Kiss1 Neurons Research Articles

SAT-405 Activation of Kiss1 Neurons in the Posterodorsal Medial Amygdala Stimulates Gonadotropin Release via the Release of Kisspeptin in Female Mice

Reproduction is regulated by a complex neuronal network the precise components of which are still unknown. Kisspeptins, secreted from Kiss1 neurons in the arcuate nucleus (Kiss1ARC) and anteroventral periventricular/periventricular (Kiss1AVPV/PeN) nuclei, have been directly linked to the release of GnRH from the hypothalamus and hence, LH from the anterior pituitary. However, there is a population of Kiss1 neurons in the posterodorsal medial amygdala (Kiss1MePD) whose contribution to GnRH/LH stimulation has not yet been described. Here, we aimed to determine whether the release of kisspeptin and/or other components, besides kisspeptin, within the Kiss1MePD neuron, can stimulate LH release into the peripheral circulation. In this regard, we used a chemogenetic approach to specifically activate Kiss1MePD neurons in adult Kiss1Cre/+(heterozygous state) females which we studied in parallel to Kiss1Cre/Cre(Kiss1 knock-out state) littermates (n=5/group). Females received bilateral stereotaxic injections of an adeno-associated virus (pAAV) encoding a Cre-driven Gq-coupled excitatory DREADD (pAAV5/hSyn-DIO-hm3Dq:mCherry; titer 3x1012 genome copies per ml; 1 µl per hemisphere). Following infection, mice were given 3 weeks for recovery and maximum expression of the AAV vector. On day 1 of the experiment, animals were administered an ip bolus injection of vehicle saline (0.9% NaCl; day 1) and then hM3D receptors were activated by ip injection of its agonist, clozapine N-oxide (CNO; 10 mg/kg dissolved in saline; day 2). Blood samples were collected just before saline or CNO treatment (0) and then every 15 min for 90 min. Kiss1Cre/+mice expressing hM3Dq:mCherry in the MePD and treated with CNO to activate the Kiss1MePD neurons, showed an increase in LH within 30 min after the injection (P=0.0107) compared to animals receiving saline treatment, which was sustained for a further 30 min before returning to basal levels. Furthermore, no alteration in LH was observed in Kiss1Cre/Cre(i.e., Kiss1 KO) animals treated with either saline or CNO indicating that kisspeptin is the only component within the Kiss1MePD neuron that has the ability to stimulate LH release. Analysis following the completion of pharmacological studies demonstrated that mCherry expression was evident in 89% of the targeted Kiss1MePD neurons. Interestingly, mCherry labeled projections were observed in the ARC and specifically in close contact with Kiss1ARC neurons. Thus, in the female mouse, Kiss1MePD neurons have the ability to stimulate the gonadotropic axis but only when kisspeptin is present. Furthermore, this study suggests that this action is, at least in part, via the activation of Kiss1ARC neurons which, in turn, stimulate GnRH/LH release. Overall, our data provide insight into the potential mechanism via which amygdala regulated social cues can enhance reproductive function.

MON-LB082 Elevated Expression of KISSR Is Associated with Secretory Function of Functioning Gonadotroph Adenomas

Background: Gonadotroph adenomas are identified as the most common histological subtype of pituitary adenomas. But overwhelming majority of them are hormonally silent and act as non-functioning pituitary adenomas that presenting only with mass effects. The rest few named functioning gonadotroph adenomas (FGAs) can secrete biologically active gonadotropins and lead to characteristic abnormities of the hypophysial-gonadal axis function, such as isosexual precocious puberty in children, ovarian hyperstimulation and menstrual irregularity in premenopausal women and testicular enlargement in men. The mechanism remains unknown why FGAs have different secretory function from the clinical non-functioning ones which are morphologically same. Kisspeptin secreted by KISS1 neurons on hypothalamus can stimulate GnRH secretion when combines with its receptor KISS1R. Objective: To investigate whether kisspeptin and KISS1R are of effect for the secretory function of FGAs. Methods: we studied 18 clinically and histologically verified pituitary adenoma tissue samples: Group I(3 FGAs including 2 females and 1 male) ,Group II(12 NFPAs with immunohistochemistry positive for FSH and/or LH)and Group III(3 NFPAs with immunohistochemistry negative for FSH and LH). The mRNA expression levels of Kisspeptin and KISS1R were measured by RT-qPCR and the protein expression of KISS1R were determined by Western bolt. We also sequenced KISS1R gene in sera of all 3 FGAs patients and 2 female FGA stissue samples. The DNA methylation analyses of KISS1R promoter region in PA tissues were also performed. Results: We found that the KISS1R mRNA level was significantly different among three groups(p<0.05): Group I was highest,which was 1.8 times as Group II and 8.5 times as Group III. But there was no difference of Kisspeptin mRNA level among the groups. Western bolt showed that the KISS1R protein level was also significantly up-regulated in FGA group than the other two groups(p<0.05). A missense change c.1091T>A in exon 5 of KISS1R gene was found in the both female FGA pituitary adenoma DNA while no mutations of this gene were found in the serum samples of 3 FGA patients. No DNA methylation differences of KISS1R promoter region were observed among the three groups. Conclusion: We speculate that the elevated expression of KISS1R could play a role in the secretion function of FGAs. The expression of KISS1R might need to achieve to a certain threshold that could bring the secretory ability of FGAs. Sources of Research Support: The National Key Research and Development Program of China” (No. 2016YFC0901501) and CAMS Innovation Fund for Medical Science (CAMS-2016-I2M-1-002) awarded to HJZ. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

