The ontogeny of kisspeptin receptor in the uterine contractions in rats: Its possible role in the quiescence of non-pregnant and pregnant uteri.

The objectives of this study were to investigate the effects of KISS1 94-121 fragment on the contractility of non-pregnant and pregnant rat uteri, and to determine the uterine and myometrial expressions of Kiss1r. Uterine muscle strips were obtained from non-pregnant Sprague-Dawley rats in oestrous phase and from pregnant rats on gestational days 5, 15, 18, 20 or 22. The in vitro contractility measurements were carried out in an isolated organ bath in the presence of KISS1 94-121. Experiments with 5-day pregnant tissues were also performed in the presence of kisspeptin-234 trifluoroacetate. The mRNA and protein expressions of Kiss1r were measured by RT-PCR and Western blot analysis, while localizations of receptors were defined by fluorescent immunohistochemistry. KISS1 94-121 induced a dose-dependent relaxation both in non-pregnant and pregnant intact and endometrium-denuded uteri. A gradual decrease was found in the uterine expressions of Kiss1r mRNA and protein towards the end of the gestational period, and it was further confirmed by the immunohistochemical results. The significant majority of Kiss1r is found in the myometrium, however the few endometrial Kiss1r also influences the uterine contractions. The relaxing effect of kisspeptin is continuously reduced towards the end of gestational period in parallel with the reduction of Kiss1r expression. Our results suggest a putative role of kisspeptin in the maintenance of uterine quiescence that may have significance in miscarriage or preterm contractions.

Kisspeptin receptor agonist has therapeutic potential for female reproductive disorders.

BACKGROUNDKisspeptin is a key regulator of hypothalamic gonadotropin-releasing hormone (GnRH) neurons and is essential for reproductive health. A specific kisspeptin receptor (KISS1R) agonist could significantly expand the potential clinical utility of therapeutics targeting the kisspeptin pathway. Herein, we investigate the effects of a KISS1R agonist, MVT-602, in healthy women and in women with reproductive disorders.METHODSWe conducted in vivo and in vitro studies to characterize the action of MVT-602 in comparison with native kisspeptin-54 (KP54). We determined the pharmacokinetic and pharmacodynamic properties of MVT-602 (doses 0.01 and 0.03 nmol/kg) versus KP54 (9.6 nmol/kg) in the follicular phase of healthy women (n = 9), and in women with polycystic ovary syndrome (PCOS; n = 6) or hypothalamic amenorrhea (HA; n = 6). Further, we investigated their effects on KISS1R-mediated inositol monophosphate (IP1) and Ca2+ signaling in cell lines and on action potential firing of GnRH neurons in brain slices.RESULTSIn healthy women, the amplitude of luteinizing hormone (LH) rise was similar to that after KP54, but peaked later (21.4 vs. 4.7 hours; P = 0.0002), with correspondingly increased AUC of LH exposure (169.0 vs. 38.5 IU∙h/L; P = 0.0058). LH increases following MVT-602 were similar in PCOS and healthy women, but advanced in HA (P = 0.004). In keeping with the clinical data, MVT-602 induced more potent signaling of KISS1R-mediated IP1 accumulation and a longer duration of GnRH neuron firing than KP54 (115 vs. 55 minutes; P = 0.0012).CONCLUSIONTaken together, these clinical and mechanistic data identify MVT-602 as having considerable therapeutic potential for the treatment of female reproductive disorders.TRIAL REGISTRATIONInternational Standard Randomised Controlled Trial Number (ISRCTN) Registry, ISRCTN21681316.FUNDINGNational Institute for Health Research and NIH.

Role for Kisspeptin and Neurokinin B in Regulation of Luteinizing Hormone and Testosterone Secretion in the Fetal Sheep.

Evidence suggests that the hypothalamic-pituitary-gonadal (HPG) axis is active during the critical period for sexual differentiation of the ovine sexually dimorphic nucleus, which occurs between gestational day (GD) 60 and 90. Two possible neuropeptides that could activate the fetal HPG axis are kisspeptin and neurokinin B (NKB). We used GD85 fetal lambs to determine whether intravenous administration of kisspeptin-10 (KP-10) or senktide (NKB agonist) could elicit luteinizing hormone (LH) release. Immunohistochemistry and fluorescent in situ hybridization (FISH) were employed to localize these peptides in brains of GD60 and GD85 lamb fetuses. In anesthetized fetuses, KP-10 elicited robust release of LH that was accompanied by a delayed rise in serum testosterone in males. Pretreatment with the GnRH receptor antagonist (acyline) abolished the LH response to KP-10, confirming a hypothalamic site of action. In unanesthetized fetuses, senktide, as well as KP-10, elicited LH release. The senktide response of females was greater than that of males, indicating a difference in NKB sensitivity between sexes. Gonadotropin-releasing hormone also induced a greater LH discharge in females than in males, indicating that testosterone negative feedback is mediated through pituitary gonadotrophs. Kisspeptin and NKB immunoreactive cells in the arcuate nucleus were more abundant in females than in males. Greater than 85% of arcuate kisspeptin cells costained for NKB. FISH revealed that the majority of these were kisspeptin/NKB/dynorphin (KNDy) neurons. These results support the hypothesis that kisspeptin-GnRH signaling regulates the reproductive axis of the ovine fetus during the prenatal critical period acting to maintain a stable androgen milieu necessary for brain masculinization.

Kisspeptin and Glucose Homeostasis.

