Aspartame Intake Delayed Puberty Onset in Female Offspring Rats and Girls.

SCOPE: The disturbance of the hypothalamic-pituitary-gonadal (HPG) axis, gut microbiota (GM) community, and short-chain fatty acids (SCFAs) is a triggering factor for pubertal onset. The study investigates the effects of the long-term intake of aspartame on puberty and GM in animals and humans. METHODS AND RESULTS: Aspartame-fed female offspring rats result in vaginal opening time prolongation, serum estrogen reduction, and serum luteinizing hormone elevation. , 60 mg kg-1 aspartame treatment decreases the mRNA levels of gonadotropin-releasing hormone (GnRH), Kiss1, and G protein-coupled receptor 54 (GPR54), increases the mRNA level of RFamide-related peptide-3 (RFRP-3), and decreases the expression of GnRH neurons in the hypothalamus. Significant differences in relative bacterial abundance at the genus levels and decreased fecal SCFA levels are noted by 60 mg kg-1 aspartame treatment. Among which, Escherichia-Shigella is negatively correlated with several SCFAs. In girls, high-dose aspartame consumption decreases the risk of precocious puberty. CONCLUSIONS: Aspartame reduces the chance of puberty occurring earlier than usual in female offspring and girls. Particularly, 60 mg kg-1 aspartame-fed female offspring delays pubertal onset through the dysregulation of HPG axis and GM composition by inhibiting the Kiss1/GPR54 system and inducing the RFRP-3. An acceptable dose of aspartame should be recommended during childhood.

Effect of nourishing and purging fire Chinese herbal mixture on delaying light-induced premature puberty in rats.

OBJECTIVE: To elucidate the mechanism of the nourishing Yin and purging fire Chinese herbal mixture (NYPF) in delaying light-induced premature puberty in rats. METHODS: Twenty-one days old female Sprague-Dawley rats were randomly assigned to normal group (N), long light exposure group (L), NYPF and normal saline group (NS). Rats in the L, NYPF and NS groups were exposed to 16 h: 350 lux light/8 h: dark, while rats in the N group were exposed to 12 h: 50 lux light/12 h: dark. NYPF and normal saline was administered to the rats in the NYPF group or NS group, respectively, from day 21. Five rats in every group were sacrificed at 9 p.m. on day 28 (P28), on the day when rat's vulva opened in the L group (L-VO), on the day when the first estrous interphase occurred in rats of L group (L-E1), and on the day when the second estrous interphase occurred in rats of L group (L-E2), respectively. RESULITS: On day 34, all rats in the L group, 80% of rats in the NS group, 40% of rats in the N group, and 20% of rats in the NYPF group showed complete opening of the vulva. At P28, mRNA level of hypothalamic kisspeptin (Kiss-1) in the L group was significantly higher than that in the N group (P < 0.05). The rats in the L and NS groups had significantly lower hypothalamic arginine-phenylalanine-amide (RFamide)-related peptide 3 (RFRP-3) mRNA levels than those in the N group (P < 0.05), whereas RFRP-3 mRNA level was significantly higher in the NYPF group than that in the L group (P < 0.05). At L-VO, the ovarian index of the L and NS groups was significantly higher than that of the N group (P < 0.05) and estradiol (E2) level of the NYPF group was significantly lower than that of the N and NS groups (P < 0.05); hypothalamic Kiss-1 mRNA level in the L and NS groups was significantly higher than that in the N and NYPF groups (P < 0.05), whereas hypothalamic RFRP-3 mRNA level in the L, NYPF, and NS groups was significantly lower than that in the N group (P < 0.05). At L-E1, E2 level of the L and NS groups was significantly higher than that of the N group (P < 0.01), whereas it was significantly lower in the NYPF group than that of the N, L, and NS groups (P < 0.01), and serum luteinizing hormone level of the L and NS groups was significantly higher than that of the N group (P < 0.05); levels of serum melatonin and ovarian melatonin receptor 1 (MT-1) mRNA in the L, NYPF, and NS groups were significantly lower than those in the N group (P < 0.05). At L-E2, the uterine organ index of the NYPF group was significantly lower than that of the L group (P < 0.05); and ovarian MT-1 mRNA level of the L and NS groups was significantly lower than that in the N group (P < 0.05). CONCLUSIONS: NYPF can delay puberty onset in rats exposed to strong light for a prolonged duration, and regulation of the gene expression of Kiss-1 and RFRP-3 in the hypothalamus has been suggested as one of the mechanisms.

