Kisspeptin/neurokinin B/dynorphin Neurons Research Articles

30 Central Regulation of Kndy Neurons During Undernutrition in Sheep

Abstract Adequate energy intake is essential for reproduction. While we have known for decades that feed restriction inhibits gonadotropin-releasing hormone/luteinizing hormone (GnRH/LH) secretion, the central mechanisms that govern this reduction during undernutrition remain to be fully elucidated. The past few years, we have focused on examining central mechanisms whereby chronic feed restriction in young sheep results in the inhibition of LH secretion. This presentation will focus on our current understanding of how feed restriction impacts kisspeptin/neurokinin B/dynorphin (KNDy) neurons in young sheep and will give a glimpse into our next few years as we examine how input to KNDy neurons could play a key role in regulating reproduction during undernutrition.

FRI280 Partial Loss Of KNDy Neurons In Prenatally Androgen Treated Female Rats Alters The LH Secretion And Ovarian Morphology In A Model Of Polycystic Ovary Syndrome

Abstract Disclosure: N.S. Aquino: None. A. Campideli-Santana: None. L.M. Antunes: None. R. Araújo-Lopes: None. K.S. Silva: None. P.C. Henriques: None. D.D. Gusmão: None. M.P. Bernuci: None. R.E. Szawka: None. A.M. dos Reis: None. Polycystic Ovary Syndrome (PCOS) is a high-prevalent endocrine dysfunction, causing metabolic disorders and infertility. Women with PCOS show a disrupted hypothalamus-pituitary-gonadal (HPG) axis, which results in increased luteinizing hormone (LH) secretion, LH pulse frequency, and LH/follicle stimulating hormone (FSH) ratio. These changes are related to an impaired negative-feedback effect of ovarian steroids, estradiol and progesterone, on the gonadotrophin releasing-hormone (GnRH) neurons. Because the kisspeptin/neurokinin-B/dynorphin (KNDy) neurons of the arcuate (ARC) nucleus are responsible for mediating the ovarian steroid negative feedback, we investigated whether the ablation of KNDy neurons would change the LH secretion and ovarian morphology in a rat model of PCOS. Pregnant dams were treated with dihydrotestosterone (DHT, 3 mg/day; s.c) from days 16 to 19 of pregnancy. Three months after birth, the prenatally androgen-treated (PNA) offspring received intra-ARC stereotaxic injections of the neurokinin-3 receptor agonist conjugated with saporin (NK3-SAP) to induce the lesion of KNDy neurons and were submitted to serial blood sampling for LH measurement. PNA NK3-SAP rats had a moderate loss of ARC neurokinin-B-immunoreactive neurons (between 50-70%) compared with the Blank-SAP-injected PNA group. The PNA Blank-SAP rats displayed LH pulse frequency, pulse amplitude, and mean LH levels similar to control rats on diestrus. Remarkably, the partial loss of KNDy neurons increased the LH pulse amplitude and mean LH without changing the pulse frequency. Histological analysis was performed to investigate the outcome of the partial loss of KNDy neurons on the ovarian morphology in PNA rats. The numbers of primordial, primary, and secondary health follicles were decreased in PNA Blank-SAP compared with diestrus rats. Notably, the number of primordial follicles was partially restored in NK3-SAP rats. On the other hand, neither the number of atretic follicles nor the number of corpora lutea was changed in PNA Blank or NK3-SAP rats, indicating no change in the ovulatory function. Therefore, we provide evidence that KNDy neurons negatively modulate LH release and LH pulse amplitude in PNA rats. Moreover, the results suggest that KNDy neurons are involved in the reduction of the primordial follicle pool and ovarian reserve in PNA rats. Financial Support: Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Brazilian National Council for Scientific and Technological Development (CNPq), British Society for Neuroendocrinology (BSN). Presentation: Friday, June 16, 2023

Deletion of Growth Hormone Secretagogue Receptor in Kisspeptin Neurons in Female Mice Blocks Diet-Induced Obesity.

