The interplay between kisspeptin and endocannabinoid systems modulates male hypothalamic and gonadic control of reproduction in vivo.

Introduction: Male reproduction is under the control of the hypothalamus-pituitary-gonadal (HPG) axis. The endocannabinoid system (ECS) and the kisspeptin system (KS) are two major signaling systems in the central and peripheral control of reproduction, but their possible interaction has been poorly investigated in mammals. This manuscript analyzes their possible reciprocal modulation in the control of the HPG axis. Materials and methods: Adolescent male rats were treated with kisspeptin-10 (Kp10) and endocannabinoid anandamide (AEA), the latter alone or in combination with the type 1 cannabinoid receptor (CB1) antagonist rimonabant (SR141716A). The hypothalamic KS system and GnRH expression, circulating sex steroids and kisspeptin (Kiss1) levels, and intratesticular KS and ECS were evaluated by immunohistochemical and molecular methods. Non-coding RNAs (i.e., miR145-5p, miR-132-3p, let7a-5p, let7b-5p) were also considered. Results: Circulating hormonal values were not significantly affected by Kp10 or AEA; in the hypothalamus, Kp10 significantly increased GnRH mRNA and aromatase Cyp19, Kiss1, and Kiss1 receptor (Kiss1R) proteins. By contrast, AEA treatment affected the hypothalamic KS at the protein levels, with opposite effects on the ligand and receptor, and SR141716A was capable of attenuating the AEA effects. Among the considered non-coding RNA, only the expression of miR145-5p was positively affected by AEA but not by Kp10 treatment. Localization of Kiss1+/Kiss1R+ neurons in the arcuate nucleus revealed an increase of Kiss1R-expressing neurons in Kp10- and AEA-treated animals associated with enlargement of the lateral ventricles in Kp10-treated animals. In the brain and testis, the selected non-coding RNA was differently modulated by Kp10 or AEA. Lastly, in the testis, AEA treatment affected the KS at the protein levels, whereas Kp10 affected the intragonadal levels of CB1 and FAAH, the main modulator of the AEA tone. Changes in pubertal transition-related miRNAs and the intratesticular distribution of Kiss1, Kiss1R, CB1, and CB2 following KP and AEA treatment corroborate the KS-ECS crosstalk also showing that the CB1 receptor is involved in this interplay. Conclusion: For the first time in mammals, we report the modulation of the KS in both the hypothalamus and testis by AEA and revealed the KP-dependent modulation of CB1 and FAAH in the testis. KP involvement in the progression of spermatogenesis is also suggested.

Treadmill exercise has healing effects on obesity-induced sexual behavior disorder through kisspeptin and kiss1R expression in male rats.

The basic objective of this study was to examine the possible effects of treadmill exercise on obesity-related sexual behavior disorder in obese male rats and the role of kisspeptin in this effect. The rats were separated from their mothers at the age of 3 weeks, and classified into four groups as Control (C): normal diet-sedentary group, Exercise (E): normal diet-exercise group, Obese (O): high-fat diet-sedentary group, Obese + Exercise (O+E): high-fat diet-exercise grouSexual behavioral testing was conducted in the rats. At the end of the study, brain samples were taken from the animals for gene expression analyses. The treadmill exercise caused a significant increase in the O+E Group compared to the O Group in kisspeptin and kiss1R gene expression and in EF, ML, IL, MF, IF, III, EL, PEI, IR1, MFT, IFT, IRT sexual behavior parameters (p<0.05), and a significant decrease in ML, IL, III, EL sexual behavior parameters (p<0.05). Treadmill exercise caused a significant decrease in EF, ML, IL, MF, IF, III, EL, PEI, IR1, MFT, IFT, IRT sexual behavior parameters and kisspeptin and kiss1R gene expression in the hypothalamus, hippocampus, prefrontal cortex and corpus striatum in E Group compared to C Group (p<0.05), and a significant increase in ML, IL, III, EL sexual behavior parameters (p<0.05). Based on this effect, we believe that it is caused by an increase in kisspeptin and kiss1R expression in the hypothalamus, hippocampus, prefrontal cortex and corpus striatum. In conclusion, treadmill exercise-induced kisspeptin secretion may increase GnRH secretion and cause hypothalamo-pituitary gonadal axis activation and ameliorative effect on deteriorated sexual function.

