GPR54 Research Articles

Regulation of prostate cancer by hormone-responsive G protein-coupled receptors.

Regulation of prostate cancer by androgen and androgen receptor (AR), and blockade of AR signaling by AR antagonists and steroidogenic enzyme inhibitors have been extensively studied. G protein-coupled receptors (GPCRs) are a family of membrane receptors that regulate almost all physiological processes. Nearly 40% of FDA-approved drugs in the market target GPCRs. A variety of GPCRs that mediate reproductive function have been demonstrated to be involved in the regulation of prostate cancer. These GPCRs include gonadotropin-releasing hormone receptor, luteinizing hormone receptor, follicle-stimulating hormone receptor, relaxin receptor, ghrelin receptor, and kisspeptin receptor. We highlight here GPCR regulation of prostate cancer by these GPCRs. Further therapeutic approaches targeting these GPCRs for the treatment of prostate cancer are summarized.

The expression of kisspeptin and its receptor in the domestic cat ovary and uterus in different stages of the ovarian cycle

Kisspeptin is well known for its indispensable role in the regulation of reproduction, mainly through controlling the release of GnRH at the hypothalamic level. Recent studies have shown that kisspeptin and the kisspeptin receptor are expressed in the ovary and uterus, indicating an additional local function in reproduction at the extra-hypothalamic level. In this study, we aimed: (1) to investigate the localization pattern of kisspeptin and its receptor in the domestic cat ovary and uterus throughout the ovarian cycle using immunohistochemistry; and (2) to compare the relative expression of ovarian Kiss1 mRNA levels at different ovarian stages with qPCR analysis. Ovaries and uteri were collected and classified into three ovarian stages (inactive, follicular and luteal stages (n = 7 in each stage)) according to the ovarian morphology, vaginal cytology and serum progesterone. Kisspeptin immunoreactivity (Kp-IR) and kisspeptin receptor immunoreactivity (KpR-IR) were present in the ovaries and uteri at all ovarian stages, with no notable differences in the localization patterns between the ovarian stages. In the ovary, Kp-IR and KpR-IR were present in various ovarian compartments, including the follicles at all classes and the corpus luteum (CL). In the follicles, Kp-IR and KpR-IR were present in the oocytes, granulosa cells and theca cells. Kp-IR was also detected in the follicular fluid of antral follicles. In CL, a strong intensity of Kp-IR was present in the periphery CL of development/maintenance, with a relatively fainter intensity in the central CL. By contrast, KpR-IR was present in both peripheral and central CL at the same intensity. In the uterus, Kp-IR and KpR-IR were present in the uterine glands, myometrium and perimetrium. The relative ovarian Kiss1 mRNA level was higher in the follicular stage than in the luteal stage (P < 0.05). We concluded that kisspeptin and its receptor are present in the cat ovary and uterus, suggesting possible local functions of kisspeptin at the extra-hypothalamic level, such as folliculogenesis, oocyte survival and uterine adenogenesis.

Biological Significance of Kisspeptin-Kiss 1 Receptor Signaling in the Habenula of Teleost Species.

Kisspeptin is a neuropeptide, encoded by kisspeptin 1 (KISS1)/Kiss1 gene, which primarily acts as the regulator of reproductive functions via its receptor, kisspeptin receptor (KissR) in vertebrates. In the brain, Kiss1 gene is mainly expressed in the hypothalamic region, but KissR gene is widely distributed throughout the brain, suggesting that kisspeptin–KissR system may be involved in not only reproductive, but also non-reproductive functions. In non-mammalian vertebrates, there are two or more kisspeptin and KissR types. The zebrafish (Danio rerio) possess two kisspeptin (Kiss1 and Kiss2) and their respective receptors [Kiss1 receptor (KissR1) and KissR2]. In the brain of zebrafish, while Kiss2 is expressed in the preoptic-hypothalamic area, Kiss1 is predominantly expressed in the habenula, an evolutionarily conserved epithalamic structure. Similarly, KissR1 is expressed only in the habenula, while KissR2 is widely distributed in the brain, suggesting that the two kisspeptin systems play specific roles in the brain. The habenular Kiss1 is involved in the modulation of the raphe nuclei and serotonin-related behaviors such as fear response in the zebrafish. This review summarizes the roles of multiple kisspeptin–KissR systems in reproductive and non-reproductive functions and neuronal mechanism, and debates the biological and evolutional significance of habenular kisspeptin–KissR systems in teleost species.