SUN-473 The Contribution of Bmal1 Expression in the Suprachiasmatic Nucleus to Female Fertility

The preovulatory gonadotropin-releasing hormone (GnRH) and resulting luteinizing hormone (LH) surge is gated to the onset of the active period in mice by a circadian and steroid signal. The temporal signal that initiates this process is believed to arrive from the suprachiasmatic nucleus (SCN), the hypothalamic circadian pacemaker. The SCN may contribute to reproduction through two discrete projections: vasoactive inhibitory peptide (VIP) neurons in the SCN project to gonadotropin-releasing hormone (GnRH) neurons in the preoptic area, and arginine vasopressin (AVP) neurons in the SCN project to kisspeptin (Kiss1) neurons in the anteroventral periventricular (AVPV). Genomic deletion of the critical circadian clock component, Bmal1, results in infertility in females. We sought to determine whether Bmal1 was critical in the SCN VIP and AVP neurons by using cre/lox technology to create three lines of mutant mice: AVP-Bmal1-/-, VIP-Bmal1-/-, and AVP-VIP-Bmal1-/-. We found no significant difference in LH levels following a kisspeptin challenge (2 mg/kg) between AVP-Bmal1-/- and Bmal1flox/flox (617.4 ± 216.9 vs 629.6 ± 110.6 pg/mL) or GnRH challenge (10 μg/kg) (1029.0 ± 246.2 vs 712.7 ± 142.6 pg/mL). Preliminary findings of an induced LH surge found AVP-Bmal1-/- and VIP-Bmal1-/- mice are able to mount a surge at the time of lights off (Bmal1flox/flox: 3 of 5; AVP-Bmal1-/-: 1 of 2, VIP-Bmal1:-/- 4 of 4; AVP-VIP-Bmal1-/- 0 of 2). Additionally, we were able to detect an endogenous surge at the time of lights off in both AVP-Bmal1-/-, VIP-Bmal1-/- mice, but not AVP-VIP-Bmal1-/-. However, AVP-Bmal1-/-, VIP-Bmal1-/-, and AVP-VIP-Bmal1-/- females carried litters to term and show no differences in the time to first litter compared to Bmal1flox/flox control (AVP-Bmal1-/-: 48 ± 4.8 vs 44.3 ± 0.3 days; VIP-Bmal1-/-: 29.6 ± 7.7 vs 31.0 ± 4.2 days; AVP-VIP-Bmal1-/-;27.7 ± 7 vs 23 ± 0 days); number of litters in 100 days (AVP-Bmal1-/-: 3.7 ±0.6 vs 4 ± 0.6; VIP-Bmal1-/-: 3.0 ± 1.0 vs 3.0 ± 0.0; AVP-VIP-Bmal1-/- 3.5 ± 0.7 vs 3.5 ± 0.7 days); or pups per litter (AVP-Bmal1-/-: 7.2 ± 0.7 vs 7.3 ± 0.7; VIP-Bmal1-/-: 6.2 ± 1.6 vs 7.6 ± 2.9; AVP-VIP-Bmal1-/- 8.1 ± 0.9 vs 9 ± 1.4 pups). Overall, these findings suggest that Bmal1 expression in the SCN AVP or VIP neurons is not required for fertility in a 12:12 light:dark cycle.