The gut peptide, ghrelin, mediates energy homeostasis and reproduction by acting through its receptor, growth hormone secretagogue receptor (GHSR), expressed in hypothalamic neurons in the arcuate (ARC). We have shown 17β-estradiol (E2) increases Ghsr expression in Kisspeptin/Neurokinin B/Dynorphin (KNDy) neurons, enhancing sensitivity to ghrelin. We hypothesized that E2-induced Ghsr expression augments KNDy sensitivity in a fasting state by elevating ghrelin to disrupt energy expenditure in females. We produced a Kiss1-GHSR knockout to determine the role of GHSR in ARC KNDy neurons. We found that changes in ARC gene expression with estradiol benzoate (EB) treatment were abrogated by the deletion of GHSR and ghrelin abolished these differences. We also observed changes in metabolism and fasting glucose levels. Additionally, knockouts were resistant to body weight gain on a high fat diet (HFD). Behaviorally, we found that knockouts on HFD exhibited reduced anxiety-like behavior. Furthermore, knockouts did not refeed to the same extent as controls after a 24 h fast. Finally, in response to cold stress, knockout females had elevated metabolic parameters compared to controls. These data indicate GHSR in Kiss1 neurons modulate ARC gene expression, metabolism, glucose homeostasis, behavior, and thermoregulation, illustrating a novel mechanism for E2 and ghrelin to control Kiss1 neurons.

Opioidergic pathways and kisspeptin in the regulation of female reproduction in mammals.

Endogenous opioid peptides have attracted attention as critical neuropeptides in the central mechanism regulating female reproduction ever since the discovery that arcuate dynorphin neurons that coexpress kisspeptin and neurokinin B (NKB), which are also known as kisspeptin/neurokinin B/dynorphin (KNDy) neurons, play a role as a master regulator of pulsatile gonadotropin-releasing hormone (GnRH) release in mammals. In this study, we first focus on the role of dynorphin released by KNDy neurons in the GnRH pulse generation. Second, we provide a historical overview of studies on endogenous opioid peptides. Third, we discuss how endogenous opioid peptides modulate tonic GnRH/gonadotropin release in female mammals as a mediator of inhibitory internal and external cues, such as ovarian steroids, nutritional status, or stress, on reproduction. Then, we discuss the role of endogenous opioid peptides in GnRH surge generation in female mammals.

Synergistic Control of KNDy Neuronal Influence on Energy Balance by Ghrelin and Estradiol

The gut peptide, ghrelin, mediates negative energy homeostasis and the neuroendocrine control of energy homeostasis by acting through its receptor, growth hormone secretagogue receptor (GHSR). GHSR, expressed in hypothalamic Kisspeptin/Neurokinin B/Dynorphin (KNDy) neurons in the arcuate (ARC), is well known to regulate energy balance. We have previously shown 17-beta-estradiol (E2) robustly increases Ghsr expression in KNDy neurons, enhancing their sensitivity to ghrelin. We hypothesize that E2-induced increase in GHSR expression augments KNDy sensitivity in a fasting state by elevating ghrelin to reduce energy expenditure in females. We developed a Kiss1-specific GHSR knockout to determine the role of GHSR in ARC KNDy neurons and fed them either a low-fat diet (LFD) or a high-fat diet (HFD). Knockout (experimental) females were resistant to HFD in terms of body weight gain, adiposity, and food intake compared to HFD-fed controls. HFD-fed experimental females also exhibited slower glucose clearance compared to HFD-fed controls. Experimental females, regardless of diet, exhibited elevated fasting (5h) glucose. Metabolic rates (V.O2, V.CO2) and energy expenditure (heat) were not different. Respiratory Exchange Ratio (RER) was elevated in LFD-fed females, indicating the utilization of carbohydrates over fat for energy. Further meal pattern analysis revealed a reduction in meal duration in HFD-fed females, but elevated meal frequency, while HFD-fed experimental females exhibited a reduced meal size. In two separate meal pattern experiments, experimental and control females were fasted for 24h and refed or injected with ghrelin (I.P. 1mg/kg) or saline. We observed a striking delay in refeeding behavior in experimental females compared to controls during the refeeding period after fasting. After injection, control females responded to ghrelin with a rapid and sustained increase in food intake which was blunted in experimentals. Collectively, these data suggest that GHSR activation in KNDy neurons modulates metabolism, glucose homeostasis, and feeding behavior, illustrating a novel mechanism for E2 and ghrelin to synergistically control KNDy neuronal output and their subsequent behavioral and physiological outcomes.

