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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Effect of estrogen on the expression of GnRH and kisspeptin in the hypothalamus of rats during puberty

The aim of this study was to assess whether changes in kisspeptin and GnRH levels could be attributed to sex steroids at puberty onset. We used the ovariectomy (OVX) model in rats treated with 17β-estradiol (E₂; OVX + E₂), or oil (OVX + oil), and in intact rats treated with E₂ (intact + E₂) or oil only (intact + oil) to determine gene expression changes of Kiss1 and Gnrh1 in the hypothalamus and protein expression of kisspeptin and GnRH in the different areas of the hypothalamus. In the intact + E₂ and OVX + E₂ rats on the day of the onset of puberty, GnRH-immunoreactive (ir) cell numbers decreased (P < 0.05) in the arcuate nucleus but were increased in the preoptic area; Kisspeptin-ir cells increased (P < 0.05) in the arcuate nucleus, periventricular nucleus, and preoptic area; no difference (P > 0.05) was found in the paraventricularis nucleus for GnRH-ir or kisspeptin-ir cells. Additionally, levels of Kiss1 and Gnrh1 messenger RNA in the hypothalamus were significantly higher (P < 0.05) in the OVX + E₂ or intact + E₂ rats than in the OVX + oil or intact + oil animals, respectively. In the OVX + oil rats, OVX significantly increased (P < 0.05) levels of Gnrh1 and Kiss1 messenger RNA and the expression of GnRH and kisspeptin in the hypothalamus compared to intact + oil animals. These results suggest that kisspeptin and GnRH play major roles in modulating the activity of estrogen circuits at the onset of puberty.     

西红花酸治疗DHT诱导的PCOS小鼠的机制研究

目的:观察西红花酸对双氢睾酮(Dihydrotestosterone,DHT)诱导的多囊卵巢综合症(Polycystic ovarian syndrome,PCOS)小鼠的疗效并探讨其作用机制。方法:妊娠15-18天给予孕鼠皮下注射DHT诱导子代雌鼠PCOS模型。待子鼠8周龄后,随机选择一半数量的PCOS小鼠连续4周西红花酸灌胃,作为西红花酸给药组(n=18)。给药期间检测体重和动情周期,待小鼠16周龄左右,通过眼球取血后处死,取出下丘脑、卵巢。采用HE染色观察卵巢组织的病理改变;ELISA试剂盒检测血清中雌二醇(Estradiol,E2)、睾酮(Testosterone,T)、孕酮(Progesterone,P4)、促黄体生成素(Luteinizing hormone,LH)、卵泡刺激素(Follicle-stimulating hormone,FSH);采用免疫组化、Western blot和实时荧光定量PCR法检测下丘脑的前腹侧视旁核(Anteroventral periventricular,AVPV)、弓状核(Arcuate,ARC)的kisspeptin以及视前区(Preoptic area,POA)里Gn RH的表达水平。结果:与对照组相比,PCOS小鼠卵巢与体重的比值上升了22.56%±6.77%,动情周期延长,卵巢内出现大的空泡,闭锁卵泡数量增加了138.74%±33.22%,窦状卵泡、成熟窦状卵泡和黄体数量分别减少了38.80%±4.69%、56.35%±7.32%和63.77%±7.25%,血清中E2、P4和FSH水平分别降低了40.99%±2.69%、56.91%±5.25%、和38.80%±4.69%,而T、LH水平分别升高了43.23%±4.70%和148.46%±28.16%,下丘脑中AVPV中kisspeptin神经元表达减少,ARC中kisspeptin神经元表达增多,POA中Gn RH神经元减少,而以上情况能够被西红花酸改善。结论:西红花酸分别通过促进和抑制下丘脑AVPV核和ARC核团的kisspeptin表达改善PCOS的病理变化。     

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.     

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.     

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     

Intermittent fasting ameliorates di-(2-ethylhexyl) phthalate-induced precocious puberty in female rats: A study of the hypothalamic–pituitary–gonadal axis

Di-(2-ethylhexyl) phthalate has been reported to interfere with the development and function of animal reproductive systems. However, hardly any studies provide methods to minimize or prevent the adverse effects of DEHP on reproduction. The energy balance state of mammals is closely related to reproductive activities, and the reproductive axis can regulate reproductive activities according to changes in the body's energy balance state. In this study, the effects of every other day fasting (EODF), as a way of intermittent fasting, on preventing the precocious puberty induced by DEHP in female rats was studied. EODF significantly improved the advancement of vaginal opening age (as the markers of puberty onset) and elevated serum levels of luteinizing hormone and estradiol (detected by ELISA) induced by 5 mg kg^?1 DEHP exposure (D5). The mRNA and western blot results showed that the EODF could minimized the increase of gonadotropin-releasing hormone expression induced by DEHP exposure. The administration of DEHP could elevate the levels of kisspeptin protein and the number of kisspeptin-immunoreactive neurons in anteroventral periventricular nucleu, and this increase was diminished considerably by EODF treatment. In contrast, the D5 and D0 groups showed no remarkable difference in the level of Kiss1 expression in arcuate nucleus, whereas the D5 + EODF group had a remarkable decrease in kisspeptin expression as compared with the other two groups. Our results indicated that EODF might inhibit the acceleration of puberty onset induced by DEHP exposure via HPG axis.     

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.     

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.     

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.     

Inhibitory action of tongue sole LPXRFa,the piscine ortholog of gonadotropin-inhibitory hormone,on the signaling pathway induced by tongue sole kisspeptin in COS-7 cells transfected with their cognate receptors

Kisspeptin (Kiss) acts as a positive regulator of reproduction by acting on gonadotropes and gonadotropin-releasing hormone (GnRH) neurons. Despite its functional significance, the intricate web of intracellular signal transduction pathways in response to Kiss is still far from being fully understood in teleosts. Accordingly, we investigated the molecular mechanism of Kiss action and its possible interaction with LPXRFa signaling in this study. In vitro functional analysis revealed that synthetic tongue sole Kiss2 decapeptide increased the cAMP responsive element-dependent luciferase (CRE-luc) activity in COS-7 cells transfected with its cognate receptor, while this stimulatory effect was markedly reduced by two inhibitors of the adenylate cyclase (AC)/protein kinase A (PKA) pathway. Similarly, Kiss2 also significantly stimulated serum responsive element-dependent luciferase (SRE-luc) activity, whereas this stimulatory effect was evidently attenuated by two inhibitors of the phospholipase C (PLC)/protein kinase C (PKC) pathway. In addition, LPXRFa-2 suppressed Kiss2-elicited CRE-luc activity in a dose-dependent manner. Taken together, Kiss2 utilizes both AC/PKA and PLC/PKC pathways to exert its functions via its cognate receptor and LPXRFa may antagonize the action of Kiss2 by inhibiting kisspeptin signaling. As far as we know, this study is the first to characterize the half-smooth tongue sole kisspeptin and LPXRFa signaling pathway in COS-7 cells transfected with their cognate receptors and provides novel information on the interaction between LPXRFa system and kisspeptin system in teleosts.     

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 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.