesis and Adulthood in Zebrafish (Danio rerio)

Kisspeptin1 (product of the Kiss1 gene) is the key neuropeptide that gates puberty and maintains fertility by regulating the gonadotropin-releasing hormone (GnRH) neuronal system in mammals. Inactivating mutations in Kiss1 and the kisspeptin receptor (GPR54/Kiss1r) are associated with pubertal failure and infertility. Kiss2, a paralogous gene for kiss1, has been recently identified in several vertebrates including zebrafish. Using our transgenic zebrafish model system in which the GnRH3 promoter drives expression of emerald green fluorescent protein, we investigated the effects of kisspeptins on development of the GnRH neuronal system during embryogenesis and on electrical activity during adulthood. Quantitative PCR showed detectable levels of kiss1 and kiss2 mRNA by 1 day post fertilization, increasing throughout embryonic and larval development. Early treatment with Kiss1 or Kiss2 showed that both kisspeptins stimulated proliferation of trigeminal GnRH3 neurons located in the peripheral nervous system. However, only Kiss1, but not Kiss2, stimulated proliferation of terminal nerve and hypothalamic populations of GnRH3 neurons in the central nervous system. Immunohistochemical analysis of synaptic vesicle protein 2 suggested that Kiss1, but not Kiss2, increased synaptic contacts on the cell body and along the terminal nerve-GnRH3 neuronal processes during embryogenesis. In intact brain of adult zebrafish, whole-cell patch clamp recordings of GnRH3 neurons from the preoptic area and hypothalamus revealed opposite effects of Kiss1 and Kiss2 on spontaneous action potential firing frequency and membrane potential. Kiss1 increased spike frequency and depolarized membrane potential, whereas Kiss2 suppressed spike frequency and hyperpolarized membrane potential. We conclude that in zebrafish, Kiss1 is the primary stimulator of GnRH3 neuronal development in the embryo and an activator of stimulating hypophysiotropic neuron activities in the adult, while Kiss2 plays an additional role in stimulating embryonic development of the trigeminal neuronal population, but is an RFamide that inhibits electrical activity of hypophysiotropic GnRH3 neurons in the adult.     

Sex-specific changes in the expression of kisspeptin, kisspeptin receptor, gonadotropins and gonadotropin receptors in the Senegalese sole (Solea senegalensis) during a full reproductive cycle

Kisspeptin is thought to have a major role in the control of the onset of puberty in vertebrates. However, our current understanding of its function in fish and how it integrates with other hormones is incomplete due to the high diversity of this group of animals and a still limited amount of available data. This study examined the temporal and spatial changes in expression of kisspeptin, gonadotropins and their respective receptors in the Senegalese sole during a full reproductive cycle. Kiss2 and kiss2r expression was determined by qRT-PCR in the forebrain and midbrain while expression of fshβ and lhβ was determined in the pituitary and fshr and lhr in the gonads. Plasma levels of testosterone (T), 11-ketotestosterone (11-KT) and estradiol-17β were measured by ELISA and gonadal maturation was assessed histologically. In males, kiss2 and kiss2r expression in the brain areas examined was highest towards the end of winter, just before the spawning season, which took place the following spring. This coincided with maximum levels of pituitary fshβ and lhβ, plasma T and 11-KT and the highest number of maturing fish. However, these associations were not evident in females, since the highest expression of kiss2, kiss2r and gonadotropins were observed in the fall, winter or spring, depending upon the variable and tissue considered. Taken together, these data show not only temporal and spatial, but also sex-specific differences in the expression of kisspeptin and its receptor. Thus, while expression of kiss2 in Senegalese sole males agrees with what one would expect according to its proposed role as a major regulator of the onset of reproduction, in females the situation was not so clear, since kiss2 and kiss2r expression was highest either before or during the spawning season.     

The kisspeptin/gonadotropin-releasing hormone pathway and molecular signaling of puberty in fish