Kisspeptin/Neurokinin B/Dynorphin (KNDy) cells as integrators of diverse internal and external cues: evidence from viral-based monosynaptic tract-tracing in mice

Neurons in the hypothalamic arcuate nucleus (ARC) that co-express kisspeptin, neurokinin B and dynorphin (KNDy cells) are essential for mammalian reproduction as key regulators of gonadotropin-releasing hormone (GnRH) secretion. Although multiple endogenous and exogenous signals act indirectly via KNDy neurons to regulate GnRH, the identity of upstream neurons that provide synaptic input to this subpopulation is unclear. We used rabies-mediated tract-tracing in transgenic Kiss1-Cre mice combined with whole-brain optical clearing and multiple-label immunofluorescence to create a comprehensive and quantitative brain-wide map of neurons providing monosynaptic input to KNDy cells, as well as identify the estrogen receptor content and peptidergic phenotype of afferents. Over 90% of monosynaptic input to KNDy neurons originated from hypothalamic nuclei in both male and female mice. The greatest input arose from non-KNDy ARC neurons, including proopiomelanocortin-expressing cells. Significant female-dominant sex differences in afferent input were detected from estrogen-sensitive hypothalamic nuclei critical for reproductive endocrine function and sexual behavior in mice, indicating KNDy cells may provide a unique site for the coordination of sex-specific behavior and gonadotropin release. These data provide key insight into the structural framework underlying the ability of KNDy neurons to integrate endogenous and environmental signals important for the regulation of reproductive function.

Prenatal Testosterone Exposure Alters GABAergic Synaptic Inputs to GnRH and KNDy Neurons in a Sheep Model of Polycystic Ovarian Syndrome.

Prenatal testosterone (T)-treated female sheep display reproductive deficits similar to women with polycystic ovarian syndrome (PCOS), including an increase in LH pulse frequency due to actions of the central GnRH pulse generator. In this study, we used multiple-label immunocytochemistry to investigate the possibility of changes in the γ-aminobutyric acid (GABA) neurotransmitter system at two key components of the GnRH pulse generator in prenatal T-treated sheep: kisspeptin/neurokinin B/dynorphin (KNDy) neurons of the arcuate nucleus, and GnRH neurons in the preoptic area (POA) and mediobasal hypothalamus (MBH). We observed a significant decrease and increase, respectively, in the number of GABAergic synapses onto POA and MBH GnRH neurons in prenatal T-treated ewes; additionally, there was a significant increase in the number of GABAergic inputs onto KNDy neurons. To determine the actions of GABA on GnRH and KNDy neurons, we examined colocalization with the chloride transporters NKCC1 and KCC2, which indicate stimulatory or inhibitory activation of neurons by GABA, respectively. Most GnRH neurons in both POA and MBH colocalized NKCC1 cotransporter whereas none contained the KCC2 cotransporter. Most KNDy neurons colocalized either NKCC1 or KCC2, and 28% of the KNDy population contained NKCC1 alone. Therefore, we suggest that, as in the mouse, GABA in the sheep is stimulatory to GnRH neurons, as well as to a subset of KNDy neurons. Increased numbers of stimulatory GABAergic inputs to both MBH GnRH and KNDy neurons in prenatal T-treated animals may contribute to alterations in steroid feedback control and increased GnRH/LH pulse frequency seen in this animal model of PCOS.

SAT-426 Rabies-Mediated Monosynaptic Tract-Tracing of Sexually Dimorphic Estrogen-Sensitive Afferents to KNDy Neurons in the Mouse