Kisspeptin modulates pain sensitivity of CFLP mice

Kisspeptin, a hypothalamic neuropeptide, is a member of the RF-amide family, which have been known to modify pain sensitivity in rodents. The aim of the present study was to investigate the effect of kisspeptin-13 (KP-13), an endogenous derivative of kisspeptin, on nociception in adult male and female CFLP mice and the possible interaction of KP-13 with morphine on nociception.Mice were injected with different doses of KP-13, 30, 60 and 120 min after of which the nociceptive sensitivity were assessed via the tail-flick test. To investigate the receptor involved in the mediation a kisspeptin receptor antagonist (KP-234) pretreatment was applied before KP-13 administration. Furthermore, we investigated the effect of KP-13 on the acute antinociceptive effect of morphine, on acute morphine tolerance and on naloxone-precipitated withdrawal. Last, the Von Frey test was used in order to assess KP-13′s effect on mechanical nociception.Our results showed that KP-13 decreased the nociceptive threshold of both males and females independent of sex, which was prevented by KP-234. Furthermore, KP-13 treatment depressed the acute antinociceptive effect of morphine and attenuated the development of morphine tolerance. KP-13 also induced a mechanical hypersensitivity. These data underlie kisspeptin's hyperalgesic action and argues for the role of kisspeptin receptor 1 in the mediation of its action. Furthermore, our results suggest that central KP-13 administration can modify the acute effects of morphine.

The Emerging Role(s) for Kisspeptin in Metabolism in Mammals.

Kisspeptin was initially identified as a metastasis suppressor. Shortly after the initial discovery, a key physiologic role for kisspeptin emerged in the regulation of fertility, with kisspeptin acting as a neurotransmitter via the kisspeptin receptor, its cognate receptor, to regulate hypothalamic GnRH neurons, thereby affecting pituitary–gonadal function. Recent work has demonstrated a more expansive role for kisspeptin signaling in a variety of organ systems. Kisspeptin has been revealed as a significant player in regulating glucose homeostasis, feeding behavior, body composition as well as cardiac function. The direct impact of kisspeptin on peripheral metabolic tissues has only recently been recognized. Here, we review the emerging endocrine role of kisspeptin in regulating metabolic function. Controversies and current limitations in the field as well as areas of future studies toward kisspeptin’s diverse array of functions will be highlighted.

Normosmic idiopathic hypogonadotrophic hypogonadism due to a rare KISS1R gene mutation

SummaryHypogonadotrophic hypogonadism is due to impaired or reduced gonadotrophin secretion from the pituitary gland. In the absence of any anatomical or functional lesions of the pituitary or hypothalamic gland, the hypogonadotrophic hypogonadism is referred to as idiopathic hypogonadotrophic hypogonadism (IHH). We present a case of a young lady born to consanguineous parents who was found to have IHH due to a rare gene mutation.Learning points:The genetic basis of a majority of cases of IHH remains unknown.IHH can have different clinical endocrine manifestations.Patients can present late to the healthcare service because of unawareness and stigmata associated with the clinical features.Family members of affected individuals can be affected to varying degrees.

Functional examination of novel kisspeptin phosphinic peptides.

Kisspeptins acting on their cognate G protein-coupled receptor, kisspeptin receptor, play important roles in the suppression of cancer cell metastasis and regulation of the reproductive system, and therefore are important for therapeutic intervention. All native functional human kisspeptins (kisspeptin-54, kisspsptin-14 and kisspeptin-13) share the 10 amino acids of kisspeptin-10 at their C-terminus (45–54). However, they are inactivated rapidly by matrix metalloproteinases (MMPs) through the cleavage of the peptide bond between glycine51 and leucine52, which limits their clinical applications. Development of MMP-resistant analogues of kisspeptins may provide better therapeutic outputs. In the present study, two kisspeptin phosphinic peptides were designed and synthesized, and their ability to induce phosphorylation of ERK1/2 through kisspeptin receptor and their inhibition on MMP-2 and MMP-9 whose activity correlates with cancer metastasis were assessed. The results showed that one analogue, phosphinic kisspeptin R isomer (PKPR), exhibited kisspeptin receptor-agonistic activity and also inhibitory activity on MMP-2, indicating that PKPR may serve as a lead for the further development of kisspeptin analogues for therapeutic purpose.

