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.     

Effect of a postnatal high-fat diet exposure on puberty onset,estrous cycle regularity,and kisspeptin expression in female rats

Kisspeptin, encoded by Kiss1, plays a key role in pubertal maturation and reproduction as a positive upstream regulator of the hypothalamic-pituitary-gonadal (HPG) axis. To examine the role of high-fat diet (HFD) on puberty onset, estrous cycle regularity, and kisspeptin expression, female rats were exposed to HFD in distinct postnatal periods. Three groups of rats were exposed to HFD containing 60% energy from fat during the pre-weaning period (postnatal day (PND) 1–16, HFD PND 1–16), post-weaning period (HFD PND 21–34), or during both periods (HFD PND 1–34). Puberty onset, evaluated by vaginal opening, was monitored on days 30–34. Leptin, estradiol (E₂), Kiss1 mRNA levels, and number of kisspeptin-immunoreactive cells in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) were measured at day 34. Body weight increased only in rats exposed to HFD during post-weaning period, whereas the timing of vaginal opening was unaffected in all three groups. Leptin, Kiss1 mRNA levels, and number of kisspeptin-immunoreactive cells at day 34 were not affected by HFD. Additionally, the estrous cycle regularity was monitored in rats exposed to HFD for 40 days from weaning. Leptin, E₂, and Kiss1 mRNA levels in the AVPV and ARC were measured after the HFD exposure. Thirty-three percent of rats exposed to HFD exhibited irregular estrous cycles and a two-fold increase in leptin. By contrast, E₂ level and Kiss1 mRNA levels were not affected by the treatment. These data show that postnatal HFD exposure induced irregular estrous cycles, but had no effect on puberty onset or kisspeptin.     

Collapsin response mediator protein 4 affects the number of tyrosine hydroxylase‐immunoreactive neurons in the sexually dimorphic nucleus in female mice

In the sexually dimorphic anteroventral periventricular nucleus (AVPV) of the hypothalamus, females have a greater number of tyrosine hydroxylase‐immunoreactive (TH‐ir) and kisspeptin‐immunoreactive (kisspeptin‐ir) neurons than males. In this study, we used proteomics analysis and gene‐deficient mice to identify proteins that regulate the number of TH‐ir and kisspeptin‐ir neurons in the AVPV. Analysis of protein expressions in the rat AVPV on postnatal day 1 (PD1; the early phase of sex differentiation) using two‐dimensional fluorescence difference gel electrophoresis followed by MALDI‐TOF‐MS identified collapsin response mediator protein 4 (CRMP4) as a protein exhibiting sexually dimorphic expression. Interestingly, this sexually differential expressions of CRMP4 protein and mRNA in the AVPV was not detected on PD6. Prenatal testosterone exposure canceled the sexual difference in the expression of Crmp4 mRNA in the rat AVPV. Next, we used CRMP4‐knockout (CRMP4‐KO) mice to determine the in vivo function of CRMP4 in the AVPV. Crmp4 knockout did not change the number of kisspeptin‐ir neurons in the adult AVPV in either sex. However, the number of TH‐ir neurons was increased in the AVPV of adult female CRMP4‐KO mice as compared with the adult female wild‐type mice. During development, no significant difference in the number of TH‐ir neurons was detected between sexes or genotypes on embryonic day 15, but a female‐specific increase in TH‐ir neurons was observed in CRMP4‐KO mice on PD1, when the sex difference was not yet apparent in wild‐type mice. These results indicate that CRMP4 regulates the number of TH‐ir cell number in the female AVPV. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 502–517, 2013     