SAT-407 Evidence for Impaired Kisspeptin Signaling During Metabolic Stress in Female Mice

Stress impairs reproduction, at least in part, by suppressing gonadotropin secretion. One common metabolic stressor is hypoglycemia, which occurs in a variety of pathologies and in healthy people, particularly athletes. Insulin-induced hypoglycemia (IIH) is a repeatable, reliable, and quantifiable acute model of metabolic stress that inhibits pulsatile luteinizing hormone (LH) secretion, though little is known about the underlying mechanism. We have observed that IIH robustly suppresses LH pulses in female mice, which could be mediated by either a pituitary or hypothalamic effect. In the present study, we performed a kisspeptin challenge to determine whether downstream cells involved in LH pulsatility are impaired during IIH. Adult mice (C57/BL6, n = 8/group) were ovariectomized and acclimated to handling for tail-tip blood collection and i.p. injection. On the day of each challenge, mice were fasted for 5 hr and then received an i.p. injection of either saline or insulin (0.75mU/kg). All animals then received an injection of kisspeptin (2μg/g), 45 min later. This time point represents a consistent period of LH pulse suppression following IIH. Frequent blood samples were collected for 30 min before and after kisspeptin injection. Two-weeks later, the experiment was repeated with each mouse receiving the alternate treatment (i.e. insulin or saline) in a cross-over design. Mean LH concentrations were significantly increased after kisspeptin injection in animals treated with saline (saline, pre: 3.8 ± 0.6 vs. post: 7.8 ± 0.5 ng/ml). Interestingly, the LH response to kisspeptin in animals treated with insulin was also markedly increased (insulin, pre: 3.2 ± 0.6 vs. post: 6.4 ± 0.3 ng/ml), but not significantly different from animals treated with saline. We conclude from this study that impaired GnRH or gonadotrope cell function is not the primary cause of reduced LH secretion in this model of metabolic stress. Since kisspeptin neurons in the arcuate nucleus likely form the GnRH/LH pulse generator, we next determined whether arcuate kisspeptin cells were inhibited during IIH. For this purpose, we used a mouse model expressing GFP in kisspeptin cells (Kiss1-Cre-GFP). Mice were fasted and injected with insulin or saline, as above, and fixed neural tissue was collected for assessment of c-Fos immunoreactivity in arcuate Kiss1 cells. Two hours following saline injection ~20% of Kiss1 neurons contained c-Fos. In contrast, only 6.6 ± 0.5% of Kiss1 neurons contained c-Fos in animals treated with insulin. Thus, reduced gonadotropin secretion in IIH may be mediated by an inhibition of the ARC kisspeptin cells. Together these data support the hypothesis that inhibition of LH pulses by metabolic stress is mediated by neural factors including arcuate kisspeptin cells. Future work will investigate kisspeptin cell afferents that convey the inhibitory signal initiated by metabolic stressors.

SAT-415 Action of Neurotensin, CRH, And RFRP-3 in E2-Induced Negative Feedback Control: Studies Using a Mouse Arc Hypothalamic Cell Model

The recently established immortalized hypothalamic cell model mHypoA-55 possesses characteristics similar to those of Kiss-1 neurons in the arcuate nucleus (ARC) region of the hypothalamus. Here, we show that Kiss-1 gene expression in these cells was downregulated by 17β-estradiol (E2) under certain conditions. Both neurotensin (NT) and corticotropin-releasing hormone (CRH) were expressed in these cells and upregulated by E2. Stimulation of mHypoA-55 cells with NT and CRH significantly decreased Kiss-1 mRNA expression. A mammalian gonadotropin-inhibitory hormone homolog, RFamide-related peptide-3 (RFRP-3), was also found to be expressed in mHypoA-55 cells, and RFRP-3 expression in these cells was increased by exogenous melatonin stimulation. E2 stimulation also upregulated RFRP-3 expression in these cells. Stimulation of mHypoA-55 cells with RFRP-3 significantly increased the expression of NT and CRH. Furthermore, melatonin stimulation resulted in the increase of both NT and CRH mRNA expression in mHypoA-55 cells. On the other hand, in experiments using mHypoA-50 cells, which were originally derived from hypothalamic neurons in the anteroventral periventricular nucleus, Kiss-1 gene expression was upregulated by both NT and CRH, although E2 increased both NT and CRH expression, similarly to the mHypoA-55 cells. Our observations using the hypothalamic ARC cell model mHypoA-55 suggest that NT and CRH have inhibitory effects on Kiss-1 gene expression under the influence of E2 in association with RFRP-3 expression. Thus, these neuropeptides might be involved in E2-induced negative feedback mechanisms.