Enhancing β‐cell Sensitivity to Kisspeptin via Linkage to GLP‐1

Glucagon Like Peptide‐1 (GLP‐1) and Kisspeptin (KP) receptors are co‐expressed on pancreatic β‐cells, and activation of both receptors potentiate glucose stimulated insulin secretion (GSIS), although they appear to work via different downstream signaling pathways. Unlike GLP‐1, KP has a low affinity for receptor activation (EC50 20 nM vs &gt;1 uM) making it difficult to use as a drug without significant side effects. We developed a bivalent ligand composed of GLP‐17–36 and KP‐10 binding domains linked together, to evaluate if linking KP to a high affinity ligand would promote its activity at low concentrations by presumably increasing its local membrane concentration after GLP‐1 binding. This feature may also provide some specificity for targeting to β‐cells which express both receptors. The effects of KP‐10, GLP‐1 and the KP‐10/GLP‐1 bivalent analog on insulin secretion from the INS 832/3 cell line were investigated. KP‐10 by itself had no effect on insulin secretion in the absence of glucose, but more than doubled GSIS (basal, 0.23 +/− 0.07; GSIS 0.60 +/− 0.02 ng/min) with an EC50 of ~25 μM. At saturating doses, KP‐10 (100 μm) and GLP‐1 (100 nM) had similar effects on GSIS, and together as monomers had an additive effect (0.79 +/− 0.07 (ng/min) consistent with activation of independent downstream signaling pathways. The dimer also did not activate secretion in the absence of glucose, but doubled GSIS much like the individual monomers (0.64 +/− 0.01 ng/min); that is, an additive response was not obvious. This finding suggests that either the KP‐10 moiety is not binding or is not active in the bivalent ligand, or that GLP‐1 activity is diminished in the bivalent construct such that the combined effect of GLP‐1/KP‐10 is diminished when compared to the additive effect or the monomers. We have evidence from previous studies that binding of the GLP‐1 moiety within a bivalent construct is decreased relative to monomeric GLP‐11, suggesting an additive response may be present. Currently, we are determining the ability of GLP‐1/KP‐10 to initiate downstream signaling from both ligands to resolve the question: is KP active in the GLP‐1/KP at concentrations where monomeric KP‐10 is not.Support or Funding InformationFunded by the American Diabetes Association and Juvenile Diabetes Research FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Stimulation of growth hormone by kisspeptin antagonists in ewes.

Kisspeptin signalling is indispensable for fertility, stimulating gonadotropin-releasing hormone (GnRH) secretion and mediating gonadal steroid feedback on GnRH neurons. Moreover, kisspeptin neurons have been implicated in other non-reproductive neuroendocrine roles. Kisspeptin appears to also regulate growth hormone secretion but much of the data appear contradictory. We sought to clarify a potential role of kisspeptin in growth hormone (GH) regulation by examining the effect of kisspeptin antagonists on GH secretion in ewes under various physiological conditions. Our data show clear and robust increases in GH secretion following lateral ventricle or third ventricle infusion of kisspeptin antagonists p-234 and p-271 in either ovariectomized or anestrous ewes. Central infusion of kisspeptin-10 had no effect on GH secretion. To determine the level at which kisspeptin may influence GH secretion, we examined expression of the cognate kisspeptin receptor, GPR54, in pituitary cells and showed by immunocytochemistry that the majority of somatotropes express GPR54 while expression was largely negative in other pituitary cells. Overall, we have demonstrated that blocking kisspeptin signalling by antagonists stimulates GH secretion in ewes and that this is likely mediated by inhibiting endogenous kisspeptin activation of GPR54 expressed on somatotropes. The findings suggest that endogenous kisspeptin inhibits GH secretion through GPR54 expressed on somatotropes.

Kisspeptin Receptor GPR54 Promotes Adipocyte Differentiation and Fat Accumulation in Mice.