Leptin Signaling in Kiss1 Neurons Arises after Pubertal Development

The adipocyte-derived hormone leptin is required for normal pubertal maturation in mice and humans and, therefore, leptin has been recognized as a crucial metabolic cue linking energy stores and the onset of puberty. Several lines of evidence have suggested that leptin acts via kisspeptin expressing neurons of the arcuate nucleus to exert its effects. Using conditional knockout mice, we have previously demonstrated that deletion of leptin receptors (LepR) from kisspeptin cells cause no puberty or fertility deficits. However, developmental adaptations and system redundancies may have obscured the physiologic relevance of direct leptin signaling in kisspeptin neurons. To overcome these putative effects, we re-expressed endogenous LepR selectively in kisspeptin cells of mice otherwise null for LepR, using the Cre-loxP system. Kiss1-Cre LepR null mice showed no pubertal development and no improvement of the metabolic phenotype, remaining obese, diabetic and infertile. These mice displayed decreased numbers of neurons expressing Kiss1 gene, similar to prepubertal control mice, and an unexpected lack of re-expression of functional LepR. To further assess the temporal coexpression of Kiss1 and Lepr genes, we generated mice with the human renilla green fluorescent protein (hrGFP) driven by Kiss1 regulatory elements and crossed them with mice that express Cre recombinase from the Lepr locus and the R26-tdTomato reporter gene. No coexpression of Kiss1 and LepR was observed in prepubertal mice. Our findings unequivocally demonstrate that kisspeptin neurons are not the direct target of leptin in the onset of puberty. Leptin signaling in kisspeptin neurons arises only after completion of sexual maturation.     

Kisspeptin Signaling Is Required for the Luteinizing Hormone Response in Anestrous Ewes following the Introduction of Males

The introduction of a novel male stimulates the hypothalamic-pituitary-gonadal axis of female sheep during seasonal anestrus, leading to the resumption of follicle maturation and ovulation. How this pheromone cue activates pulsatile secretion of gonadotropin releasing hormone (GnRH)/luteinizing hormone (LH) is unknown. We hypothesised that pheromones activate kisspeptin neurons, the product of which is critical for the stimulation of GnRH neurons and fertility. During the non-breeding season, female sheep were exposed to novel males and blood samples collected for analysis of plasma LH profiles. Females without exposure to males served as controls. In addition, one hour before male exposure, a kisspeptin antagonist (P-271) or vehicle was infused into the lateral ventricle and continued for the entire period of male exposure. Introduction of a male led to elevated mean LH levels, due to increased LH pulse amplitude and pulse frequency in females, when compared to females not exposed to a male. Infusion of P-271 abolished this effect of male exposure. Brains were collected after the male effect stimulus and we observed an increase in the percentage of kisspeptin neurons co-expressing Fos, by immunohistochemistry. In addition, the per-cell expression of Kiss1 mRNA was increased in the rostral and mid (but not the caudal) arcuate nucleus (ARC) after male exposure in both aCSF and P-271 treated ewes, but the per-cell content of neurokinin B mRNA was decreased. There was also a generalized increase in Fos positive cells in the rostral and mid ARC as well as the ventromedial hypothalamus of females exposed to males. We conclude that introduction of male sheep to seasonally anestrous female sheep activates kisspeptin neurons and other cells in the hypothalamus, leading to increased GnRH/LH secretion.     

Quantification of Rat Kisspeptin Using a Novel Radioimmunoassay

Kisspeptin is a hypothalamic peptide hormone that plays a pivotal role in pubertal onset and reproductive function. Previous studies have examined hypothalamic kisspeptin mRNA expression, either through in situ hybridisation or real-time RT-PCR, as a means quantifying kisspeptin gene expression. However, mRNA expression levels are not always reflected in levels of the translated protein. Kisspeptin-immunoreactivity (IR) has been extensively examined using immunohistochemistry, enabling detection and localisation of kisspeptin perikaya in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV). However, quantification of kisspeptin-IR remains challenging. We developed a specific rodent radioimmunoassay assay (RIA) capable of detecting and quantifying kisspeptin-IR in rodent tissues. The RIA uses kisspeptin-10 as a standard and radioactive tracer, combined with a commercially available antibody raised to the kisspeptin-10 fragment. Adult female wistar rat brain samples were sectioned at 300 µm and the ARC and AVPV punch micro-dissected. Brain punches were homogenised in extraction buffer and assayed with rodent kisspeptin-RIA. In accord with the pattern of kisspeptin mRNA expression, kisspeptin-IR was detected in both the ARC (47.1±6.2 fmol/punch, mean±SEM n = 15) and AVPV (7.6±1.3 fmol/punch, mean±SEM n = 15). Kisspeptin-IR was also detectable in rat placenta (1.26±0.15 fmol/mg). Reverse phase high pressure liquid chromatography analysis showed that hypothalamic kisspeptin-IR had the same elution profile as a synthetic rodent kisspeptin standard. A specific rodent kisspeptin-RIA will allow accurate quantification of kisspeptin peptide levels within specific tissues in rodent experimental models.     