Recapitulating thyroid cancer histotypes through engineering embryonic stem cells.

Thyroid carcinoma (TC) is the most common malignancy of endocrine organs. The cell subpopulation in the lineage hierarchy that serves as cell of origin for the different TC histotypes is unknown. Human embryonic stem cells (hESCs) with appropriate in vitro stimulation undergo sequential differentiation into thyroid progenitor cells (TPCs-day 22), which maturate into thyrocytes (day 30). Here, we create follicular cell-derived TCs of all the different histotypes based on specific genomic alterations delivered by CRISPR-Cas9 in hESC-derived TPCs. Specifically, TPCs harboring BRAFV600E or NRASQ61R mutations generate papillary or follicular TC, respectively, whereas addition of TP53R248Q generate undifferentiated TCs. Of note, TCs arise by engineering TPCs, whereas mature thyrocytes have a very limited tumorigenic capacity. The same mutations result in teratocarcinomas when delivered in early differentiating hESCs. Tissue Inhibitor of Metalloproteinase 1 (TIMP1)/Matrix metallopeptidase 9 (MMP9)/Cluster of differentiation 44 (CD44) ternary complex, in cooperation with Kisspeptin receptor (KISS1R), is involved in TC initiation and progression. Increasing radioiodine uptake, KISS1R and TIMP1 targeting may represent a therapeutic adjuvant option for undifferentiated TCs.

Hypothalamic kisspeptin and kisspeptin receptors: Species variation in reproduction and reproductive behaviours.

Kisspeptin, encoded by the KISS1 gene, was first discovered as a potential metastasis suppressor gene. The prepro-kisspeptin precursor is cleaved into shorter mature bioactive peptides of varying sizes that bind to the G protein-coupled receptor GPR54 (=KISS1R). Over the last two decades, multiple types of Kiss and KissR genes have been discovered in mammalian and non-mammalian vertebrate species, but they are remarkably absent in birds. Kiss neuronal populations are distributed mainly in the hypothalamus. The KissRs are widely distributed in the brain, including the hypothalamic and non-hypothalamic regions, such as the hippocampus, amygdala, and habenula. The role of KISS1-KISS1R in humans and Kiss1-Kiss1R in rodents is associated with puberty, gonadal maturation, and the reproductive axis. However, recent gene deletion studies in zebrafish and medaka have provided controversial results, suggesting that the reproductive role of kiss is dispensable. This review highlights the evolutionary history, localisation, and significance of Kiss-KissR in reproduction and reproductive behaviours in mammalian and non-mammalian vertebrates.

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.

Role of Kisspeptin in Regulation of Reproductive and Immune Reactions.

The work is focused on physiological role of the hormone kisspeptin produced by neurons of the hypothalamus anterior zone, which is a key regulator of reproduction processes. Role of the hormone in transmission of information on metabolic activity and induction of the secretion of gonadotropin-releasing hormone (GnRH) by the hypothalamus that determines gestation processes involving fertilization, placentation, fetal development, and child birth is considered. The literature data on molecular mechanisms and effects of kisspeptin on reproductive system including puberty initiation are summarized and analyzed. In addition, attention is paid to hormone-mediated changes in the cardiovascular system in pregnant women. For the first time, the review examines the effect of kisspeptin on functional activity of immune system cells presenting molecular mechanisms of the hormone signal transduction on the level of lymphoid cells that lead to the immune tolerance induction. In conclusion, a conceptual model is presented that determines the role of kisspeptin as an integrator of reproductive and immune functions during pregnancy.

Thermoneutral conditions correct the obese phenotype in female, but not male, Kiss1r knockout mice.