Estradiol controls reproductive status and sexual behavior by acting upon a variety of neurochemical systems in the brain to mediate the sexually dimorphic release of gonadotrophins. However, the neural pathways that transmit steroid hormone information to gonadotropin-releasing hormone (GnRH) neurons are unclear. Kisspeptin/Neurokinin B/Dynorphin (KNDy) neurons of the arcuate nucleus (ARC) are critical for reproductive status and implicated to relay a number of peripheral signals, including gonadal steroids, to regulate GnRH secretion. We recently reported the distribution of primary afferents to KNDy neurons (Moore et al, International Congress for Neuroendocrinology, 2018), however, it is not known if KNDy neurons receive synaptic input from steroid hormone-sensitive cells. To address this, we utilized rabies-mediated tract-tracing to unequivocally determine the distribution of estradiol-sensitive monosynaptic inputs to KNDy neurons. Adult male (n=6) and female (n=4) Kiss1-Cre mice were stereotaxically injected in the ARC with Cre-dependent adenoassociated viruses (AAVs) to express avian receptor protein with GFP (TVA-GFP) and rabies glycoprotein (G) in ARC kisspeptin (KNDy) neurons. After three weeks, the glycoprotein-deleted rabies virus (RVdG) containing mCherry was injected in the same location. RVdG exclusively infects KNDy cells expressing TVA, and, as G is required for rabies to cross transynaptically, RVdG crosses into primary afferents of KNDy neurons to express mCherry. As RVdG induces toxicity in cells, a time-point trial determined mCherry-positive cells retain protein expression after 5 days. Mice were perfused and coronal sections immunolabeled for GFP to identify KNDy cells, mCherry to visualize KNDy afferents and estrogen receptor alpha (ERα). Immunoreactivity was mapped using fluorescent microscopy and slide scanning software (MBFbioscience) and dual labeled mCherry- and ERα- positive cells analyzed using confocal microscopy. Male and female mice were statistically compared using students t tests. Dual-labeled cells were detected within the bed nucleus of the stria terminalis and the hypothalamic rostral periventricular area, medial preoptic nucleus (MPON), ventromedial and anterior hypothalamus, ARC and premammillary nucleus. Compared to male mice, females exhibited a significantly higher percentage of dual-labeled cells in the periventricular nucleus (PeN, 46.7±16.4% in females, 7.8±5.1% in males, p<0.05) and MPON (34.3±7.2% in females, 10.9±6% in males, p<0.05). The PeN and MPON are critical for reproductive endocrine function and sexual behavior in the mouse, respectively. Together, these data indicate that sexually dimorphic estradiol-sensitive neuronal populations in the rostral hypothalamus may directly relay steroid hormone signals to KNDy neurons to regulate GnRH secretion and perhaps other functions.

OR33-6 The Neurokinin 3 Receptor Antagonist, Fezolinetant, Is Effective in Treatment of Menopausal Vasomotor Symptoms: A Randomized, Placebo-Controlled, Double-Blind, Dose-Ranging Study

Vasomotor symptoms (VMS; eg, hot flashes, night sweats) are caused by a loss of thermoregulatory control. The thermoregulatory center in the brain is innervated by kisspeptin/neurokinin B/dynorphin (KNDy) neurons. The activity of KNDy neurons is stimulated by neurokinin 3 receptor (NK3R) activation and inhibited by estrogen negative-feedback. Declining estrogen levels at menopause disturb this balance, leading to VMS. Fezolinetant is an antagonist at NK3R that acts to moderate KNDy neuron function for the treatment of VMS (1). The aim of the current trial was to evaluate fezolinetant dose levels and treatment regimen in VMS. In this phase 2b, double-blind trial (NCT03192176), menopausal women age >40−65 y with moderate/severe VMS (≥50/wk) were randomized to one of the following: fezolinetant 15, 30, 60, or 90 mg BID or 30, 60, or 120 mg QD or placebo (PBO). Coprimary efficacy outcomes were frequency and severity of moderate/severe VMS at wk 4 and 12. Secondary efficacy outcomes included changes in moderate/severe VMS frequency and severity by study wk. Of 356 women randomized, 352 received ≥1 dose study drug (safety population; mean [SD] age: 54.6 [4.7] y; 73% white) and 287 (81%) completed the study: PBO: 37/44 (84%); fezolinetant: 250/312 (80%). The most common reasons for discontinuation were withdrawal of consent (6.7%) and AEs (5.9%). All fezolinetant dose groups demonstrated reductions in VMS frequency and severity at wk 4 and 12. Significant dose responses were observed in all coprimary efficacy endpoints. The differences from PBO in least squares (LS) mean changes from baseline in moderate/severe VMS frequency per day at wk 4 were −1.9, −3.0, −2.8, and −3.5 for 15, 30, 60, and 90 mg BID, respectively, and −2.3, −3.0, and −2.4 for 30, 60, and 120 mg QD, respectively, with a common SE of 0.8 (all P<.05). The differences from PBO in LS mean change from baseline in moderate/severe VMS frequency per day at wk 12 were −1.8, −2.1, −2.3, and −2.6 for 15, 30, 60, and 90 mg BID, respectively, and −2.1, −2.6, and −2.1 for 30, 60, and 120 mg QD, respectively, with a common SE of ~0.7 (all P<.05). Dose response was apparent in frequency and severity as early as at wk 1. The majority of TEAEs were mild or moderate in severity. There were no deaths or serious treatment-related TEAEs. This study demonstrates that fezolinetant is a safe and effective nonhormonal treatment for rapid reduction of moderate/severe menopausal VMS. 1Fraser GL, et al. Clinical evaluation of the NK3 receptor antagonist fezolinetant (a.k.a. ESN364) for the treatment of menopausal hot flashes [abstract OR16-5]. Endocr Rev. 2017;38(3).