Physiological role of metastin/kisspeptin in regulating gonadotropin-releasing hormone (GnRH) secretion in female rats

Various studies have attempted to unravel the physiological role of metastin/kisspeptin in the control of gonadotropin-releasing hormone (GnRH) release. A number of evidences suggested that the population of metastin/kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) is involved in generating a GnRH surge to induce ovulation in rodents, and thus the target of estrogen positive feedback. Females have an obvious metastin/kisspeptin neuronal population in the AVPV, but males have only a few cell bodies in the nucleus, suggesting that the absence of the surge-generating mechanism or positive feedback action in males is due to the limited AVPV metastin/kisspeptin neuronal population. On the other hand, the arcuate nucleus (ARC) metastin/kisspeptin neuronal population is considered to be involved in the regulation of tonic GnRH release. The ARC metastin/kisspeptin neurons show no sex difference in their expression, which is suppressed by gonadal steroids in both sexes. Thus, the ARC population of metastin/kisspeptin neurons is a target of estrogen negative feedback action on tonic GnRH release. The lactating rat model provided further evidence indicating that ARC metastin/kisspeptin neurons are involved in GnRH pulse generation, because pulsatile release of luteinizing hormone (LH) is profoundly suppressed by suckling stimulus and the LH pulse suppression is well associated with the suppression of ARC metastin/kisspeptin and KiSS-1 gene expression in lactating rats.     

Insulin and Leptin Signaling Interact in the Mouse Kiss1 Neuron during the Peripubertal Period

Reproduction requires adequate energy stores for parents and offspring to survive. Kiss1 neurons, which are essential for fertility, have the potential to serve as the central sensors of metabolic factors that signal to the reproductive axis the presence of stored calories. Paradoxically, obesity is often accompanied by infertility. Despite excess circulating levels of insulin and leptin, obese individuals exhibit resistance to both metabolic factors in many neuron types. Thus, resistance to insulin or leptin in Kiss1 neurons could lead to infertility. Single deletion of the receptors for either insulin or the adipokine leptin from Kiss1 neurons does not impair adult reproductive dysfunction. However, insulin and leptin signaling pathways may interact in such a way as to obscure their individual functions. We hypothesized that in the presence of genetic or obesity-induced concurrent insulin and leptin resistance, Kiss1 neurons would be unable to maintain reproductive function. We therefore induced a chronic hyperinsulinemic and hyperleptinemic state in mice lacking insulin receptors in Kiss1 neurons through high fat feeding and examined the impact on fertility. In an additional, genetic model, we ablated both leptin and insulin signaling in Kiss1 neurons (IR/LepR^Kiss mice). Counter to our hypothesis, we found that the addition of leptin insensitivity did not alter the reproductive phenotype of IR^Kiss mice. We also found that weight gain, body composition, glucose and insulin tolerance were normal in mice of both genders. Nonetheless, leptin and insulin receptor deletion altered pubertal timing as well as LH and FSH levels in mid-puberty in a reciprocal manner. Our results confirm that Kiss1 neurons do not directly mediate the critical role that insulin and leptin play in reproduction. However, during puberty kisspeptin neurons may experience a critical window of susceptibility to the influence of metabolic factors that can modify the onset of fertility.     