Kisspeptin, a neuropeptide that activates gonadotropin-releasing hormone (GnRH) neurons, has also been implicated as a regulator of energy balance. Kisspeptin receptor (Kiss1r) knockout (KO) mice display an obese phenotype in adulthood compared to wild-type (WT) controls due to reduced energy expenditure. Additionally, experimental evidence shows that the temperature of typical rodent housing conditions (22 °C) increases the metabolism of mice above basal levels. Female Kiss1r KO mice show reduced core temperature and impaired temperature adaptation to an acute cold challenge, suggesting their temperature homeostasis processes are altered. The present study examined the phenotype of gonadectomised Kiss1r KO mice at both sub-thermoneutral and thermoneutral temperature (22 °C and 30 °C). Our results confirmed the obese phenotype in Kiss1r KO mice at 22 °C, and revealed a sexually dimorphic effect of thermal neutrality on the phenotype. In female KO mice, the obesity observed at 22 °C was attenuated at 30 °C. Plasma leptin levels were higher in KO than WT female mice at 22 °C (P < 0.001) but not at 30 °C. Importantly, the expression of Ucp1 mRNA in brown adipose tissue was lower in KO mice compared to WT mice at 22 °C (P < 0.05), but not different from WT at 30 °C. In male KO mice, a metabolic phenotype was observed at 22 °C and 30 °C. These results provide further evidence for kisspeptin-mediated regulation of adiposity via altered energy expenditure. Moreover, thermoneutral housing alleviated the obese phenotype in female Kiss1r KO mice, compared to WT, indicating the impairment in these mice may relate to an inability to adapt to the chronic cold stress that is experienced at 22 °C.

Cloning, characterisation and expression profile of kisspeptin1 and the kisspeptin1 receptor in the hypothalamic-pituitary-ovarian axis of Chinese alligator Alligator sinensis during the reproductive cycle.

Kisspeptin1 (Kiss1), a product of the Kiss1 gene, plays an important role in the regulation of reproduction in vertebrates by activating the Kiss1 receptor (Kiss1R) and its coexpression with gonadotrophin-releasing hormone (GnRH) in GnRH neurons. The purpose of this study was to clone the Kiss1 and Kiss1R genes found in the brain of Alligator sinensis and to explore their relationship with reproduction. The full-length cDNA of Kiss1 is 816bp, the open reading frame (ORF) is 417bp and the gene encodes a 138-amino acid precursor protein. The full-length cDNA of Kiss1R is 2348bp, the ORF is 1086bp and the gene encodes a 361-amino acid protein. Quantitative polymerase chain reaction showed that, except for Kiss1R expression in the hypothalamus, the expression of Kiss1 and Kiss1Rduring the reproductive period of A. sinensis was higher than that in the hypothalamus, pituitary gland and ovary during the hibernation period. The changes in GnRH2 mRNA in the hypothalamus were similar to those of GnRH1 and peaked during the reproductive period. This study confirms the existence of Kiss1 and Kiss1R in A. sinensis and the findings strongly suggest that Kiss1 and Kiss1R may participate in the regulation of GnRH secretion in the hypothalamus of alligators during the reproductive period. Furthermore, this is the first report of the full-length cDNA sequences of Kiss1 and Kiss1R in reptiles.

Expression of genes for Kisspeptin (KISS1), Neurokinin B (TAC3), Prodynorphin (PDYN), and gonadotropin inhibitory hormone (RFRP) across natural puberty in ewes.