KNDy Cells Revisited.

In the past decade since kisspeptin/neurokinin B/dynorphin (KNDy) cells were first identified in the mammalian hypothalamus, a plethora of new research has emerged adding insights into the role of this neuronal population in reproductive neuroendocrine function, including the basis for GnRH pulse generation and the mechanisms underlying the steroid feedback control of GnRH secretion. In this mini-review, we provide an update of evidence regarding the roles of KNDy peptides and their postsynaptic receptors in producing episodic GnRH release and assess the relative contribution of KNDy neurons to the "GnRH pulse generator." In addition, we examine recent work investigating the role of KNDy neurons as mediators of steroid hormone negative feedback and review evidence for their involvement in the preovulatory GnRH/LH surge, taking into account species differences that exist among rodents, ruminants, and primates. Finally, we summarize emerging roles of KNDy neurons in other aspects of reproductive function and in nonreproductive functions and discuss critical unresolved questions in our understanding of KNDy neurobiology.

Three-dimensional imaging of KNDy neurons in the mammalian brain using optical tissue clearing and multiple-label immunocytochemistry

Kisspeptin/Neurokinin B/Dynorphin (KNDy) neurons of the arcuate nucleus (ARC) play a key role in the regulation of fertility. The ability to detect features of KNDy neurons that are essential for fertility may require three-dimensional (3D) imaging of the complete population. Recently developed protocols for optical tissue clearing permits 3D imaging of neuronal populations in un-sectioned brains. However, these techniques have largely been described in the mouse brain. We report 3D imaging of the KNDy cell population in the whole rat brain and sheep hypothalamus using immunolabelling and modification of a solvent-based clearing protocol, iDISCO. This study expands the use of optical tissue clearing for multiple mammalian models and provides versatile analysis of KNDy neurons across species. Additionally, we detected a small population of previously unreported kisspeptin neurons in the lateral region of the ovine mediobasal hypothalamus, demonstrating the ability of this technique to detect novel features of the kisspeptin system.

Nitric oxide-heat shock protein axis in menopausal hot flushes: neglected metabolic issues of chronic inflammatory diseases associated with deranged heat shock response.

Although some unequivocal underlying mechanisms of menopausal hot flushes have been demonstrated in animal models, the paucity of similar approaches in humans impedes further mechanistic outcomes. Human studies might show some as yet unexpected physiological mechanisms of metabolic adaptation that permeate the phase of decreased oestrogen levels in both symptomatic and asymptomatic women. This is particularly relevant because both the severity and time span of hot flushes are associated with increased risk of chronic inflammatory disease. On the other hand, oestrogen induces the expression of heat shock proteins of the 70 kDa family (HSP70), which are anti-inflammatory and cytoprotective protein chaperones, whose expression is modulated by different types of physiologically stressful situations, including heat stress and exercise. Therefore, lower HSP70 expression secondary to oestrogen deficiency increases cardiovascular risk and predisposes the patient to senescence-associated secretory phenotype (SASP) that culminates in chronic inflammatory diseases, such as obesities, type 2 diabetes, neuromuscular and neurodegenerative diseases. This review focuses on HSP70 and its accompanying heat shock response (HSR), which is an anti-inflammatory and antisenescent pathway whose intracellular triggering is also oestrogen-dependent via nitric oxide (NO) production. The main goal of the manuscript was to show that the vasomotor symptoms that accompany hot flushes may be a disguised clue for important neuroendocrine alterations linking oestrogen deficiency to the anti-inflammatory HSR. Results from our own group and recent evidence on hypothalamic control of central temperature guided a search on PubMed and Google Scholar websites. Oestrogen elicits rapid production of the vasodilatory gas NO, a powerful activator of HSP70 expression. Whence, part of the protective effects of oestrogen over cardiovascular and neuroendocrine systems is tied to its capacity of inducing the NO-elicited HSR. The hypothalamic areas involved in thermoregulation (infundibular nucleus in humans and arcuate nucleus in other mammals) and whose neurons are known to have their function altered after long-term oestrogen ablation, particularly kisspeptin-neurokinin B-dynorphin neurons, (KNDy) are the same that drive neuroprotective expression of HSP70 and, in many cases, this response is via NO even in the absence of oestrogen. From thence, it is not illogical that hot flushes might be related to an evolutionary adaptation to re-equip the NO-HSP70 axis during the downfall of circulating oestrogen. Understanding of HSR could shed light on yet uncovered mechanisms of menopause-associated diseases as well as on possible manipulation of HSR in menopausal women through physiological, pharmacological, nutraceutical and prebiotic interventions. Moreover, decreased HSR indices (that can be clinically determined with ease) in perimenopause could be of prognostic value in predicting the moment and appropriateness of starting a HRT.