SAT-LB070 Tachykinins Are Essential for the Induction of the Preovulatory LH Surge in Mice

Tachykinins (TACs) result from the differential processing of two genes in rodents, Tac1 (encoding substance P (SP) and neurokinin A (NKA)) and Tac2 (encoding NKB). TACs exert their effects in the hypothalamus by acting on different G protein-coupled receptors (GPCRs): NK1R, the receptor of SP, NK2R, the receptor of NKA, and NK3R, the receptor of NKB. We and others have documented a stimulatory role for all these tachykinins in the release of GnRH in the presence of physiological circulating levels of sex steroids. Important cross-reactivity between the TAC ligand-receptor systems has been documented and suggested as a possible cause for the reversal of hypogonadal NKB-signaling deficient patients. In order to test the possible redundancy in the TAC systems, we generated a mouse model of congenital removal of all TACs (Tac1KO/Tac2KO, aka TACKO). This model revealed a sexually dimorphic effect of TACs on puberty onset and fertility. TACKO male mice (n= 14) displayed delayed puberty onset compared with wildtype (WT) littermates, resembling Tac1KO and Tac2KO mice separately, but retained normal fertility. By contrast, TACKO females presented a slight delay in the timing of vaginal opening (VO) -a marker of puberty onset—, compared to controls, but none of the females (n=10) presented signs of first estrus within 30 days after VO, indicating a failure to undergo complete sexual maturation. This was supported by a profound reproductive impairment, as only 20% of the TACKO females that were mated with fertile WT males for 8 weeks delivered pups (and these litters presented a significantly reduced number of pups and significantly larger parturition latency than controls). To test the ability of TACKO females to mount a normal LH surge, a similar cohort of TACKO females (n = 5) was subjected to an LH surge inducing protocol, which evidenced the absence of a detectable preovulatory surge in KO mice. Overall, these data suggest a novel role for TACs as essential players in the induction of the preovulatory LH surge in females, through a redundant mechanism that is only evident in the absence of all TACs, but not in the absence of Tac1 or Tac2, separately. These data further suggest that while TACs participate in the timing of puberty onset in both sexes, they are dispensable for the tonic (pulsatile) release of GnRH/LH as evidenced by the normal fertile phenotype of male TACKO mice. Because preoptic area Kiss1 neurons do not coexpress TACs, these findings strongly support an additional role for caudal hypothalamic (arcuate and ventromedial nuclei) neurons in the control of ovulation. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

SAT-425 NKB Signaling in the Posterodorsal Medial Amygdala Stimulates Gonadotropin Release in a Kisspeptin-Independent Manner in Female Mice

Female reproduction is regulated by a complex signaling network, the function of which is dependent on the level of circulating estrogens. Neurokinin B (NKB) colocalizes with kisspeptin in Kiss1 neurons of the arcuate nucleus (ARC). The autosynaptic action of NKB via the neurokinin 3 receptor (NK3R) in these neurons induces kisspeptin release in the presence of sex steroids. This has led us to believe that NKB stimulates LH secretion in a kisspeptin-dependent manner despite the latter mechanism having been investigated in the persistent hypogonadal state [e.g. Kiss1r knock out (KO) mice and agonadal monkeys]. Here, we aimed to assess whether in the presence of estradiol, senktide, a NK3R specific agonist, can stimulate LH release in a kisspeptin-independent manner. Adult WT female mice were ovariectomized (OVX) or OVX and estradiol-treated (OVX+E2) and studied in parallel to Kiss1KO or Kiss1KO+E2 littermates (n=10/group). Intracerebroventricular (icv) injections of senktide (600pmol) were performed and blood samples were collected before and 25 min after icv injection. Interestingly, the presence of E2 in Kiss1KO females resulted in LH increase like that observed in WT animals. Subsequently, we aimed to locate the brain area which senktide may be acting to stimulate LH release in Kiss1KO+E2 mice. We mapped the distribution of NK3R in the mouse hypothalamus and moreover investigated the anatomical relationship of NK3R and GnRH expression with dual-label immunohistochemistry as a likely pathway of the kisspeptin-independent stimulation of LH release exerted by NKB. We observed no instances of co-expression between NK3R and GnRH cell bodies (>100 cells analyzed from a total of 16 mice) and only minimal close appositions between the two proteins in the ARC and medial septum. Thus, the kisspeptin-independent action of NKB cannot be attributed to direct stimulation of NK3R located on GnRH neurons. Interestingly, we observed that NK3R immunopositive cells in the posterodorsal medial amygdala (MePD), unlike the ARC, were upregulated in the presence of E2. Next, WT OVX+E2 and Kiss1KO+E2 females were injected with senktide (600pmol; n=5/group) in the ARC, the AVPV/PeN or the MePD; areas found to contain NK3R-expressing neurons, and blood samples were collected before and then every 15 min for 45 min. Senktide significantly stimulated LH secretion in all three areas in WT OVX+E2, but only in the MePD of Kiss1KO+E2 mice. Conversely, in the absence of E2, Kiss1KO females injected with senktide in the MePD did not show any alteration in LH, mimicking the results we obtained after icv administration. These data demonstrate that activation of NK3R in the amygdala stimulates LH release in a kisspeptin independent manner in the presence of estrogen. This novel pathway for LH release, that bypasses the kisspeptin neuron, may play an important role in linking amygdala regulated social cues with gonadotropin release.