GPR54, Kisspeptin-1 receptor (KISS1R), a member of rhodopsin family, plays a critical role in puberty development and has been proposed to be involved in regulation of energy metabolism. This study aims to explore the function of GPR54 in adipogenesis, lipid metabolism, and obesity in addition to its effect through hormones. Results showed that when fed a high-fat diet, the weight growth of castrated or ovariectomized Gpr54−/− mice was significantly slower than that of WT control, together with a lower triglyceride concentration. The ratio of white adipose tissue was lower, and average size of adipocytes was smaller in Gpr54−/− mice. Meanwhile, there were less adipose tissue macrophages (ATMs), especially pro-inflammatory macrophages. Expression of inflammatory related genes also indicated that inflammatory response caused by obesity was not as drastic in Gpr54−/− mice as in WT mice. Liver triglyceride in Gpr54−/− mice was reduced, especially in female mice. On the other hand, oil drop formation was accelerated when hepatocytes were stimulated by kisspeptin-10 (Kp-10). Primary mesenchymal stem cells (MSCs) of Gpr54−/− mice were less likely to differentiate into adipocytes. When stimulated by Kp-10, 3T3-L1 cell differentiation into adipocytes was accelerated and triglyceride synthesis was significantly promoted. These data indicated that GPR54 could affect obesity development by promoting adipocyte differentiation and triglyceride accumulation. To further elucidate the mechanism, genes related to lipid metabolism were analyzed. The expression of genes involved in lipid synthesis including PPARγ, ACC1, ADIPO, and FAS was significantly changed in Gpr54−/− mice. Among them PPARγ which also participate in adipocyte differentiation displayed a marked reduction. Moreover, phosphorylation of ERK, which involved in GPR54 signaling, was significantly decreased in Gpr54−/− mice, suggesting that GPR54 may promote lipid synthesis and obesity development by activating MAP kinase pathway. Therefore, in addition to the involvement in hormone regulation, our study demonstrated that GPR54 directly participates in obesity development by promoting adipocyte differentiation and fat accumulation. This provided evidence of involvement of GPR54 in lipid metabolism, and revealed new potentials for the identification and development of novel drug targets for metabolic diseases.

Research progress of height improvement in central precocious puberty

Central precocious puberty(CPP)is a disease that results from the premature activation of hypothalamic-pituitary-gonadal axis(before 8 years in girls and 9 years in boys). The CPP children show abnormally early appearance of secondary sexual characteristics.Genetic factors, ethnicity, environmenal factors have been involved in the pathogenesis of CPP.Gonadotropin-releasing hormone analogues(GnRHa)are the most common drugs for CPP.The factors of GnRHa treatment on height improvement include the age of puberty onset and the age of treatment initiation.The CPP girls diagnosed and treated before the age of 6 have the best effect, so CPP children need early treatment.Adequate treatment duration is an important factor to ensure height improvement, at least 2 years.The CPP children that are younger according to the bone age have more favorable outcomes of height improvement.By delaying epiphyseal closure, aromatase inhibitors can improve the height of CPP boys.KISS1、KISS1R 、MKRN3 and other genes are related to CPP , early identification of CPP genes can play a guiding role in early intervention.For the CPP girls treated with GnRHa, when the growth rate is less than 4cm/year, recombinant human growth hormone can effectively improve adult height.Appropriate aerobic exercise can also improve height.This article reviews the factors and strategies of height improvement in central precocious puberty. Key words: Central precocious puberty; Height improvement

The effects of interaction of Dopaminergic and Kisspeptin neural pathways on Ghrelin secretion in rats