Effects of Season and Estradiol on KNDy Neuron Peptides, Colocalization With D2 Dopamine Receptors, and Dopaminergic Inputs in the Ewe.

Seasonal reproduction in sheep is primarily due to a dramatic increase in the ability of estradiol (E2) to inhibit the pulsatile secretion of gonadotropin-releasing hormone (GnRH) during the nonbreeding season [anestrus (ANS)]. Recent findings suggest that kisspeptin/neurokinin B/dynorphin (KNDy) neurons of the arcuate nucleus (ARC) play a key role in conveying this negative feedback influence, with dopaminergic projections from the retrochiasmatic area acting upon KNDy cells to decrease kisspeptin release and thus inhibit GnRH pulses. However, several questions remain unanswered: (1) Are the coexpressed KNDy peptides, neurokinin B (NKB) and dynorphin, under seasonal regulation similar to kisspeptin? (2) Are seasonal changes in these peptides and their colocalization of D2 dopamine receptors (D2Rs) steroid dependent? and (3) Do KNDy neurons receive direct input from dopaminergic terminals? We used dual- and triple-label immunofluorescence to analyze brain sections through the ARC of ovariectomized (OVX) and OVX plus E2 ewes perfused during either the breeding season or ANS. Results showed (1) steroid-dependent and steroid-independent seasonal changes in kisspeptin and NKB, but not dynorphin, immunoreactivity; (2) increased D2R coexpression during ANS that was dependent on the presence of E2; and (3) evidence that KNDy cells receive direct contact from dopaminergic terminals and that this input increases during ANS. These results support the hypothesis that dopamine acts to inhibit GnRH secretion in ANS by directly suppressing the activity of ARC KNDy neurons, and implicate NKB as well as kisspeptin in seasonal shifts in E2-negative feedback in the sheep.

Prenatal testosterone exposure decreases colocalization of insulin receptors in kisspeptin/neurokinin B/dynorphin and agouti-related peptide neurons of the adult ewe.

Insulin serves as a link between the metabolic and reproductive systems, communicating energy availability to the hypothalamus and enabling reproductive mechanisms. Adult Suffolk ewes prenatally exposed to testosterone (T) display an array of reproductive and metabolic dysfunctions similar to those seen in women with polycystic ovarian syndrome (PCOS), including insulin resistance. Moreover, prenatal T treatment alters neuropeptide expression in KNDy (co-expressing kisspeptin, neurokinin B/dynorphin) and agouti-related peptide (AgRP) neurons in the arcuate nucleus, two populations that play key roles in the control of reproduction and metabolism, respectively. In this study, we determined whether prenatal T treatment also altered insulin receptors in KNDy and AgRP neurons, as well as in preoptic area (POA) kisspeptin, pro-opiomelanocortin (POMC), and gonadotropin-releasing hormone (GnRH) neurons of the adult sheep brain. Immunofluorescent detection of the beta subunit of insulin receptor (IRβ) revealed that KNDy, AgRP and POMC neurons, but not GnRH or POA kisspeptin neurons, colocalize IRβ in control females. Moreover, prenatal T treatment decreased the percentage of KNDy and AgRP neurons that colocalized IRβ, consistent with reduced insulin sensitivity. Administration of the anti-androgen drug, Flutamide, during prenatal T treatment, prevented the reduction in IRβ colocalization in AgRP, but not in KNDy neurons, suggesting that these effects are programmed by androgenic and oestrogenic actions, respectively. These findings provide novel insight into the effects of prenatal T treatment on hypothalamic insulin sensitivity and raise the possibility that decreased insulin receptors, specifically within KNDy and AgRP neurons, may contribute to the PCOS-like phenotype of this animal model.