The mechanisms underlying the initiation of puberty in fish are poorly understood, and whether the Kiss1 receptor (Kiss1r; previously designated G protein-coupled receptor 54; GPR54) and its ligands, kisspeptins, play a significant role, as has been established in mammals, is not yet known. We determined (via real-time PCR) temporal patterns of expression in the brain of kiss1r, gnrh2, and gnrh3 and a suite of related genes in the hypothalamo-pituitary-gonadal (HPG) axis and analyzed them against the timing of gonadal germ cell development in male and female fathead minnow (Pimephales promelas). Full- or partial-length cDNAs for kiss1r (736 bp), gnrh2 (698 bp), and gnrh3 (804 bp) cloned from fathead minnow were found to be expressed only in the brain, testis, and ovary of adult fish. Localization of kiss1r, gnrh2, and gnrh3 within the brain provided evidence for their physiological roles and a likely hypophysiotropic role for GnRH3 in this species (which, like other cyprinids, does not appear to express gnrh1). In both sexes, kiss1r expression in the brain increased at the onset of puberty and reached maximal expression in males when spermatagonia type B appeared in the testis and in females when cortical alveolus-stage oocytes first appeared in the ovary, the timings of which differed for the two sexes. However, kiss1r expression was considerably lower during more advanced stages of spermatogenesis and oogenesis. The expression of kiss1r closely aligned with that of the gnrh genes (gnrh3 in particular), suggesting the Kiss1r/kisspeptin system in fish has a similar role in puberty to that occurring in mammals, and this hypothesis was supported by the induction of gnrh3 (2.25-fold) and kiss1r (1.5-fold) in early-mid pubertal fish injected with mammalian kisspeptin-10 (2 nmol/g wet weight). An intriguing finding, and contrasting that in mammals, was an elevated expression of esr1, ar, and cyp19a2 (genes involved in sex steroid signaling) in the brain at the onset of puberty, and in females slightly in advance of the elevation in the expression of kiss1r.     

Neuroanatomical Evidence That Kisspeptin Directly Regulates Isotocin and Vasotocin Neurons

Neuropeptide kisspeptin has been suggested to be an essential central regulator of reproduction in response to changes in serum gonadal steroid concentrations. However, in spite of wide kisspeptin receptor distribution in the brain, especially in the preoptic area and hypothalamus, the research focus has mostly been confined to the kisspeptin regulation on GnRH neurons. Here, by using medaka whose kisspeptin (kiss1) neurons have been clearly demonstrated to be regulated by sex steroids, we analyzed the anatomical distribution of kisspeptin receptors Gpr54-1 and Gpr54-2. Because the both receptors were shown to be activated by kisspeptins (Kiss1 and Kiss2), we analyzed the anatomical distribution of the both receptors by in situ hybridization. They were mainly expressed in the ventral telencephalon, preoptic area, and hypothalamus, which have been suggested to be involved in homeostatic functions including reproduction. First, we found gpr54-2 mRNA expression in nucleus preopticus pars magnocellularis and demonstrated that vasotocin and isotocin (Vasopressin and Oxytocin ortholog, respectively) neurons express gpr54-2 by dual in situ hybridization. Given that kisspeptin administration increases serum oxytocin and vasopressin concentration in mammals, the present finding are likely to be vertebrate-wide phenomenon, although direct regulation has not yet been demonstrated in mammals. We then analyzed co-expression of kisspeptin receptors in three types of GnRH neurons. It was clearly demonstrated that gpr54-expressing cells were located adjacent to GnRH1 neurons, although they were not GnRH1 neurons themselves. In contrast, there was no gpr54-expressing cell in the vicinities of neuromodulatory GnRH2 or GnRH3 neurons. From these results, we suggest that medaka kisspeptin neurons directly regulate some behavioral and neuroendocrine functions via vasotocin/isotocin neurons, whereas they do not regulate hypophysiotropic GnRH1 neurons at least in a direct manner. Thus, direct kisspeptin regulation of GnRH1 neurons proposed in mammals may not be the universal feature of vertebrate kisspeptin system in general.     

The vasoactive potential of kisspeptin-10 in the peripheral vasculature

Splice products of the Kiss1 protein (kisspeptins) have been shown to be involved in a diverse range of functions, including puberty, metastasis and vasoconstriction in large human arteries. Circulating Kisspeptin-10 (Kp-10) plasma levels are low in normal individuals but are elevated during various disease states as well as pregnancy. Here, we investigated the potential of Kp-10, the shortest biologically active kisspeptin, to influence microvascular effects, concentrating on the cutaneous vasculature. Kp-10 caused a dose-dependent increase in oedema formation (0.3-10 nmol/injection site), assessed by Evans Blue albumin dye extravasation, in the dorsal skin of CD1 mice. Oedema formation was shown to be inhibited by the histamine H(1) receptor antagonist mepyramine. The response was characterised by a ring of pallor at the injection site in keeping with vasoconstrictor activity. Therefore, changes in dorsal skin blood flow were assessed by clearance of intradermally injected (99m)technetium. Kp-10 was found to significantly reduce clearance, in keeping with decreased blood flow and providing further evidence for vasoconstrictor activity. The decreased clearance was partially inhibited by co-treatment with the cyclo-oxygenase inhibitor indomethacin. Finally evidence for the kisspeptin receptor gene (Kiss1R), but not the kisspeptin peptide gene (Kiss1), mRNA expression was observed in heart, aorta and kidney samples from normal and angiotensin II induced hypertensive mice, with similar mRNA levels observed in each. We have evidence for two peripheral vasoactive roles for kisspeptin-10. Firstly, plasma extravasation indicative of ability to induce oedema formation and secondly decreased peripheral blood flow, indicating microvascular constriction. Thus Kp-10 has vasoactive properties in the peripheral microvasculature.     