Kisspeptin promotes follicular development through its effects on modulation of P450 aromatase expression and steroidogenesis in sheep ovarian follicles.

The present study was conducted to ascertain whether the role of kisspeptin in promoting in vitro development of preantral follicles was through the regulation of P450 aromatase gene expression and steroidogenesis in sheep. Accordingly, the cumulus cells and oocytes were collected from different development stages of preantral follicles grown in vivo and cultured in vitro in TCM199B (Group I), TCM199B + KP (10 µg/mL) (Group II) and Standard medium + KP (10 µg/mL). To measure the steroid (Estradiol-17ß; E2 and Progesterone; P4 ) synthesis through ELISA, spent culture medium was collected separately from the same in vitro groups. E2 synthesis in the spent medium collected from all the three groups showed an increasing trend from PFs' exposed to respective culture media for 3 min to 2-day culture stage but decreased thereafter till 6-day culture stage. This is followed by a sharp increase in E2 concentration in the spent medium collected after in vitro maturation. However, P4 synthesis in group III followed increased pattern as the development progressed from PFs' exposed to culture medium for 3 min to in vitro maturation stage. The steroid production was observed at all stages of in vitro development in altered supplemented conditions. The steroid synthesis in the spent medium was highest in the 6 day cultured PFs' in Standard medium + KP matured in vitro for 24 h. Therefore, supplementation of kisspeptin along with other growth factors promoted steroid production in cultured preantral follicles far better than in other media.

Single neonatal estrogen implant sterilizes female animals by decreasing hypothalamic KISS1 expression.

Reproductive sterilization by surgical gonadectomy is strongly advocated to help manage animal populations, especially domesticated pets, and to prevent reproductive behaviors and diseases. This study explored the use of a single-injection method to induce sterility in female animals as an alternative to surgical ovariohysterectomy. The idea was based on our recent finding that repetitive daily injection of estrogen into neonatal rats disrupted hypothalamic expression of Kisspeptin (KISS1), the neuropeptide that triggers and regulates pulsatile secretion of GnRH. Neonatal female rats were dosed with estradiol benzoate (EB) either by daily injections for 11 days or by subcutaneous implantation of an EB-containing silicone capsule designed to release EB over 2-3 weeks. Rats treated by either method did not exhibit estrous cyclicity, were anovulatory, and became infertile. The EB-treated rats had fewer hypothalamic Kisspeptin neurons, but the GnRH-LH axis remained responsive to Kisspeptin stimulation. Because it would be desirable to use a biodegradable carrier that is also easier to handle, an injectable EB carrier was developed from PLGA microspheres to provide pharmacokinetics comparable to the EB-containing silicone capsule. A single neonatal injection of EB-microspheres at an equivalent dosage resulted in sterility in the female rat. In neonatal female Beagle dogs, implantation of an EB-containing silicone capsule also reduced ovarian follicle development and significantly inhibited KISS1 expression in the hypothalamus. None of the treatments produced any concerning health effects, other than infertility. Therefore, further development of this technology for sterilization in domestic female animals, such as dogs and cats is worthy of investigation.

Is seminal nerve growth factor-induced luteinizing hormone release in camelids mediated at the hypothalamus?

In brief: Seminal nerve growth factor induces ovulation in camelids by influencing the secretion of gonadotrophin-releasing hormone (GnRH) into the portal vessels of the pituitary gland. We show that the nerve growth factor-induced release of GnRH is not mediated directly through interaction with hypothalamic neurons. Abstract: Ovulation in camelids is triggered by seminal nerve growth factor (NGF). The mechanism of action of NGF appears to occur via the central nervous system. In this study, we tested the hypothesis that NGF acts in the hypothalamus to induce GnRH release. To determine if NGF-induced ovulation is associated with a rise in NGF concentrations in the cerebrospinal fluid (CSF), llamas were i) mated with an urethrostomized male, ii) mated with intact male, or given intrauterine iii) seminal plasma or i.v.) saline (Experiment 1). To characterize the luteinizing hormone (LH) response after central vs peripheral administration, llamas were treated with saline (negative control) or NGF either by i.v. or intracerebroventricular (ICV) administration (Experiment 2). To determine the role of kisspeptin, the effect of ICV infusion of a kisspeptin receptor antagonist on NGF-induced LH secretion and ovulation was tested in llamas (Experiment 3). In Experiment 1, a surge in circulating concentrations of LH was detected only in llamas mated with an intact male and those given intrauterine seminal plasma, but no changes in CSF concentrations of NGF were detected. In Experiment 2, peripheral administration (i.v.) of NGF induced an LH surge and ovulation, whereas no response was detected after central (ICV) administration. In Experiment 3, the kisspeptin receptor antagonist had no effect on the LH response to NGF. In conclusion, results did not support the hypothesis that NGF-induced ovulation is mediated via a trans-synaptic pathway within the hypothalamus, but rather through a releasing effect on tanycytes at the median eminence.