Immunohistochemical characterization of the arcuate kisspeptin/neurokinin B/dynorphin (KNDy) and preoptic kisspeptin neuronal populations in the hypothalamus during the estrous cycle in heifers.

Elucidating the physiological mechanisms that control reproduction is an obvious strategy for improving the fertility of cattle and developing new agents to control reproductive functions. The present study aimed to identify kisspeptin neurons in the bovine hypothalamus, clarifying that a central mechanism is also present in the cattle brain, as kisspeptin is known to play an important role in the stimulation of gonadotropin-releasing hormone (GnRH)/gonadotropin secretion in other mammals. To characterize kisspeptin neurons in the bovine hypothalamus, the co-localizations of kisspeptin and neurokinin B (NKB) or kisspeptin and dynorphin A (Dyn) were examined. Hypothalamic tissue was collected from Japanese Black or Japanese Black × Holstein crossbred cows during the follicular and luteal phases. Brain sections, including the arcuate nucleus (ARC) and the preoptic area (POA), were dual immunostained with kisspeptin and either NKB or Dyn. In the ARC, both NKB and Dyn were co-localized in kisspeptin neurons during both the follicular and luteal phases, demonstrating the presence of kisspeptin/NKB/Dyn-containing neurons, referred to as KNDy neurons, in cows. In the POA, no co-localization of kisspeptin with either NKB or Dyn was detected. Kisspeptin expression in the follicular phase was higher than that in the luteal phase, suggesting that kisspeptin expression in the POA is positively controlled by estrogen in cows. The kisspeptin neuronal populations in the ARC and POA likely play important roles in regulating the GnRH pulse and surge, respectively, in cows.

The role of kisspeptin signalling in the hypothalamic-pituitary-gonadal axis--current perspective.

The discovery of kisspeptins in the recent past remoulded current understanding of the neuroendocrine axis relating to the regulation of human puberty and reproduction. Kisspeptins have been recognised to act upstream of GnRH and have been shown to play a vital role in the control of the hypothalamic-pituitary-gonadal axis via regulation of gonadotrophin secretion, onset of puberty, and control of fertility. KNDy (kisspeptin/neurokinin-B/dynorphin) neurons have been suggested to modulate GnRH pulsatile secretion, which is required to support reproductive function in both sexes. They have also been involved in mediating both positive and negative sex steroid feedback signals to GnRH neurons and serve as a vital connection between reproduction and metabolic status of the body. When kisspeptin is administered to healthy humans, and in patients with reproductive disorders, it strongly and directly stimulates GnRH and subsequent LH secretion and enhances LH pulse frequency. These observations suggest that kisspeptins are a potential novel therapeutic approach for treating disorders with either pathologically reduced or augmented gonadotrophins pulsatile secretion and is currently a focus of translational research. Kisspeptins have also been identified in several peripheral reproductive organs, indicating their role in modulation of ovarian function, embryo implantation, and placentation, but a great deal of work remains to be done to explore further in this regard, and the evidence is only available from studies done on animal models. In this review we will mainly focus on current available evidence related to the role of kisspeptins in controlling GnRH pulse frequency, specifically their role in puberty, fertility, and reproduction. We will also be appraising other factors that regulate the kiSS1/Kisspeptin/GPR-54 system.