Kisspeptin and the seasonal control of reproduction in hamsters

Reproduction is a complex and energy demanding function. When internal and external conditions might impair reproductive success (negative energy balance, stress, harsh season) reproductive activity has to be repressed. Recent evidence suggests that these inhibitory mechanisms operate on Kiss1-expressing neurons, which were recently shown to be implicated in the regulation of GnRH release. Hamsters are seasonal rodents which are sexually active in long photoperiod and quiescent in short photoperiod. The photoperiodic information is transmitted to the reproductive system by melatonin, a pineal hormone whose secretion is adjusted to night length. The photoperiodic variation in circulating melatonin has been shown to synchronize reproductive activity with seasons, but the mechanisms involved in this effect of melatonin were so far unknown. Recently we have observed that Kiss1 mRNA level in the arcuate nucleus of the Syrian hamster is lower in short photoperiod, when animals are sexually quiescent. Notably, intracerebroventricular infusion of Kiss1 gene product, kisspeptin, in hamsters kept in short photoperiod is able to override the inhibitory photoperiod and to reactivate sexual activity. The inhibition of Kiss1 expression in short photoperiod is driven by melatonin because pinealectomy prevents decrease in Kiss1 mRNA level in short photoperiod and melatonin injection in long photoperiod down regulates Kiss1 expression. Whether melatonin acts directly on arcuate Kiss1 expressing neurons or mediates its action via interneurons is the subject of the current investigations.     

Expression of Kisspeptins and Kiss Receptors Suggests a Large Range of Functions for Kisspeptin Systems in the Brain of the European Sea Bass

This study, conducted in the brain of a perciform fish, the European sea bass, aimed at raising antibodies against the precursor of the kisspeptins in order to map the kiss systems and to correlate the expression of kisspeptins, kiss1 and kiss2, with that of kisspeptin receptors (kiss-R1 and kiss-R2). Specific antibodies could be raised against the preprokiss2, but not the preoprokiss1. The data indicate that kiss2 neurons are mainly located in the hypothalamus and project widely to the subpallium and pallium, the preoptic region, the thalamus, the pretectal area, the optic tectum, the torus semicircularis, the mediobasal medial and caudal hypothalamus, and the neurohypophysis. These results were compared to the expression of kiss-R1 and kiss-R2 messengers, indicating a very good correlation between the wide distribution of Kiss2-positive fibers and that of kiss-R2 expressing cells. The expression of kiss-R1 messengers was more limited to the habenula, the ventral telencephalon and the proximal pars distalis of the pituitary. Attempts to characterize the phenotype of the numerous cells expressing kiss-R2 showed that neurons expressing tyrosine hydroxylase, neuropeptide Y and neuronal nitric oxide synthase are targets for kisspeptins, while GnRH1 neurons did not appear to express kiss-R1 or kiss-R2 messengers. In addition, a striking result was that all somatostatin-positive neurons expressed-kissR2. These data show that kisspeptins are likely to regulate a wide range of neuronal systems in the brain of teleosts.     

Reproductive functions of kisspeptin and Gpr54 across the life cycle of mice and men

The reproductive phenotypes of nearly two dozen patients with mutations in GPR54 have been reported, as have the phenotypes of four mouse lines mutant for Gpr54 and two lines mutant for Kiss1. These phenotypes demonstrate that kisspeptin/Gpr54 function is required at all phases of the life cycle when the secretion of gonadotropin-releasing hormone (GnRH) is robust. Furthermore, there is phenotypic variability ranging from severe hypogonadism to partial sexual development. Collectively, these findings suggest that kisspeptin and Gpr54 serve as an essential conduit for relaying developmental information to the GnRH neuron.