Mapping neuronal inputs to Kiss1 neurons in the arcuate nucleus of the mouse.

The normal function of the mammalian reproductive axis is strongly influenced by physiological, metabolic and environmental factors. Kisspeptin neuropeptides, encoded by the Kiss1 gene, are potent regulators of the mammalian reproductive axis by stimulating gonadodropin releasing hormone secretion from the hypothalamus. To understand how the reproductive axis is modulated by higher order neuronal inputs we have mapped the afferent circuits into arcuate (ARC) Kiss1 neurons. We used a transgenic mouse that expresses the CRE recombinase in Kiss1 neurons for conditional viral tracing with genetically modified viruses. CRE-mediated activation of these viruses in Kiss1 neurons allows the virus to move transynaptically to label neurons with primary or secondary afferent inputs into the Kiss1 neurons. Several regions of the brain showed synaptic connectivity to arcuate Kiss1 neurons including proopiomelanocortin neurons in the ARC itself, kisspeptin neurons in the anteroventral periventricular nucleus, vasopressin neurons in the supraoptic and suprachiasmatic nuclei, thyrotropin releasing neurons in the paraventricular nucleus and unidentified neurons in other regions including the subfornical organ, amygdala, interpeduncular nucleus, ventral premammilary nucleus, basal nucleus of stria terminalis and the visual, somatosensory and piriform regions of the cortex. These data provide an insight into how the activity of Kiss1 neurons may be regulated by metabolic signals and provide a detailed neuroanatomical map for future functional studies.

Estrogen signaling in arcuate Kiss1 neurons suppresses a sex-dependent female circuit promoting dense strong bones

Central estrogen signaling coordinates energy expenditure, reproduction, and in concert with peripheral estrogen impacts skeletal homeostasis in females. Here, we ablate estrogen receptor alpha (ERα) in the medial basal hypothalamus and find a robust bone phenotype only in female mice that results in exceptionally strong trabecular and cortical bones, whose density surpasses other reported mouse models. Stereotaxic guided deletion of ERα in the arcuate nucleus increases bone mass in intact and ovariectomized females, confirming the central role of estrogen signaling in this sex-dependent bone phenotype. Loss of ERα in kisspeptin (Kiss1)-expressing cells is sufficient to recapitulate the bone phenotype, identifying Kiss1 neurons as a critical node in this powerful neuroskeletal circuit. We propose that this newly-identified female brain-to-bone pathway exists as a homeostatic regulator diverting calcium and energy stores from bone building when energetic demands are high. Our work reveals a previously unknown target for treatment of age-related bone disease.

NKB signaling in the posterodorsal medial amygdala stimulates gonadotropin release in a kisspeptin-independent manner in female mice.

This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Metabolic regulation of female puberty via hypothalamic AMPK–kisspeptin signaling