Expression of particular genes in hypothami of ewes was measured across the natural pubertal transition by in situ hybridization. The ewes were allocated to three groups (n = 4); prepubertal, postpubertal and postpubertally gonadectomized (GDX). Prepubertal sheep were euthanized at 20 weeks of age and postpubertal animals at 32 weeks. GDX sheep were also euthanized at 32 weeks, 1 week after surgery. Expression of KISS1, TAC3, PDYN in the arcuate nucleus (ARC), RFRP in the dorsomedial hypothalamus and GNRH1 in the preoptic area was quantified on a cellular basis. KISS1R expression by GNRH1 cells was quantified by double-label in situ hybridization. Across puberty, detectable KISS1 cell number increased in the caudal ARC and whilst PDYN cell numbers were low, numbers increased in the rostral ARC. TAC3 expression did not change but RFRP expression/cell was reduced across puberty. There was no change across puberty in the number of GNRH1 cells that expressed the kisspeptin receptor (KISS1R). GDX shortly after puberty did not increase expression of any of the genes of interest. We conclude that KISS1 expression in the ARC increases during puberty in ewes and this may be a causative factor in the pubertal activation of the reproductive axis. A reduction in expression of RFRP may be a factor in the onset of puberty, removing negative tone on GNRH1 cells. The lack of changes in expression of genes following GDX suggest that the effects of gonadal hormones may differ in young and mature animals.

Expression of Kisspeptin and its receptor in the hypothalamus of cyclic and acyclic buffalo (Bubalus bubalis).

Kisspeptin (Kiss1), neurokinin-B (NKB) and dynorphin (Dyn) neurons regulate the surge and pulsatile centres of gonadotropin releasing hormone (GnRH) in the hypothalamus and are modulated by the ovarian steroids. Accordingly, we studied the temporospatial expression of Kiss1, its receptor and other genes that regulate GnRH in the preoptic area (POA) and arcuate (ARC) regions of hypothalamus at different phases of bubaline estrous cycle. Brain of buffalo (n = 32) was collected immediately after exsanguination and categorized into early luteal (EL), mid luteal (ML), follicular (FL) stages and acyclic (n = 8/group). Total RNA was extracted from the POA and ARC of each stage and real time PCR amplification of Kiss1, Kiss1r, NKB, NKB receptor (NKBR), Dyn, Dyn receptor (OPRK1), GnRH1, ERα, PR, LEPR and GHSR was done using GAPDH as endogenous control and acyclic stage as calibrator group. Further, immunolocalization of Kiss1 and Kiss1r was done on the hypothalamus. In the POA, significant up-regulation of Kiss1 and NKB with a concomitant down-regulation of Dyn transcripts was recorded at FL stage. There was, however, down-regulation of Kiss1 and Kiss1r during the EL perhaps due to the loss of estradiol as a consequence of ovulation. On the other hand, in the ARC, there was a significant up-regulation of Kiss1 and Dyn at FL and ML, while NKB transcript was consistently down-regulated at any stage of estrous cycle. In the POA, expression of ERα was not modulated; however, PR was down-regulated in the EL. In the ARC, the ERα expression was significantly up-regulated in the EL, whereas, PR was moderately expressed irrespective of the stage of estrous cycle. The immunolocalization study revealed the presence of Kiss1 and Kiss1r in the POA and ARC in the cyclic buffalo with relative abundance at FL. The transcriptional profile of the genes suggests that there is estrous cycle stage specific expression of Kiss1, Kiss1r and other GnRH regulating genes in the POA and ARC regions of hypothalamus in the buffalo. Up-regulation of Kiss1r in the POA during ML and ARC during EL indicates the involvement of kisspeptinergic system in the regulation of low LH pulse frequencies during the early and mid luteal phases in the cyclic buffalo.

The Impact of Morphine on Reproductive Activity in Male Rats Is Regulated by Rf-Amid-Related Peptide-3 and Substance P Adjusting Hypothalamic Kisspeptin Expression.