Kiss1 gene expression, sperm indices and testicular histopathology following the administration of Hibiscus sabdariffa in rats.

OBJECTIVE: This study investigated the expression of Kiss1 gene on the testis and the blood of Wistar rats, following the administration of methanolic extract of Hibiscus Sabdariffa (MEHS). METHODS: Fifteen (15) rats with an average weight of 204g were randomly divided into three (3) groups (A-C). Group A was given no treatment and served as the normal control group. Groups B and C were orally administered 200mg/kg and 400mg/kg of MEHS, respectively. The extract was administered once a day for 21 days. RESULTS: There was a significant increase in the relative testicular weight in group B and C compared to the control group (p=0.035). There was no significant difference in the sperm parameters, reproductive hormones, and antioxidant levels in all the treatment groups when compared to the control group (p>0.05). There is a significantly lower expression intensity of the Kiss1 gene in the blood in groups B (p=0.000) and C (p=0.017), compared to the control group. There is no difference in the relative intensity of Kiss1 gene expression in the testis of all the experimental groups (p=0.173). CONCLUSIONS: MEHS caused no histopathological changes on the testis at both doses. MEHS shows the potential of downregulating the expression of the Kiss1 gene in the blood. However, this effect lacks a regulatory mechanism on the reproductive hormones, sperm parameters, testicular morphology, and antioxidative levels.

Novel therapeutic avenues for kisspeptin.

Kisspeptin is a hypothalamic neuropeptide that acts via the hypothalamus to stimulate hypothalamic gonadotrophin-releasing hormone secretion and downstream gonadotrophin release. In health, kisspeptin induces normal puberty and modulates ovulation in healthy women. Hypothalamic kisspeptin expression is reduced in several functional reproductive disorders; thus, treating such conditions with kisspeptin is conceptually attractive. Recent studies have demonstrated that kisspeptin can induce a more physiological degree of oocyte maturation during in vitro fertilisation treatment that can reduce the risk of potentially life-threatening complications such as ovarian hyperstimulation syndrome seen with human chorionic gonadotrophin. Furthermore, chronic use of kisspeptin could potentially restore reproductive health in females with hypothalamic amenorrhoea, treat hyposexual drive disorder in otherwise healthy males and has potential indications in polycystic ovary syndrome, osteoporosis and metabolic dysfunction-associated fatty liver disease. Finally, kisspeptin analogues could potentially overcome some of the pharmacological challenges associated with the natural forms of kisspeptin such as short duration of action and development of tachyphylaxis.

Kiss1-dependent and independent release of luteinizing hormone and testosterone in perinatal male rats.

Prenatal and postnatal biphasic increases in plasma testosterone levels derived from perinatal testes are considered critical for defeminizing/masculinizing the brain mechanism that regulates sexual behavior in male rats. Hypothalamic kisspeptin neurons are indispensable for stimulating GnRH and downstream gonadotropin, as well as the consequent testicular testosterone production/release in adult male rats. However, it is unclear whether kisspeptin is responsible for the increase in plasma testosterone levels in perinatal male rats. The present study aimed to investigate the role of Kiss1/kisspeptin in generating perinatal plasma LH and the consequent testosterone increase in male rats by comparing the plasma testosterone and LH profiles of wild-type (Kiss1+/+) and Kiss1 knockout (Kiss1-/-) male rats. A biphasic pattern of plasma testosterone levels, with peaks in the prenatal and postnatal periods, was found in both Kiss1+/+ and Kiss1-/- male rats. Postnatal plasma testosterone and LH levels were significantly lower in Kiss1-/- male rats than in Kiss1+/+ male rats, whereas the levels in the prenatal embryonic period were comparable between the genotypes. Exogenous kisspeptin challenge significantly increased plasma testosterone and LH levels and the number of c-Fos-immunoreactive GnRH neurons in neonatal Kiss1-/- and Kiss1+/+ male rats. Kiss1 and Gpr54 (kisspeptin receptor gene) were found in the testes of neonatal rats, but kisspeptin treatment failed to stimulate testosterone release in the cultured testes of both genotypes. These findings suggest that postnatal, but not prenatal, testosterone increase in male rats is mainly induced by central kisspeptin-dependent stimulation of GnRH and consequent LH release.