Conditions of metabolic distress, from malnutrition to obesity, impact, via as yet ill-defined mechanisms, the timing of puberty, whose alterations can hamper later cardiometabolic health and even life expectancy. AMP-activated protein kinase (AMPK), the master cellular energy sensor activated in conditions of energy insufficiency, has a major central role in whole-body energy homeostasis. However, whether brain AMPK metabolically modulates puberty onset remains unknown. We report here that central AMPK interplays with the puberty-activating gene, Kiss1, to control puberty onset. Pubertal subnutrition, which delayed puberty, enhanced hypothalamic pAMPK levels, while activation of brain AMPK in immature female rats substantially deferred puberty. Virogenetic overexpression of a constitutively active form of AMPK, selectively in the hypothalamic arcuate nucleus (ARC), which holds a key population of Kiss1 neurons, partially delayed puberty onset and reduced luteinizing hormone levels. ARC Kiss1 neurons were found to express pAMPK, and activation of AMPK reduced ARC Kiss1 expression. The physiological relevance of this pathway was attested by conditional ablation of the AMPKα1 subunit in Kiss1 cells, which largely prevented the delay in puberty onset caused by chronic subnutrition. Our data demonstrate that hypothalamic AMPK signaling plays a key role in the metabolic control of puberty, acting via a repressive modulation of ARC Kiss1 neurons in conditions of negative energy balance.

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.

SIRT1 mediates obesity- and nutrient-dependent perturbation of pubertal timing by epigenetically controlling Kiss1 expression

Puberty is regulated by epigenetic mechanisms and is highly sensitive to metabolic and nutritional cues. However, the epigenetic pathways mediating the effects of nutrition and obesity on pubertal timing are unknown. Here, we identify Sirtuin 1 (SIRT1), a fuel-sensing deacetylase, as a molecule that restrains female puberty via epigenetic repression of the puberty-activating gene, Kiss1. SIRT1 is expressed in hypothalamic Kiss1 neurons and suppresses Kiss1 expression. SIRT1 interacts with the Polycomb silencing complex to decrease Kiss1 promoter activity. As puberty approaches, SIRT1 is evicted from the Kiss1 promoter facilitating a repressive-to-permissive switch in chromatin landscape. Early-onset overnutrition accelerates these changes, enhances Kiss1 expression and advances puberty. In contrast, undernutrition raises SIRT1 levels, protracts Kiss1 repression and delays puberty. This delay is mimicked by central pharmacological activation of SIRT1 or SIRT1 overexpression, achieved via transgenesis or virogenetic targeting to the ARC. Our results identify SIRT1-mediated inhibition of Kiss1 as key epigenetic mechanism by which nutritional cues and obesity influence mammalian puberty.

Estradiol-Dependent and -Independent Stimulation of Kiss1 Expression in the Amygdala, BNST, and Lateral Septum of Mice.

Kisspeptin, encoded by Kiss1, activates reproduction by stimulating GnRH neurons. Although most Kiss1 neurons are located in the hypothalamus, smaller Kiss1 populations also reside in the medial amygdala (MeA), bed nucleus of the stria terminalis (BnST), and lateral septum (LS). However, very little is known about the regulation and function of these extra-hypothalamic Kiss1 neurons. This study focused on the roles and interactions of two signaling factors, estradiol (E2) and GABA, known to stimulate and inhibit, respectively, extra-hypothalamic Kiss1 expression. First, using estrogen receptor (ER)α knockout (KO) and βERKO mice, we demonstrated that Kiss1 in both the BnST and LS is stimulated by E2, as occurs in the MeA, and that this E2 upregulation occurs via ERα, but not ERβ. Second, using GABABR KO and wild-type mice, we determined that whereas E2 normally increases extra-hypothalamic Kiss1 levels, such upregulation by E2 is further enhanced by the concurrent absence of GABABR signaling in the MeA and LS, but not the BnST. Third, we demonstrated that when GABABR signaling is absent, the additional removal of gonadal sex steroids does not abolish Kiss1 expression in the MeA and BnST, and in some cases the LS. Thus, Kiss1 expression in these extra-hypothalamic regions is not solely dependent on E2 stimulation. Finally, we demonstrated a significant positive correlation between Kiss1 levels in the MeA, BnST, and LS, but not between these regions and the hypothalamus (anteroventral periventricular nucleus/periventricular nucleus). Collectively, our findings indicate that both E2 and GABA independently regulate all three extra-hypothalamic Kiss1 populations, but their regulatory interactions may vary by brain region and additional yet-to-be-identified factors are likely involved.

Estrogenic-dependent glutamatergic neurotransmission from kisspeptin neurons governs feeding circuits in females.