Obviously, opiates (e.g., morphine) are associated with the suppression and dysfunction of reproductive axis. It has been reported that substance P (SP) and RF-amid-related peptide-3 (RFRP-3) can exhibit anti-opioid effects in some regions of the nervous system. Moreover, SP and RFRP-3 are deemed as neuropeptides which exert modulatory and regulatory impacts on the function of the reproductive axis. The precise interactions of morphine with SP or RFRP-3 on the parameters of the reproductive activity, however, are not fully known. The present study was aimed to determine the impacts of the interaction of morphine either with SP or RFRP-3 on the hormonal and behavioral parameters of reproductive activity in male rats. In addition, it was aimed at determining whether the effects of these interactions rely on kisspeptin/G protein coupled receptor 54 (GPR54) pathway as the main upstream pulse generator and the mediator of the function of many inputs of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) system or not. Altogether, the resulted data from the sexual behavior tests, radioimmunoassay of LH/testosterone, and real-time quantitative PCR for the assessment of the expression of hypothalamic Kiss1, Gpr54, and Gnrh1 genes following concomitant administration of morphine with SP or RFRP-3 revealed that the suppressing effects of morphine on the parameters of reproductive axis activity can be affected by the administration of either RFRP-3 or SP. It is advocated that SP and RFRP-3, by the modulation of the expression of hypothalamic Kiss1, can possibly antagonize the effects of morphine on GnRH/LH system and sexual behavior.

Kiss1 and its receptor: molecular characterization and immunolocalization in the hypothalamus and corpus luteum of the buffalo.

ABSTARCT The neuropeptide kisspeptin (Kp) through its receptor Kiss1r regulates the HPG axis by controlling GnRH release. Since buffalo is a seasonal breeder with problems of delayed puberty and postpartum anestrus, we characterized the Kiss1 and Kiss1r and investigated the immunolocalization in the hypothalamus and corpus luteum (CL). Kiss1 and Kiss1r genes were amplified from gDNA covering the coding region, cloned and sequenced with accession numbers MF168937 and MG820539, respectively. The Kiss1 DNA sequence had two exonic segment contained coding sequence (cds); 408 bp encoding a predicted protein of 136 aa with conservation of Kp-10 and shared 94.5-98.3% identity with ruminants. Kiss1r DNA sequence consisted of five exons with a cds of 1134 bp encoding a protein of 378 aa. Phylogenetic analysis of Kiss1 and Kiss1r revealed that it formed a monophyletic clade with cattle, which branched from sheep and goat. Immunofluorescence study revealed the presence of Kiss1 and Kiss1r in the neuronal soma and perinuclear area of preoptic and arcuate regions of the hypothalamus and luteal cells of the CL. This is the first report on molecular characterization of bubaline Kiss1 and Kiss1r genes that confirmed the presence of conserved Kp-10 like other ruminants and kisspeptinergic system is present in the hypothalamus and CL.

Neonatal and juvenile exposure to perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS): Advance puberty onset and kisspeptin system disturbance in female rats.

Perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS) are widespread and persistent chemicals in the environment, and limited data about their effects on puberty development are available. In order to explore the effects of neonatal and juvenile PFOA/PFOS exposure on puberty maturation, female rats were injected with PFOA or PFOS at 0.1, 1 and 10 mg/kg/day during postnatal day (PND) 1-5 or 26-30. The day of vaginal opening (VO) and first estrus were significantly advanced in 10 mg/kg PFOA, 1 and 10 mg/kg PFOS groups after neonatal and juvenile exposure. Besides, neonatal PFOA/PFOS exposure increased body weight and anogenital distance (AGD) in a non-dose-dependent manner. Estradiol and luteinizing hormone levels were also increased with more frequent occurrences of irregular estrous cycles in 0.1 and 1 mg/kg PFOA/PFOS exposure groups. Although no altered ovarian morphology was observed, follicles numbers were reduced in neonatal groups. Kiss1, Kiss1r and ERα mRNA expressions were downregulated after two periods' exposure in the hypothalamic anteroventral periventricular (AVPV) and arcuate (ARC) nuclei. PFOA/PFOS exposure also suppressed kisspeptin fiber intensities, especially at the high dose. In conclusion, neonatal and juvenile are critical exposure periods, during which puberty maturation may be vulnerable to environmental exposure of PFOA/PFOS, and kisspeptin system plays a key role during these processes.