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.     

An eGFP-expressing subpopulation of growth hormone secretagogue receptor cells are distinct from kisspeptin,tyrosine hydroxylase,and RFamide-related peptide neurons in mice

Ghrelin acts on the growth hormone secretagogue receptor (GHSR) in the brain to elicit changes in physiological functions. It is associated with the neural control of appetite and metabolism, however central ghrelin also affects fertility. Central ghrelin injection in rats suppresses luteinizing hormone (LH) concentrations and pulse frequency. Although ghrelin suppresses LH and regulates kisspeptin mRNA in the anteroventral periventricular/periventricular nucleus (AVPV/PeN), there is no neuroanatomical evidence linking GHSR neural circuits to kisspeptin neurons. In this study, we first determined coexpression of GHSR and GnRH neurons using a GHSR-eGFP reporter mouse line. Using dual-label immunohistochemistry, we saw no coexpression. GHSR-eGFP expressing cells were present in the AVPV/PeN and over 90% of these expressed estrogen receptor-α (ERα). Despite this, we observed no evidence of GHSR-eGFP/kisspeptin coexpressing neurons in the AVPV/PeN. To further examine the phenotype of GHSR-eGFP cells in the AVPV/PeN, we determined coexpression with tyrosine hydroxylase (TH) and showed virtually no coexpression in the AVPV/PeN (<2%). We also observed no coexpression of GHSR-eGFP and RFamide-related peptide-3 (RFRP3) neurons in the dorsomedial hypothalamic nucleus. Importantly, we observed that approximately half of the GHSR-eGFP cells in the AVPV coexpressed Ghsr mRNA (as determined by in situ hybridization) so these data should be interpreted accordingly. Although ghrelin influences the hypothalamic reproductive axis, our data using a GHSR-eGFP reporter suggests ghrelin regulates neurons expressing ERα but does not directly act on GnRH, kisspeptin, TH, or RFRP3 neurons, as little or no GHSR-eGFP coexpression was observed.     

Expression and Function of Kisspeptin during Mouse Decidualization

Background

Plasma kisspeptin levels dramatically increased during the first trimester of human pregnancy, which is similar to pregnancy specific glycoprotein-human chorionic gonadotropin. However, its particular role in the implantation and decidualization has not been fully unraveled. Here, the study was conducted to investigate the expression and function of kisspeptin in mouse uterus during early pregnancy and decidualization.

Methodology/Principal Findings

Quantitative PCR results demonstrated that Kiss1 and GPR54 mRNA levels showed dynamic increase in the mouse uterus during early pregnancy and artificially induced decidualization in vivo. KISS-1 and GPR54 proteins were spatiotemporally expressed in decidualizing stromal cells in intact pregnant females, as well as in pseudopregnant mice undergoing artificially induced decidualization. In the ovariectomized mouse uterus, the expression of Kiss1 mRNA was upregulated after progesterone or/and estradiol treatment. Moreover, in a stromal cell culture model, the expression of Kiss1 and GPR54 mRNA gradually rise with the progression of stromal cell decidualization, whereas the attenuated expression of Kiss1 using small interfering RNA approaches significantly blocked the progression of stromal cell decidualization.

Conclusion

our results demonstrated that Kiss1/GPR54 system was involved in promoting uterine decidualization during early pregnancy in mice.     

Inverse age-related changes between hypothalamic NPY and KISS1 gene expression during pubertal initiation in male rhesus monkey