The neuropeptides tachykinin2 (Tac2) and kisspeptin (Kiss1) in hypothalamic arcuate nucleus Kiss1 (Kiss1ARH) neurons are essential for pulsatile release of GnRH and reproduction. Since 17β-estradiol (E2) decreases Kiss1 and Tac2 mRNA expression in Kiss1ARH neurons, the role of Kiss1ARH neurons during E2-driven anorexigenic states and their coordination of POMC and NPY/AgRP feeding circuits have been largely ignored. Presently, we show that E2 augmented the excitability of Kiss1ARH neurons by amplifying Cacna1g, Hcn1 and Hcn2 mRNA expression and T-type calcium and h-currents. E2 increased Slc17a6 mRNA expression and glutamatergic synaptic input to arcuate neurons, which excited POMC and inhibited NPY/AgRP neurons via metabotropic receptors. Deleting Slc17a6 in Kiss1 neurons eliminated glutamate release and led to conditioned place preference for sucrose in E2-treated KO female mice. Therefore, the E2-driven increase in Kiss1 neuronal excitability and glutamate neurotransmission may play a key role in governing the motivational drive for palatable food in females.

Action of neurotensin, corticotropin-releasing hormone, and RFamide-related peptide-3 in E2-induced negative feedback control: studies using a mouse arcuate nucleus hypothalamic cell model.

The recently established immortalized hypothalamic cell model mHypoA-55 possesses characteristics similar to those of Kiss-1 neurons in the arcuate nucleus (ARC) region of the hypothalamus. Here, we show that Kiss-1 gene expression in these cells was downregulated by 17β-estradiol (E2) under certain conditions. Both neurotensin (NT) and corticotropin-releasing hormone (CRH) were expressed in these cells and upregulated by E2. Stimulation of mHypoA-55 cells with NT and CRH significantly decreased Kiss-1 mRNA expression. A mammalian gonadotropin-inhibitory hormone homolog, RFamide-related peptide-3 (RFRP-3), was also found to be expressed in mHypoA-55 cells, and RFRP-3 expression in these cells was increased by exogenous melatonin stimulation. E2 stimulation also upregulated RFRP-3 expression in these cells. Stimulation of mHypoA-55 cells with RFRP-3 significantly increased the expression of NT and CRH. Furthermore, melatonin stimulation resulted in the increase of both NT and CRH mRNA expression in mHypoA-55 cells. On the other hand, in experiments using mHypoA-50 cells, which were originally derived from hypothalamic neurons in the anteroventral periventricular nucleus, Kiss-1 gene expression was upregulated by both NT and CRH, although E2 increased both NT and CRH expression, similarly to the mHypoA-55 cells. Our observations using the hypothalamic ARC cell model mHypoA-55 suggest that NT and CRH have inhibitory effects on Kiss-1 gene expression under the influence of E2 in association with RFRP-3 expression. Thus, these neuropeptides might be involved in E2-induced negative feedback mechanisms.

The Role of Kiss1 Neurons As Integrators of Endocrine, Metabolic, and Environmental Factors in the Hypothalamic-Pituitary-Gonadal Axis.

Kisspeptin–GPR54 signaling in the hypothalamus is required for reproduction and fertility in mammals. Kiss1 neurons are key regulators of gonadotropin-releasing hormone (GnRH) release and modulation of the hypothalamic–pituitary–gonadal (HPG) axis. Arcuate Kiss1 neurons project to GnRH nerve terminals in the median eminence, orchestrating the pulsatile secretion of luteinizing hormone (LH) through the intricate interaction between GnRH pulse frequency and the pituitary gonadotrophs. Arcuate Kiss1 neurons, also known as KNDy neurons in rodents and ruminants because of their co-expression of neurokinin B and dynorphin represent an ideal hub to receive afferent inputs from other brain regions in response to physiological and environmental changes, which can regulate the HPG axis. This review will focus on studies performed primarily in rodent and ruminant species to explore potential afferent inputs to Kiss1 neurons with emphasis on the arcuate region but also considering the rostral periventricular region of the third ventricle (RP3V). Specifically, we will discuss how these inputs can be modulated by hormonal, metabolic, and environmental factors to control gonadotropin secretion and fertility. We also summarize the methods and techniques that can be used to study functional inputs into Kiss1 neurons.

Expression of hypothalamic kisspeptin, neurokinin B, and dynorphin A neurons attenuates in female Zucker fatty rats.