Dopamine, kisspeptin and ghrelin are important factors for regulating the reproduction and food intake. Finding the effective central or peripheral factors on ghrelin secretion attracted the attention of most researches. In the present experimental study, the effects of dopamine, kisspeptin and the GPR54 receptor signaling pathway role in the mediating the dopamine effects was determined on ghrelin secretion. Forty Wistar male rats weighing 220-250g in 8 groups received saline, 1nmol kisspeptin, 5, 15 or 45 microgram dopamine hydrochloride, simultaneous injections of 1nmol peptide 234 and kisspeptin, 15microgram dopamine and kisspeptin or peptide234 and 15microgram dopamine via third cerebral ventricle respectively. Blood samples were collected via tail vein. Mean serum ghrelin concentrations were determined by rat ghrelin kit and ELISA method. Kisspeptin significantly decreased mean serum concentration compared to saline group, while 15 or 45 microgram dopamine significantly increased mean serum ghrelin level compared to saline group. Kisspeptin significantly blocked the stimulatory effects of dopamine on ghrelin secretion compared to dopamine group. Dopaminergic and kisspeptin/GPR54 signaling pathways may interact to control the ghrelin secretion at hypothalamic level. Stimulatory effects of dopamine on ghrelin secretions could exert partly via decreasing the activity of hypothalamic kisspeptin neurons.

Kisspeptin/Kisspeptin Receptor System in the Ovary.

Kisspeptins are a family of neuropeptides that are critical for initiating puberty and regulating ovulation in sexually mature females via the central control of the hypothalamic–pituitary–gonadal axis. Recent studies have shown that kisspeptin and its receptor kisspeptin receptor (KISS1R) are expressed in the mammalian ovary. Convincing evidence indicates that kisspeptins can activate a wide variety of signals via its binding to KISS1R. Experimental data gathered recently suggest a putative role of kisspeptin signaling in the direct control of ovarian function, including follicular development, oocyte maturation, steroidogenesis, and ovulation. Dysregulation or naturally occurring mutations of the kisspeptin/KISS1R system may negatively affect the ovarian function, leading to reproductive pathology or female infertility. A comprehensive understanding of the expression, actions, and underlying molecular mechanisms of this system in the human ovary is essential for novel approaches to therapeutic and diagnostic interventions in reproductive diseases and infertility.

Comparing KISS1 and GPR54 genes between the two breeds of goats with low and high prolificacy

There is increasing evidence that kisspeptin, the peptide product of KISS1 gene and its receptor (GPR54), regulate reproductive processes through actions on other organs than hypothalamus. Therefore, the study for the first time, was conducted to investigate the nucleotide sequences and mRNA expression levels of KISS1 and GPR54 genes in pituitary, endometrium, ovary and oviduct of prolific Jintang black goat (JTG) and non-prolific Tibetan goat (TBG) during pro-estrus. By RT-PCR strategy, we obtained that the KISS1 cDNA sequence was 408-bp long, encoding a putative peptide of 135 amino acids. The GPR54 cDNA sequence was 244-bp long, encoding a putative peptide of 81 amino acids. There were two base changes in KISS1, and one base change in GPR54, but these base changes did not result in amino acid substitution between these two breeds. Real-time PCR analyses revealed that both KISS1 and GPR54 genes were expressed in all four tissues examined. KISS1 was most highly expressed in pituitary (p<0.05) and its expression was greater in JTG than TBG (p<0.05). In conclusion, a high KISS1 expression level in pituitary of prolific goat breed might be associated with its great litter size.