Kisspeptin induces LH release and ovulation in an induced ovulator†.

Kisspeptin has been implicated in the ovulatory process of several species of spontaneous ovulators but in only one induced ovulator. In contrast, NGF in semen is the principal trigger of ovulation in other species of induced ovulators-camelids. We tested the hypotheses that kisspeptin induces luteinizing hormone (LH) secretion in llamas through a hypothalamic mechanism, and kisspeptin neurons are the target of NGF in its ovulation-inducing pathway. In Experiment 1, llamas were given either NGF, kisspeptin, or saline intravenously, and LH secretion and ovulation were compared among groups. All llamas treated with NGF (5/5) or kisspeptin (5/5) had an elevation of LH blood concentrations after treatment and ovulated, whereas none of the saline group did (0/5). In Experiment 2, llamas were either pretreated with a gonadotropin-releasing hormone (GnRH) receptor antagonist or saline and treated 2 h later with kisspeptin. Llamas pretreated with saline had elevated plasma LH concentrations and ovulated (6/6) whereas llamas pretreated with cetrorelix did not (0/6). In Experiment 3, we evaluated the hypothalamic kisspeptin-GnRH neuronal network by immunohistochemistry. Kisspeptin neurons were detected in the arcuate nucleus, the preoptic area, and the anterior hypothalamus, establishing synaptic contacts with GnRH neurons. We found no colocalization between kisspeptin and NGF receptors by double immunofluorescence. Functional and morphological findings support the concept that kisspeptin is a mediator of the LH secretory pathway in llamas; however, the role of kisspeptins in the NGF ovulation-inducing pathway in camelids remains unclear since NGF receptors were not detected in kisspeptin neurons in the hypothalamus.

Hypothalamic and brain stem neurochemical profile in anorectic rats after peripheral administration of kisspeptin-10 using 1 H-nmr spectroscopy in vivo.

PURPOSE: Although anorexia nervosa is classified as a psychiatric disorder associated with socio-environmental and psychological factors, a deeper insight into the dominant neurobiological basis is needed to develop a more effective approach of treatment. Given the high contribution of genetic predisposition and the underlying pathophysiology of neurohormonal circuits, it seems that pharmacological targeting of these mechanisms may provide us with better therapeutic outcomes. METHODS: 1 H-NMR spectroscopy was used to measure concentrations of the hypothalamus and brain stem metabolites in an activity-based rodent model (ABA) after subcutaneous administration of kisspeptin-10. Because anorexia mainly affects young women and often leads to hypogonadotropic-hypogonadism, we investigated the influence of this neuropeptide, which is involved in reproductive function by regulating the hypothalamic-pituitary-gonadal axis, on the ABA model development. RESULTS: Kisspeptin reinforced food consumption in an activity-based rodent model of anorexia changing a pattern of weight loss. 1 H-NMR spectroscopy of the hypothalamus and brain stem of ABA rats revealed a statistically significant change in the concentration of creatine (Cr; decreased, P = 0.030), phosphocreatine (PCr; increased, P = 0.030), γ-aminobutyric acid (GABA; decreased, P = 0.011), glutathione (GSH; increased, P = 0.011) and inositol (INS; increased, P = 0.047) compared to the control group. Subcutaneous administration of kisspeptin reversed the decrease in GABA (P = 0.018) and Cr (P = 0.030) levels in the hypothalamus as well as restored glutamate (GLU; P = 0.040) level in the brain stem. CONCLUSIONS: We suspect that kisspeptin through modulation of hypothalamic GABAergic signaling increases food intake, and thus positively alters brain metabolism.

Kisspeptin and RF9 prevent paroxetine-induced changes in some parameters of seminal vesicle fluid in the male rats.