Zucker fatty (ZF) rats are considered to be an obese model due to leptin receptor abnormality and such rats show infertility. Pulsatile gonadotropin-releasing hormone/luteinizing hormone (LH) secretion, which is important for follicular development in females, is considered to be controlled by KNDy neurons coexpressing kisspeptin, neurokinin B (NKB), and dynorphin A (DynA), encoded by Kiss1, Tac3, and Pdyn, respectively, in the hypothalamic arcuate nucleus (ARC). The purpose of this study is to examine the expression of KNDy neurons in female ZF rats by histochemical approach because pulsatile LH secretion is suppressed. Zucker lean (ZL) rats served as a control group. Animals were ovariectomized and subcutaneously implanted with a silicon tube containing estradiol to produce plasma level of estradiol during diestrus. Plasma LH levels decreased in ZF rats compared with ZL rats. The expressions of each mRNA (Kiss1, Tac3, and Pdyn) and each peptide (kisspeptin, NKB, and DynA) in the ARC significantly decreased in ZF rats compared with ZL rats. However, the number of Kiss1 neurons in the anterior ventral periventricular nucleus did not significantly differ between the two groups. These results suggest that dysfunction of leptin signaling negatively affects KNDy neurons in the ARC, resulting in reproductive dysfunction caused by suppression of the LH pulse.

Continuous Kisspeptin Restores Luteinizing Hormone Pulsatility Following Cessation by a Neurokinin B Antagonist in Female Sheep.

Pulsatile secretion of the gonadotropin-releasing hormone (GnRH) drives pulsatile secretion of the luteinizing hormone (LH), with evidence that this depends on kisspeptin (Kiss) input to GnRH neurons. Kiss administration causes acute GnRH/LH secretion, and electrophysiological data suggest that Kiss neurons may act in a phasic manner to drive GnRH secretion, but there is not definitive evidence for this. The product of the Kiss-1 gene is proteolytically cleaved to smaller products, and the 10 amino acid C-terminal product (Kiss-10) displays full bioactivity. We have shown previously that continuous delivery of Kiss-10 to anestrous ewes can cause a surge in GnRH secretion and ovulation and increases LH pulse frequency in humans. Here, we tested the hypothesis that continuous Kiss-10 delivery can support pulsatile GnRH/LH secretion in the sheep. Neurokinin B (NKB) provides positive drive to Kiss neurons, so we therefore infused an NKB antagonist (ANT-08) intracerebroventricularly to induce cessation of pulsatile GnRH/LH secretion, with or without concomitant continuous Kiss-10 infusion. ANT-08 suppressed GnRH/LH pulsatility, which was immediately restored with continuous Kiss-10 infusion. These data support the notion that Kiss-10 action is downstream of NKB signaling and that continuous Kiss-10 stimulation of GnRH neurons is sufficient to support a pulsatile pattern of GnRH/LH secretion. This offers further support to the theory that GnRH pulse generation is intrinsic to GnRH neurons and that pulsatile GnRH release can be affected with continuous stimulation by Kiss-10.

Short-Term High-Fat Diet Increases Leptin Activation of CART Neurons and Advances Puberty in Female Mice.

Leptin is a permissive factor for puberty initiation, participating as a metabolic cue in the activation of the kisspeptin (Kiss1)-gonadotropin-releasing hormone neuronal circuitry; however, it has no direct effect on Kiss1 neurons. Leptin acts on hypothalamic cocaine- and amphetamine-regulated transcript (CART) neurons, participating in the regulation of energy homeostasis. We investigated the influence of a short-term high-fat diet (HFD) on the effect of leptin on puberty timing. Kiss1-hrGFP female mice received a HFD or regular diet (RD) after weaning at postnatal day (PN)21 and were studied at PN28 and PN32. The HFD increased body weight and plasma leptin concentrations and decreased the age at vaginal opening (HFD, 32 ± 0.53 days; RD, 38 ± 0.67 days). Similar colocalization of neurokinin B and dynorphin in Kiss1-hrGFP neurons of the arcuate nucleus (ARC) was observed between the HFD and RD groups. The HFD increased CART expression in the ARC and Kiss1 messenger RNA expression in the anteroventral periventricular (AVPV)/anterior periventricular (Pe). The HFD also increased the number of ARC CART neurons expressing leptin-induced phosphorylated STAT3 (signal transducer and activator of transcription 3) at PN32. Close apposition of CART fibers to Kiss1-hrGFP neurons was observed in the ARC of both RD- and HFD-fed mice. In conclusion, these data reinforce the notion that a HFD increases kisspeptin expression in the AVPV/Pe and advances puberty initiation. Furthermore, we have demonstrated that the HFD-induced earlier puberty is associated with an increase in CART expression in the ARC. Therefore, these data indicate that CART neurons in the ARC can mediate the effect of leptin on Kiss1 neurons in early puberty induced by a HFD.