Effect of corticotropin releasing hormone and corticotropin releasing hormone antagonist on biosynthesis of gonadotropin relasing hormone and gonadotropin relasing hormone receptor in the hypothalamic-pituitary unit of follicular-phase ewes and contribution of kisspeptin.

This study aimed to determine the mechanisms governing Gonadotropin releasing hormone (GnRH) biosynthesis and luteinising hormone (LH) secretion in follicular-phase sheep after infusion of corticotropin releasing hormone (CRH) and/or CRH antagonist corticotropin releasing hormone nist (CRH-A) into the third cerebral ventricle. The study included two experimental approaches: first, we investigated the effect of CRH or CRH-A (α-helical CRH 9-41) on GnRH and GnRH receptor (GnRHR) biosynthesis in the preoptic area (POA), anterior (AH) and ventromedial hypothalamus (VMH), stalk/median eminence (SME), and on GnRHR in the anterior pituitary (AP) using an enzyme-linked immunosorbent assay (ELISA); second, we used real-time PCR to analyse the influence of CRH and CRH-A on the levels of kisspeptin (Kiss1) mRNA in POA and VMH including arcuate nucleus (VMH/ARC), and on Kiss1 receptor (Kiss1r) mRNA abundance in POA-hypothalamic structures. These analyses were supplemented by radioimmunoassay (RIA) and ELISA methods for measurement of LH and cortisol levels in the blood, respectively. Our results show that administration of CRH significantly decreased GnRH biosynthesis in the POA/hypothalamus. CRH also decreased GnRHR abundance in the hypothalamus and in the AP, but increased it in the POA. Furthermore, administration of CRH decreased plasma LH concentration and levels of Kiss1 mRNA in the POA and VMH/ARC as well as Kiss1r mRNA in these structures and in the SME. Significant increase in plasma cortisol concentration in the group treated with CRH was also observed. For CRH-A, all analysed effects were opposite to those induced by CRH. The study demonstrates that intracerebroventricular (i.c.v.) infusion of both CRH and CRH-A affects the GnRH/GnRHR biosynthesis and LH secretion in follicular-phase sheep conceivably via either central and peripheral mechanisms including Kiss1 neurons activity and cortisol signals. It has also been suggested that CRH and CRH-A infusion probably had effects directly at the AP.

Downregulation of leptin receptor and kisspeptin/GPR54 in the murine hypothalamus contributes to male hypogonadism caused by high-fat diet-induced obesity.

PURPOSE: Obesity may lead to male hypogonadism, the underlying mechanism of which remains unclear. In the present study, we established a murine model of male hypogonadism caused by high-fat diet-induced obesity to verify the following hypotheses: 1) an increased leptin level may be related to decreased secretion of GnRH in obese males, and 2) repression of kisspeptin/GPR54 in the hypothalamus, which is associated with increased leptin levels, may account for the decreased secretion of GnRH and be involved in secondary hypogonadism (SH) in obese males. METHODS: Male mice were fed high-fat diet for 19 weeks and divided by body weight gain into diet-induced obesity (DIO) and diet-induced obesity resistant (DIO-R) group. The effect of obesity on the reproductive organs in male mice was observed by measuring sperm count and spermatozoid motility, relative to testis and epididymis weight, testosterone levels, and pathologic changes. Leptin, testosterone, estrogen, and LH in serum were detected by ELISA method. Leptin receptor (Ob-R), Kiss1, GPR54, and GnRH mRNA were measured by real-time PCR in the hypothalamus. Expression of kisspeptin and Ob-R protein was determined by Western blotting. Expression of GnRH and GPR54 protein was determined by immunohistochemical analysis. RESULTS: We found that diet-induced obesity decreased spermatozoid motility, testis and epididymis relative coefficients, and plasma testosterone and luteinizing hormone levels. An increased number and volume of lipid droplets in Leydig cells were observed in the DIO group compared to the control group. Significantly, higher serum leptin levels were found in the DIO and DIO-R groups. The DIO and DIO-R groups showed significant downregulation of the GnRH, Kiss1, GPR54, and Ob-R genes. We also found decreased levels of GnRH, kisspeptin, GPR54, and Ob-R protein in the DIO and DIO-R groups. CONCLUSIONS: These lines of evidence suggest that downregulation of Ob-R and kisspeptin/GPR54 in the murine hypothalamus may contribute to male hypogonadism caused by high-fat diet-induced obesity.