118 Identification of KISS1 in the Domestic Cat

In the last decade, a substantial dataset has been acquired to demonstrate the critical role of kisspeptins, a family of neuropeptides derived from the KISS1 gene, in the reproduction system of mammalian and non-mammalian species. The KISS1 gene initial product can be cleaved into a 54-amino acid protein, and further degrade from the N-terminus into shorter but still active peptides (Kp-10, Kp-13, and Kp-14). The resulting peptides share a common C-terminal RF-amidated motif that leads to a strong binding with kisspeptin receptor. The Kp-10 peptide is well conserved between studied species and is thought to be essential and sufficient for the activation of kisspeptin receptor signalling pathways. In several species, the sequence of KISS1 has been obtained, and synthesised Kp-10 and Kp-54 have been successfully administered to induce ovulation. However, no studies have yet been performed to identify the KISS1 gene and its products in the domestic cat. Therefore, the aim of our work was to clone Felis catus KISS1 (fKISS1) and obtain its full sequence. Total RNA was isolated from the hypothalamus of a pubertal domestic cat collected postmortem and its quality was ensured using Agilent Bioanalyzer 2100 (RNA integrity number 8.2). Initial primers were designed based on the sequence deduced from the comparison of predicted domestic cat mRNA KISS1 sequence (XM_003999477.2) and published dog, human, cow, pig, goat, mouse, and rat mRNA KISS1 sequences. The partial mRNA fKISS1 sequence obtained here was used to design gene-specific primers for 5′ RACE and 3′ RACE PCR (GeneRacer Kit, Invitrogen, Carlsbad, CA, USA). For sequencing, 6 colonies were used per 5′ end and 3′ end of cloned fKISS1 cDNA. Additionally, the presence of KISS1 mRNA was checked via PCR in the ovaries, collected after ovariohysterectomy from domestic cats during inactive, follicular, and luteal ovarian stages. Nucleotide sequencing revealed that fKISS1 cDNA is 723 bp, and the open reading frame consists of 450 bp encoding a 149-amino acid polypeptide (kisspeptin precursor). Comparison of the overall amino acid sequence of the kisspeptin precursor revealed that domestic cat kisspeptin precursor exhibits similarity of 84, 78, 75, 68, 64, 56, and 52% to those of Hawaiian monk seal, pig, naked mole rat, human, mouse, musk shrew, and dog, respectively. The core sequence of kisspeptin, Kp-10, was highly conserved compared with other mammalian and non-mammalian species (100%, Hawaiian monk seal; 100%, mouse; 100%, pig; 100%, African clawed frog; 100%, naked mole rat; 100%, musk shrew; 90%, human; 90%, dog). Apart from hypothalamic tissue, fKISS1 has been identified in the ovaries on all stages. Obtained here results could be used for development of ovulation induction protocols in the endangered feline species. Moreover, a curious high similarity of kisspeptin sequence reported here to other induced ovulators may contribute to the research on ovulation mechanism in the domestic cat.

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.

Kisspeptin expression features in the arcuate and anteroventral periventricular nuclei of hypothalamus of letrozole-induced polycystic ovarian syndrome in rats.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder of the reproductive system characterized by polycystic ovaries and androgen excess. Letrozole is a nonsteroidal aromatase inhibitor that is used in experimental research to induce PCOS. Kisspeptin is an essential protein in regulation of cyclicity. Kisspeptin receptor is expressed in the hypothalamus and pituitary glands, and kisspeptin containing neurons are affected from sex steroid hormones. We aimed to investigate the number of kisspeptin-positive cells in the arcuate (Arc) and anteroventral periventricular nuclei (AVPV) of hypothalamus in the letrozole-induced PCOS. 40 female Wistar rats were divided into the proestrus control, diestrus control, proestrus vehicle, diestrus vehicle and letrozole. Animals were sacrificed after 3weeks, and sera, ovary and brain samples were harvested for further evaluations. Letrozole group had high weight gain, high numbers of ovarian follicular cysts, high levels of luteinizing hormone and testosterone and increase number of kisspeptin-positive cells in the Arc nucleus, as compared with the control groups (P≤0.05 vs. proestrus control and proestrus vehicle). Letrozole group showed a decrease in the number of kisspeptin-positive cells in the AVPV nucleus (P≤0.05 vs. proestrus control and proestrus vehicle). Our findings show that the number of kisspeptin-positive cells may be affected from letrozole, and that the changes in the number of these cells may be in favor of the appearance of PCOS features in this group.

Conformational Change in the Ligand-Binding Pocket via a KISS1R Mutation (P147L) Leads to Isolated Gonadotropin-Releasing Hormone Deficiency.