The neuroendocrine mechanism underlying the sinusoidal wave nature of gonadotropin–releasing hormone pulse generator activity from infantile to adult age still needs to be meticulously defined. Direct inhibition of kisspeptin neurons by neuropeptide Y (NPY) and close intimacy between the two rekindle the importance of these two neuropeptides controlling reproductive axis activity. Thus, the present study was undertaken to decipher simultaneous fluctuations and to profile correlative changes in the relative expression of KISS1, NPY, and their receptor genes from the mediobasal hypothalamus of infant (n = 3), juvenile, pre-pubertal, and adult (n = 4 in each stage) male rhesus monkey (Macaca mulatta) by RT-qPCR. Significant elevation (p < 0.05?0.01) in KISS1 and KISS1R and low (p < 0.05) expression in NPY and NPY1R mRNA in the adult group as compared to the pre-pubertal group was observed. Moreover, significantly high (p < 0.05) expression of NPY and NPY1R mRNA with non-significant (p> 0.05) decline in KISS1 and KISS1R in pre-pubertal animals in comparison to infants describe inverse correlative age-associated changes during pubertal development. Current findings imply that NPY may contribute as a neurobiological brake for the dormancy of kisspeptin neurons before pubertal onset, while dwindling of this brake is likely to occasion kisspeptin dependent hypothalamic–pituitary–gonadal axis activation at puberty. These findings may help in the development of clinical and therapeutic strategies to regulate fertility in humans.     

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.     

Hormonal Regulation of Opioid Peptide Neurons in the Anteroventral Periventricular Nucleus

Steroid hormones provide a means of coordinating the activity of widespread neural systems that mediate endocrine, autonomic, and somatomotor aspects of reproductive processes that are essential for the propagation of mammalian species. Because these processes are quite different in each sex, the neural pathways that control them are also sexually differentiated. The anteroventral periventricular nucleus (AVPV) of the preoptic region occupies a nodal point in sexually dimorphic forebrain circuits and appears to play a critical role in regulating gonadotropin secretion. The AVPV contains sexually dimorphic populations of opioid peptide containing neurons that display different patterns of development and are differentially regulated in adult animals by gonadal steroids. Moreover, estrogen (ER) and progesterone (PR) receptors are expressed in AVPV neurons in a transmitter-specific way, and the expression of these nuclear transacting factors is differentially regulated by sex steroids. Thus, neurons in the AVPV show distinct patterns of hormonal regulation of gene expression, and distinct hormone receptor profiles.     

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.     

The effect of different exogenous kisspeptins on sex hormones and reproductive indices of the goldfish (Carassius auratus) broodstock

Despite several studies on fish hormone therapy, finding new candidates may provide more reproductive efficiency in artificial propagation. Kisspeptins, being upstream of the hypothalamic–pituitary-gonadal axis, appear to play a key role in the reproduction process. In the present study, the effect of different variants of kisspeptide, including goldfish (Carassius auratus) kiss1 kisspeptin (Kiss1), human kisspeptin (Hkiss), and their combination (Kiss1 + H), on the reproductive indices of goldfish broodstock in comparison to Ovaprim (a typical synthetic Gnrh hormone) was investigated. Peptides (Kiss1 and Hkiss) were synthesized using a solid-phase synthesis approach. Kiss1 and Hkiss were injected at a dose of 100 μg kg⁻¹ body weight, blood samples were taken 6 h after injection and sex hormones (E2, Dhp, and 11-Kt), gonadotropins (Lh and Fsh), cortisol and reproductive indices (fecundity, fertilization and hatching percentage) were measured. The results showed a significant increase of plasma sex hormones and gonadotropins in fish treated with kisspeptins. In addition, the cortisol and lipoprotein lipase in Kiss1, Hkiss and Kiss1 + H were remarkably increased compared to Ovaprim. In conclusion, kisspeptins could be a more suitable candidate than Ovaprim for accelerating and synchronizing oocyte maturation in the fisheries industry.     

Kisspeptin Signalling in the Hypothalamic Arcuate Nucleus Regulates GnRH Pulse Generator Frequency in the Rat

Background

Kisspeptin and its G protein-coupled receptor (GPR) 54 are essential for activation of the hypothalamo-pituitary-gonadal axis. In the rat, the kisspeptin neurons critical for gonadotropin secretion are located in the hypothalamic arcuate (ARC) and anteroventral periventricular (AVPV) nuclei. As the ARC is known to be the site of the gonadotropin-releasing hormone (GnRH) pulse generator we explored whether kisspeptin-GPR54 signalling in the ARC regulates GnRH pulses.