Altered aspects of anxiety-related behavior in kisspeptin receptor-deleted male mice.

The roles of kisspeptin signaling outside the hypothalamus in the brain are unknown. We examined here the impact of Kiss1r-deletion on hippocampus-related behaviors of anxiety and spatial learning in adult male mice using two mouse models. In the first, global Kiss1r-null and control mice were gonadectomized (GDX KISS1R-KO). In the second, KISS1R signalling was rescued selectively in gonadotropin-releasing hormone neurons to generate Kiss1r-null mice with normal testosterone levels (intact KISS1R-KO). Intact KISS1R-KO rescue mice were found to spend twice as much time in the open arms of the elevated plus maze (EPM) compared to controls (P < 0.01). GDX KISS1R-KO mice showed a similar but less pronounced trend. No differences were detected between intact KISS1R-KO mice and controls in the open field test (OFT), although a marked reduction in time spent in the centre quadrant was observed for all GDX mice (P < 0.001). No effects of KISS1R deletion or gonadectomy were detected in the Morris water maze. These observations demonstrate that KISS1R signalling impacts upon anxiogenic neural circuits operative in the EPM, while gonadal steroids appear important for anxiety behaviour observed in the OFT. The potential anxiogenic role of kisspeptin may need to be considered in the development of kisspeptin analogs for the clinic.

The effects of chronic testosterone administration on hypothalamic gonadotropin-releasing hormone regulatory factors (Kiss1, NKB, pDyn and RFRP) and their receptors in female rats.

The effects of androgens on gonadotropin-releasing hormone (GnRH) secretion in females have not been fully established. To clarify the direct effects of androgens on hypothalamic reproductive factors, we evaluated the effects of chronic testosterone administration on hypothalamic GnRH regulatory factors in ovariectomized (OVX) female rats. Both testosterone and estradiol reduced the serum luteinizing hormone levels of OVX female rats, indicating that, as has been found for estrogen, testosterone suppresses GnRH secretion via negative feedback. Similarly, the administration of testosterone or estradiol suppressed the hypothalamic mRNA levels of kisspeptin and neurokinin B, both of which are positive regulators of GnRH, whereas it did not affect the hypothalamic mRNA levels of the kisspeptin receptor or neurokinin-3 receptor. On the contrary, the administration of testosterone, but not estradiol, suppressed the hypothalamic mRNA expression of prodynorphin, which is a negative regulator of GnRH. The administration of testosterone did not alter the rats' serum estradiol levels, indicating that testosterone's effects on hypothalamic factors might be induced by its androgenic activity. These findings suggest that as well as estrogen, androgens have negative feedback effects on GnRH in females and that the underlying mechanisms responsible for these effects are similar, but do not completely correspond, to the mechanisms underlying the effects of estrogen on GnRH.

Effects of N-carbamylglutamate and L-arginine on gonadotrophin-releasing hormone (GnRH) gene expression and secretion in GT1-7 cells.