Context:Kisspeptin receptor (KISS1R) is expressed in hypothalamic gonadotropin-releasing hormone neurons and responsible for pubertal onset and reproductive functions. KISS1R mutations remain a rare cause of congenital hypogonadotropic hypogonadism (CHH).Objective:The aim of this study was to identify the genetic cause of CHH in a patient and to functionally characterize a KISS1R mutation.Design:The patient was a 47-year-old Japanese man whose parents were first cousins. He lacked secondary sexual characteristics owing to normosmic CHH. Exon segments for the KISS1R gene in this patient were screened for mutations. Functional analyses were performed using HEK293 cells expressing KISS1R mutants. Molecular dynamics simulations were performed to compare the ligand-KISS1R mutant complex with those of wild-type KISS1R variants.Results:A homozygous mutation (c.440C>T, p.P147L) in KISS1R was identified. The P147L mutation did not affect either receptor expression level or subcellular localization in the recombinant expression system. Intracellular calcium measurements and cellular dielectric spectroscopy demonstrated that the P147L mutation impaired receptor function to an extent more severe than that of a previously reported L148S mutation. A receptor-ligand binding assay showed the P147L mutation causes a substantial loss of ligand-binding affinity. Molecular dynamics simulations revealed the P147L mutation decreases the contact surface area of the ligand-receptor complex in an expanded ligand-binding pocket.Conclusion:We identified a loss-of-function mutation in KISS1R associated with CHH. Our results demonstrated that the P147L mutation causes a severe phenotype and functional impairment resulting from the loss of ligand-binding affinity due to an expanded ligand-binding pocket.

Role of amygdala kisspeptin in pubertal timing in female rats.

To investigate the mechanism by which maternal obesity disrupts reproductive function in offspring, we examined Kiss1 expression in the hypothalamic arcuate (ARC) and anteroventral periventricular (AVPV) nuclei, and posterodorsal medial amygdala (MePD) of pre-pubertal and young adult offspring. Sprague-Dawley rats were fed either a standard or energy-dense diet for six weeks prior to mating and throughout pregnancy and lactation. Male and female offspring were weaned onto normal diet on postnatal day (pnd) 21. Brains were collected on pnd 30 or 100 for qRT-PCR to determine Kiss1 mRNA levels. Maternal obesity increased Kiss1 mRNA expression in the MePD of pre-pubertal male and female offspring, whereas Kiss1 expression was not affected in the ARC or AVPV at this age. Maternal obesity reduced Kiss1 expression in all three brain regions of 3 month old female offspring, but only in MePD of males. The role of MePD kisspeptin on puberty, estrous cyclicity and preovulatory LH surges was assessed directly in a separate group of post-weanling and young adult female rats exposed to a normal diet throughout their life course. Bilateral intra-MePD cannulae connected to osmotic mini-pumps for delivery of kisspeptin receptor antagonist (Peptide 234 for 14 days) were chronically implanted on pnd 21 or 100. Antagonism of MePD kisspeptin delayed puberty onset, disrupted estrous cyclicity and reduced the incidence of LH surges. These data show that the MePD plays a key role in pubertal timing and ovulation and that maternal obesity may act via amygdala kisspeptin signaling to influence reproductive function in the offspring.

Secretion of Gonadotropins in Response to a Novel Kiss-1 Receptor Agonist, RF9 in the Mare: Modulation by Estradiol-17β and Half-Life of RF9 in the Peripheral Circulation

Three experiments were conducted to investigate the effects of the kisspeptin receptor agonist, 1-adamantanecarbonyl-RF-NH2 (RF9) on the secretion of gonadotropins, the role of estradiol-17β in modulating this response, and the metabolic half-life of RF9 in the mare. During the luteal phase of the estrous cycle (experiment 1), light horse mares were treated with sequential I.V. injections of RF9 (0.2 and 0.4 mg/kg) within a 1-hour interval to determine effects on coincident secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Dose-dependent increases (P < .001) in concentrations of both FSH and LH in response to RF9 were observed. In experiment 2, the effect of estradiol-17β (estradiol) pretreatment on responsiveness to RF9 was examined in winter anovulatory mares. Mares received a single intramuscular injection of 5-mg estradiol or corn oil followed by a bolus injection of RF9 18 hours later. Estradiol pretreatment increased (P < .05) peak concentrations of LH in response to RF9. The half-life of RF9 in the circulation of mares following peripheral administration was determined in experiment 3 using liquid chromatography/mass spectrometry. Intravenously injected RF9 was rapidly cleared from the mare's circulatory system, exhibiting a half-life of approximately 40 minutes. Data indicate that RF9 action is likely mediated via the endogenous release of GnRH and controls secretion of both FSH and LH. This effect is modulated by estradiol, putatively by enhanced responsiveness of the pituitary to GnRH and/or upregulation of Kiss-1 receptor in GnRH neurons. Use of RF9 as a pharmacologic agent in the mare may be limited by its short half-life.