Methodology/Principal Findings

We examined the effects of kisspeptin-10 or a selective kisspeptin antagonist administration intra-ARC or intra-medial preoptic area (mPOA), (which includes the AVPV), on pulsatile luteinizing hormone (LH) secretion in the rat. Ovariectomized rats with subcutaneous 17β-estradiol capsules were chronically implanted with bilateral intra-ARC or intra-mPOA cannulae, or intra-cerebroventricular (icv) cannulae and intravenous catheters. Blood samples were collected every 5 min for 5–8 h for LH measurement. After 2 h of control blood sampling, kisspeptin-10 or kisspeptin antagonist was administered via pre-implanted cannulae. Intranuclear administration of kisspeptin-10 resulted in a dose-dependent increase in circulating levels of LH lasting approximately 1 h, before recovering to a normal pulsatile pattern of circulating LH. Both icv and intra-ARC administration of kisspeptin antagonist suppressed LH pulse frequency profoundly. However, intra-mPOA administration of kisspeptin antagonist did not affect pulsatile LH secretion.

Conclusions/Significance

These data are the first to identify the arcuate nucleus as a key site for kisspeptin modulation of LH pulse frequency, supporting the notion that kisspeptin-GPR54 signalling in this region of the mediobasal hypothalamus is a critical neural component of the hypothalamic GnRH pulse generator.     

Maternal hypothyroidism reduces the expression of the kisspeptin/Kiss1r system in the maternal-fetal interface of rats

Alterations of circulating and placental levels of kisspeptin have been associated with gestational diseases. However, there are still no studies on the placental and decidual expression of Kiss1 and its receptor Kiss1r in maternal hypothyroidism, which is the aim of this work. We demonstrate that the fetoplacental restriction caused by hypothyroidism in rats is associated with a reduction in the Kiss1r expression and reduced Kiss1 and Kiss1r mRNA levels in the decidua and/or placenta. This demonstrate that fetoplacental restriction in hypothyroid rats is linked with a suppression of the kisspeptin/Kiss1r system at the maternal-fetal interface.     

Fibroblast growth factor signaling deficiencies impact female reproduction and kisspeptin neurons in mice

Fibroblast growth factor (FGF) signaling is essential for the development of the gonadotropin-releasing hormone (GnRH) system. Mice harboring deficiencies in Fgf8 or Fgf receptor 1 (Fgfr1) suffer a significant loss of GnRH neurons, but their reproductive phenotypes have not been examined. This study examined if female mice hypomorphic for Fgf8, Fgfr1, or both (compound hypomorphs) exhibited altered parameters of pubertal onset, estrous cyclicity, and fertility. Further, we examined the number of kisspeptin (KP)-immunoreactive (ir) neurons in the anteroventral periventricular/periventricular nuclei (AVPV/PeV) of these mice to assess if changes in the KP system, which stimulates the GnRH system, could contribute to the reproductive phenotypes. Single hypomorphs (Fgfr1(+/-) or Fgf8(+/-)) had normal timing for vaginal opening (VO) but delayed first estrus. However, after achieving the first estrus, they underwent normal expression of estrous cycles. In contrast, the compound hypomorphs underwent early VO and normal first estrus, but had disorganized estrous cycles that subsequently reduced their fertility. KP immunohistochemistry on Postnatal Day 15, 30, and 60 transgenic female mice revealed that female compound hypomorphs had significantly more KP-ir neurons in the AVPV/PeV compared to their wild-type littermates, suggesting increased KP-ir neurons may drive early VO but could not maintain the cyclic changes in GnRH neuronal activity required for female fertility. Overall, these data suggest that Fgf signaling deficiencies differentially alter the parameters of female pubertal onset and cyclicity. Further, these deficiencies led to changes in the AVPV/PeV KP-ir neurons that may have contributed to the accelerated VO in the compound hypomorphs.