Recent studies have shown that N-carbamylglutamate (NCG) and arginine (ARG) supplementation improves reproductive performance in livestock. The objectives of the present study were to evaluate the effects of NCG and ARG on GT1-7 cell gonadotrophin-releasing hormone (GnRH) secretion, gene expression and cell proliferation. GT1-7 cells were treated in vitro with different concentrations of NCG (0-1.0mM) or ARG (0-4.0mM) in serum-free medium for 12 or 24h. For GnRH secretion and cell proliferation, GT1-7 cells were more sensitive to NCG than ARG. NCG treatment after 12h increased cell numbers and inhibited GnRH secretion in a dose-dependent manner (P<0.05), although there was no significant effect of NCG on these parameters after 24h culture. ARG treatment decreased GnRH secretion after 24h (P<0.05), whereas it had no effect after 12h. GT1-7 cells express GnRH, Kiss-1 metastasis-suppressor (Kiss1), G-protein coupled receptor 54 (GPR54), neuronal nitric oxide synthase (nNOS) and estrogen receptor α (ERα) genes. High concentrations of NCG (1.0mM) and ARG (4.0mM) inhibited (P<0.05) GnRH and nNOS mRNA abundance in GT1-7 cells. ARG treatment decreased Kiss1 and increased ERα mRNA abundance. Thus, high concentrations of NCG (1.0mM) and ARG (4.0mM) may act both directly and indirectly to regulate GnRH neuron function by downregulating genes related to GnRH synthesis and secretion to slow GnRH production while stimulating GT1-7 cell proliferation.

Ontogeny of clock and KiSS-1 metastasis-suppressor (Kiss1) gene expression in the prepubertal mouse hypothalamus.

Kisspeptin is crucial for the generation of the circadian-gated preovulatory gonadotrophin-releasing hormone (GnRH)-LH surge in female rodents, with expression in the anteroventral periventricular nucleus (AVPV) peaking in the late afternoon of pro-oestrus. Given kisspeptin expression is established before puberty, the aim of the present study was to investigate kisspeptin and clock gene rhythms during the neonatal period. Anterior and posterior hypothalami were collected from C57BL/6J mice on Postnatal Days (P) 5, 15 and 25, at six time points across 24h, for analysis of gene expression by reverse transcription-quantitative polymerase chain reaction. Expression of aryl hydrocarbon receptor nuclear translocator-like gene (Bmal1) and nuclear receptor subfamily 1, group D, member 2 (Rev-erbα) in the anterior hypothalamus (containing the suprachiasmatic nucleus) was not rhythmic at P5 or P15, but Bmal1 expression exhibited rhythmicity in P25 females, whereas Rev-erbα expression was rhythmic in P25 males. KiSS-1 metastasis-suppressor (Kiss1) expression did not exhibit time-of-day variation in the anterior (containing the AVPV) or posterior (containing the arcuate nucleus) hypothalami in female and male mice at P5, P15 or P25. The data indicate that the kisspeptin circadian peak in expression observed in the AVPV of pro-oestrous females does not manifest at P5, P15 or P25, likely due to inadequate oestrogenic stimuli, as well as incomplete development of clock gene rhythmicity before puberty.

Kisspeptin permits the sexual development of female rats with normal and precocious puberty but is not a trigger for it.

OBJECTIVE: We inferred how KISS-1/GPR54 system to involved in precocious puberty by observing hormones level during the process of precocious puberty in model and normal rats during sexual development and the estrus cycle. METHOD: Female rats were divided randomly into CPP and control groups; the former were injected with NMDA twice daily, and control groups were injected with saline. Blood and tissue samples were collected and measured during the stages of prepuberty, vaginal opening, estrus, proestrus and diestrus. RESULTS: The times of onset of puberty and sexual maturity in the CPP group were significantly earlier than in the control groups. Hypothalamic levels of KISS-1 and GPR54 gene expression, kisspeptin, luteinizing hormone, and follicle stimulating hormone started to rise before puberty. In stable estrus cycles, kisspeptin levels were the lowest during proestrus, while gonadotropin-releasing hormone (GnRH) levels rose to the highest during estrus. GnRH levels increased significantly in the estrus cycle compared with the prepubertal stage, but kisspeptin levels did not change significantly. CONCLUSION: the hypothalamic KISS-1/GPR54 system might permit the onset of puberty, but is not its primary trigger. Hormone levels were lower and gonadal maturity markers in the CPP groups were worse than in the control groups.