The aim of the study was to examine possible impacts of paroxetine and agomelatine on the levels of some components that constitute the seminal vesicle fluid. As a second purpose, it was also aimed to examine how possible negative effects induced by paroxetine on seminal vesicle fluid components were affected by kisspeptin and RF9 (an RFamide-related peptide antagonist, RFRP). Forty-two male rats, aged 21 days, divided into six groups; control, sham, paroxetine, agomelatine, paroxetine + kisspeptin and paroxetine + RF9. Paroxetine (3.6 mg/kg) and agomelatine (10 mg/kg) were administrated by oral gavage. Kisspeptin (1 nmol) and RF9 (20 nmol) were administered intracerebroventricular (i.c.v). The experiments were ended on post-natal 120 days; fructose, vitamin E, sodium, potassium and magnesium levels were measured in seminal vesicle fluid. Fructose, vitamin E, magnesium and potassium levels were significantly decreased in seminal vesicle fluid from the rats treated with paroxetine but did not show significant differences following agomelatine administration. The co-administration of kisspeptin or RF9 with paroxetine prevented the paroxetine-induced negative effects on seminal vesicle fluid components. These results suggest that reduction in sperm fertilising ability caused by changes in seminal vesicle fluid can be seen in long-term antidepressant use. RF-9 and kisspeptin might have positive effects on long-term antidepressant use-induced infertility.

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.

Centrally administered kisspeptin suppresses feeding via nesfatin-1 and oxytocin in male rats.

Kisspeptin (KP), known as a hypothalamic neuropeptide, plays a critical role in the regulation of not only reproduction but also food intake. The anorectic neuropeptides, nesfatin-1 and oxytocin (OXT), are expressed in central nervous system, particulaly in various hypothalamic nuclei, and peripheral tissue. We examined the effects of the intracerebroventricular (icv) administration of KP-10 on feeding and nesfatin-1-immunoreactive (ir) or OXT-ir neurons in the rat hypothalamus, using Fos double immunohistochemistry in male rats. Cumulative food intake was remarkably decreased 0.5-3 h after icv administration of KP-10 (6.0 µg) compared to the vehicle treated and the KP-10 (3.8 µg) treated group. The icv administration of KP-10 significantly increased the number of nesfatin-1-ir neurons expressing Fos in the supraoptic nucleus (SON), paraventricular nucleus (PVN), arcuate nucleus (ARC), dorsal raphe nucleus, locus coeruleus, and nucleus tractus solitarius. The decreased food intake induced by KP-10 was significantly attenuated by pretreatment with the icv administration of antisense RNA against nucleobindin-2. After icv administration of KP-10, the percentages of OXT-ir neurons expressing FOS were remarkably higher in the SON and PVN than for vehicle treatment. The KP-10-induced anorexia was partially abolished by pretreatment with OXT receptor antagonist (OXTR-A). The percentage of nesfatin-1-ir neurons expressing Fos-ir in the ARC was also decreased by OXTR-A pretreatment. These results indicate that central administration of KP-10 activates nesfatin-1- and OXT neurons, and may play an important role in the suppression of feeding in male rats.

Effects of Kisspeptin-10 on Hypothalamic Neuropeptides and Neurotransmitters Involved in Appetite Control.

Besides its role as key regulator in gonadotropin releasing hormone secretion, reproductive function, and puberty onset, kisspeptin has been proposed to act as a bridge between energy homeostasis and reproduction. In the present study, to characterize the role of hypothalamic kisspeptin as metabolic regulator, we evaluated the effects of kisspeptin-10 on neuropeptide Y (NPY) and brain-derived neurotrophic factor (BDNF) gene expression and the extracellular dopamine (DA), norepinephrine (NE), serotonin (5-hydroxytriptamine, 5-HT), dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIIA) concentrations in rat hypothalamic (Hypo-E22) cells. Our study showed that kisspeptin-10 in the concentration range 1 nM⁻10 µM was well tolerated by the Hypo-E22 cell line. Moreover, kisspeptin-10 (100 nM⁻10 µM) concentration independently increased the gene expression of NPY while BDNF was inhibited only at the concentration of 10 µM. Finally, kisspeptin-10 decreased 5-HT and DA, leaving unaffected NE levels. The inhibitory effect on DA and 5-HT is consistent with the increased peptide-induced DOPAC/DA and 5-HIIA/5-HT ratios. In conclusion, our current findings suggesting the increased NPY together with decreased BDNF and 5-HT activity following kisspeptin-10 would be consistent with a possible orexigenic effect induced by the peptide.