Kisspeptin has well-established critical roles in the control of reproduction and fertility. Recently, evidence has emerged that suggests kisspeptin may have additional roles in the regulation of glucose homeostasis. Conflicting reports on the effects of kisspeptin on insulin secretion in animal models have been published, which cannot be fully accounted for by the different kisspeptin isoforms and range of kisspeptin doses used in these studies. Human studies have demonstrated associations between circulating kisspeptin levels and measures of insulin secretion and insulin resistance; and the only published interventional study has confirmed kisspeptin enhances glucose-stimulated insulin secretion in humans. Further studies are required to elucidate the mechanisms underlying the effects of kisspeptin on the pancreatic ß-cell and to determine the therapeutic potential of kisspeptin receptor agonist in the treatment of disorders of glucose homeostasis.

Towards new strategies to manage livestock reproduction using kisspeptin analogs.

The discovery of the hypothalamic neuropeptide kisspeptin and its receptor (KISS1R) have dramatically improved our knowledge about the central mechanisms controlling reproduction. Kisspeptin neurons could be considered the hub where internal and external information controlling reproduction converge. The information is here elaborated and the command dispatched to GnRH neurons, the final output of the brain system controlling reproduction. Several studies have shown that in mammals administration of kisspeptin could finely modulate many aspects of reproduction from puberty to ovulation. For example in ewes kisspeptin infusion triggered ovulation during the non-breeding season and in prepubertal rat repeated injections advanced puberty onset. However, especially in livestock, the suboptimal pharmacological properties of endogenous kisspeptin, notably it short half-life and consequently its poor pharmacodynamics, fetters its use to experimental setting. To overcome this issue synthetic KISS1R agonists, mainly based on kisspeptin backbone, were created. Their more favorable pharmacological profile, longer half-life and duration of action, allowed to perform promising initial experiments for controlling ovulation and puberty. Additional experiments and further refinement of analogs would still be necessary to exploit fully the potential of targeting the kisspeptin system. Nevertheless, it is already clear that this new strategy may represent a breakthrough in the field of reproduction control.

Kisspeptin regulates steroidogenesis and spermiation in anuran amphibian.

Kisspeptin (Kp) system has a recognized role in the control of gonadotropic axis, at multiple levels. Recently, a major focus of research has been to assess any direct activity of this system on testis physiology. Using the amphibian anuran, Pelophylax esculentus, as animal model, we demonstrate - for the first time in non-mammalian vertebrate - that testis expresses both Kiss-1 and Gpr54 proteins during the annual sexual cycle and that ex vivo 17B-estradiol (E2, 10-6 M) increases both proteins over control group. Since the interstitium is the main site of localization of both ligand and receptor, its possible involvement in the regulation of steroidogenesis has been evaluated by ex vivo treatment of testis pieces with increasing doses of Kp-10 (10-9-10-6 M). Treatments have been carried out in February - when a new wave of spermatogenesis occurs - and affect the expression of key enzymes of steroidogenesis inducing opposite effects on testosterone and estradiol intratesticular levels. Morphological analysis of Kp-treated testes reveals higher number of tubules with spermatozoa detached from Sertoli cells than control group and the expression of connexin 43, the main junctional protein in testis, is deeply affected by the treatment. In spite of the effects on spermatozoa observed ex vivo, in vivo administration of Kp-10 has been unable to induce sperm release in cloacal fluid. In conclusion, we demonstrate Kp-10 effects on steroidogenesis with possible involvement in the balance between testosterone and estradiol levels, and report new Kp-10 activities on spermatozoa-Sertoli cell interaction.

Kisspeptin-10 inhibits OHSS by suppressing VEGF secretion.

The aim of the present study was to elucidate the effects of kisspeptin-10 (Kp-10) on ovarian hyperstimulation syndrome (OHSS) and its related mechanism in OHSS rat models, human umbilical vein endothelial cells (HUVECs) and human luteinized granulosa cells. OHSS is a systemic disorder with high vascular permeability (VP) and ovarian enlargement. KISS1R (KISS1 receptor) is the specific receptor of kisspeptin. The kisspeptin/KISS1R system inhibits the expression of vascular endothelial growth factor (VEGF), which is the main regulator of VP. In our study, decreased expression of Kiss1r was observed in both ovaries and lung tissue of OHSS rats. Injection of exogenous Kp-10 inhibited the increase of VP and VEGF while promoting the expression of Kiss1r in both the ovarian and lung tissue of OHSS rats. Using HUVECs, we revealed that a high level of 17-ß estradiol (E2), a feature of OHSS, suppressed the expression of KISS1R and increased VEGF and nitric oxide (NO) through estrogen receptors (ESR2). Furthermore, KISS1R mRNA also decreased in the luteinized human granulosa cells of high-risk OHSS patients, and was consistent with the results in rat models and HUVECs. In conclusion, Kp-10 prevents the increased VP of OHSS by the activation of KISS1R and the inhibition of VEGF.

Cross-Linking Furan-Modified Kisspeptin-10 to the KISS Receptor.

Chemical cross-linking is well-established for investigating protein-protein interactions. Traditionally, photo cross-linking is used but is associated with problems of selectivity and UV toxicity in a biological context. We here describe, with live cells and under normal growth conditions, selective cross-linking of a furan-modified peptide ligand to its membrane-presented receptor with zero toxicity, high efficiency, and spatio-specificity. Furan-modified kisspeptin-10 is covalently coupled to its glycosylated membrane receptor, GPR54(KISS1R). This newly expands the applicability of furan-mediated cross-linking not only to protein-protein cross-linking but also to cross-linking in situ. Moreover, in our earlier reports on nucleic acid interstrand cross-linking, furan activation required external triggers of oxidation (via addition of N-bromo succinimide or singlet oxygen). In contrast, we here show, for multiple cell lines, the spontaneous endogenous oxidation of the furan moiety with concurrent selective cross-link formation. We propose that reactive oxygen species produced by NADPH oxidase (NOX) enzymes form the cellular source establishing furan oxidation.