Hypothalamic Response to Kisspeptin-54 and Pituitary Response to Gonadotropin-Releasing Hormone Are Preserved in Healthy Older Men.

BACKGROUND: Male testosterone levels decline by 1% per year from the age of 40 years. Whilst a primary testicular deficit occurs, hypothalamic or pituitary dysregulation may also coexist. This study aimed to compare the hypothalamic response to kisspeptin-54 and the pituitary response to gonadotropin-releasing hormone (GnRH) of older men with those of young men. METHODS: Following 1 h of baseline sampling, healthy older men (n = 5, mean age 59.3 ± 2.9 years) received a 3-h intravenous infusion of either vehicle, kisspeptin-54 0.1, 0.3, or 1.0 nmol/kg/h or GnRH 0.1 nmol/kg/h, on five different study days. Serum gonadotropins and total testosterone were measured every 10 min and compared to those of young men (n = 5/group) (mean age 28.9 ± 2.0 years) with a similar body mass index (24 kg/m2) who underwent the same protocol. RESULTS: Kisspeptin-54 and GnRH significantly stimulated serum gonadotropin release in older men compared to vehicle (p < 0.001 for all groups). Gonadotropin response to kisspeptin-54 was at least preserved in older men when compared to young men. At the highest dose of kisspeptin-54 (1.0 nmol/kg/h), a significantly greater luteinising hormone (LH) (p = 0.003) response was observed in older men. The follicle-stimulating hormone (FSH) response to GnRH was increased in older men (p = 0.002), but the LH response was similar (p = 0.38). Serum testosterone rises following all doses of kisspeptin-54 (p ≤ 0.009) were reduced in older men. CONCLUSIONS: Our data suggest that healthy older men without late-onset hypo-gonadism (LOH) have preserved hypothalamic response to kisspeptin-54 and pituitary response to GnRH, but impaired testicular response. Further work is required to investigate the use of kisspeptin-54 to identify hypothalamic deficits in men with LOH.

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.

Mechanistic insights into the more potent effect of KP-54 compared to KP-10 in vivo.

Kisspeptins regulate the mammalian reproductive axis by stimulating release of gonadotrophin releasing hormone (GnRH). Different length kisspeptins (KP) are found of 54, 14, 13 or 10 amino-acids which share a common C-terminal 10-amino acid sequence. KP-54 and KP-10 have been widely used to stimulate the reproductive axis but data suggest that KP-54 and KP-10 are not equally effective at eliciting reproductive hormone secretion after peripheral delivery. To confirm this, we analysed the effect of systemic administration of KP-54 or KP-10 on luteinizing hormone (LH) secretion into the bloodstream of male mice. Plasma LH measurements 10 min or 2 hours after kisspeptin injection showed that KP-54 can sustain LH release far longer than KP-10, suggesting a differential mode of action of the two peptides. To investigate the mechanism for this, we evaluated the pharmacokinetics of the two peptides in vivo and their potential to cross the blood brain barrier (BBB). We found that KP-54 has a half-life of ~32 min in the bloodstream, while KP-10 has a half-life of ~4 min. To compensate for this difference in half-life, we repeated injections of KP-10 every 10 min over 1 hr but failed to reproduce the sustained rise in LH observed after a single KP-54 injection, suggesting that the failure of KP-10 to sustain LH release may not just be related to peptide clearance. We tested the ability of peripherally administered KP-54 and KP-10 to activate c-FOS in GnRH neurons behind the blood brain barrier (BBB) and found that only KP-54 could do this. These data are consistent with KP-54 being able to cross the BBB and suggest that KP10 may be less able to do so.

Galanin Activates G Protein Gated Inwardly Rectifying Potassium Channels and Suppresses Kisspeptin-10 Activation of GnRH Neurons.

GnRH neurons are regulated by hypothalamic kisspeptin neurons. Recently, galanin was identified in a subpopulation of kisspeptin neurons. Although the literature thoroughly describes kisspeptin activation of GnRH neurons, little is known about the effects of galanin on GnRH neurons. This study investigated whether galanin could alter kisspeptin signaling to GnRH neurons. GnRH cells maintained in explants, known to display spontaneous calcium oscillations, and a long-lasting calcium response to kisspeptin-10 (kp-10), were used. First, transcripts for galanin receptors (GalRs) were examined. Only GalR1 was found in GnRH neurons. A series of experiments was then performed to determine the action of galanin on kp-10 activated GnRH neurons. Applied after kp-10 activation, galanin 1-16 (Gal1-16) rapidly suppressed kp-10 activation. Applied with kp-10, Gal1-16 prevented kp-10 activation until its removal. To determine the mechanism by which galanin inhibited kp-10 activation of GnRH neurons, Gal1-16 and galanin were applied to spontaneously active GnRH neurons. Both inhibited GnRH neuronal activity, independent of GnRH neuronal inputs. This inhibition was mimicked by a GalR1 agonist but not by GalR2 or GalR2/3 agonists. Although Gal1-16 inhibition relied on Gi/o signaling, it was independent of cAMP levels but sensitive to blockers of G protein-coupled inwardly rectifying potassium channels. A newly developed bioassay for GnRH detection showed Gal1-16 decreased the kp-10-evoked GnRH secretion below detection threshold. Together, this study shows that galanin is a potent regulator of GnRH neurons, possibly acting as a physiological break to kisspeptin excitation.

Inhibitory action of gonadotropin-inhibitory hormone on the signaling pathways induced by kisspeptin and vasoactive intestinal polypeptide in GnRH neuronal cell line, GT1-7.

Gonadotropin-inhibitory hormone (GnIH) acts as a negative regulator of reproduction by acting on gonadotropes and gonadotropin-releasing hormone (GnRH) neurons. Despite its functional significance, the molecular mechanism of GnIH action in the target cells has not been fully elucidated. To expand our previous study on GnIH actions in gonadotropes, we investigated the potential signal transduction pathway that conveys the inhibitory action of GnIH in GnRH neurons by using the GnRH neuronal cell line, GT1-7. We examined whether GnIH inhibits the action of kisspeptin and vasoactive intestinal polypeptide (VIP), positive regulators of GnRH neurons. Although GnIH significantly suppressed the stimulatory effect of kisspeptin on GnRH release in hypothalamic culture, GnIH had no inhibitory effect on kisspeptin stimulation of serum response element and nuclear factor of activated T-cell response element activities and ERK phosphorylation, indicating that GnIH may not directly inhibit kisspeptin signaling in GnRH neurons. On the contrary, GnIH effectively eliminated the stimulatory effect of VIP on p38 and ERK phosphorylation, c-Fos mRNA expression, and GnRH release. The use of pharmacological modulators strongly demonstrated the specific inhibitory action of GnIH on the adenylate cyclase/cAMP/protein kinase A pathway, suggesting a common inhibitory mechanism of GnIH action in GnRH neurons and gonadotropes.-Son, Y. L., Ubuka, T., Soga, T., Yamamoto, K., Bentley, G. E., Tsutsui, K. Inhibitory action of gonadotropin-inhibitory hormone on the signaling pathways induced by kisspeptin and vasoactive intestinal polypeptide in GnRH neuronal cell line, GT1-7.

Kisspeptin Mediated Signaling in Cancer.

Research over the years has gradually and sequentially highlighted contributory role of hypothalamic- based kisspeptin-signaling axis as a major positive modulator of the neuroendocrinological reproductive axis in mammals. However, a series of landmark studies provided convincing evidence of role of this signaling in regulation of cancer development and progression. It is becoming progressively more understandable that loss or reduction of KISS1 expression in different human cancers correlates inversely with progression of tumor, metastasizing potential and survival. In this review we have attempted to provide an overview highlight of the most recent updates addressing metastasis- suppressing role of KISS1. We also summarize interplay of microRNA and KISS1 in cancer. The miRNA regulation of different genes is a rapidly expanding area of research however, the community lacks a deep understanding of miRNA regulation of KISS1. Recently, emerging laboratory findings have shown that KISS1 is transcriptionally controlled by TCF21 that is in turn regulated by miR-21. Therefore, there is an urgent need for further study of how miRNA directly or indirectly influences KISS1 at the posttranscriptional level. There is also a lack of evidence regarding natural agents that mediate upregulation or downregulation of KISS1. Increasing the knowledge of the KISS1/KISS1R signaling axis will be helpful in achieving personalized medicine.