The Distribution of Substance P and Kisspeptin in the Mediobasal Hypothalamus of the Male Rhesus Monkey and a Comparison of Intravenous Administration of These Peptides to Release GnRH as Reflected by LH Secretion

Substance P (SP) was recently reported to be expressed in human kisspeptin/neurokinin B/dynorphin (KNDy) neurons and to enhance KNDy neuron excitability in the mouse hypothalamus. We therefore examined (1) interactions of SP and kisspeptin in the mediobasal hypothalamus of adult male rhesus monkeys using immunofluorescence, and (2) the ability of SP to induce LH release in GnRH-primed, agonadal juvenile male monkeys. SP cell bodies were observed only occasionally in the arcuate nucleus (Arc), but more frequently dorsal to the Arc in the region of the premammillary nucleus. Castration resulted in an increase in the number of SP cell bodies in the Arc but not in the other regions. SP fibers innervated the Arc, where they were found in close apposition with kisspeptin perikarya in the periphery of this nucleus. Beaded SP axons projected to the median eminence, where they terminated in the external layer and intermingled with beaded kisspeptin axons. Colocalization of the two peptides, however, was not observed. Although close apposition between SP fibers and kisspeptin neurons suggest a role for SP in modulating GnRH pulse generator activity, i.v. injections of SP failed to elicit release of GnRH (as reflected by LH) in the juvenile monkey. Although the finding of structural interactions between SP and kisspeptin neurons is consistent with the notion that this tachykinin may be involved in regulating pulsatile GnRH release, the apparent absence of expression of SP in KNDy neurons suggests that this peptide is unlikely to be a fundamental component of the primate GnRH pulse generator.

Prenatal Testosterone Treatment Leads to Changes in the Morphology of KNDy Neurons, Their Inputs, and Projections to GnRH Cells in Female Sheep.

Prenatal testosterone (T)-treated ewes display a constellation of reproductive defects that closely mirror those seen in PCOS women, including altered hormonal feedback control of GnRH. Kisspeptin/neurokinin B/dynorphin (KNDy) neurons of the arcuate nucleus (ARC) play a key role in steroid feedback control of GnRH secretion, and prenatal T treatment in sheep causes an imbalance of KNDy peptide expression within the ARC. In the present study, we tested the hypothesis that prenatal T exposure, in addition to altering KNDy peptides, leads to changes in the morphology and synaptic inputs of this population, kisspeptin cells of the preoptic area (POA), and GnRH cells. Prenatal T treatment significantly increased the size of KNDy cell somas, whereas POA kisspeptin, GnRH, agouti-related peptide, and proopiomelanocortin neurons were each unchanged in size. Prenatal T treatment also significantly reduced the total number of synaptic inputs onto KNDy neurons and POA kisspeptin neurons; for KNDy neurons, the decrease was partly due to a decrease in KNDy-KNDy synapses, whereas KNDy inputs to POA kisspeptin cells were unaltered. Finally, prenatal T reduced the total number of inputs to GnRH cells in both the POA and medial basal hypothalamus, and this change was in part due to a decreased number of inputs from KNDy neurons. The hypertrophy of KNDy cells in prenatal T sheep resembles that seen in ARC kisspeptin cells of postmenopausal women, and together with changes in their synaptic inputs and projections to GnRH neurons, may contribute to defects in steroidal control of GnRH observed in this animal model.

Investigation of the kisspeptin/neurokinin B/dynorphin (KNDy) neurons by using N-7 cells: immortalized hypothalamic cell line

Kisspeptin, a neuropeptide hormone encoded by the Kiss-1 gene, and its receptor have been recently recognized as fundamental activators of the gonadotropic axis with key roles in the control of gonadotropin secretion, especially, in vivo studies. Moreover, recent observations show that neurokinin B (NKB) is coexpressed with kisspeptin and dynorphin in the arcuate nucleus (ARC) in hypothalamus, named the Kisspeptin/NKB/Dynorphin (KNDy) neurons. KNDy neurons are thought to drive pulsatile GnRH secretion and gonadotropin release, and estradiol negatively regulates kisspeptin expression in KNDy neurons.

Kisspeptin, Neurokinin B, and Dynorphin Act in the Arcuate Nucleus to Control Activity of the GnRH Pulse Generator in Ewes

Recent work has led to the hypothesis that kisspeptin/neurokinin B/dynorphin (KNDy) neurons in the arcuate nucleus play a key role in GnRH pulse generation, with kisspeptin driving GnRH release and neurokinin B (NKB) and dynorphin acting as start and stop signals, respectively. In this study, we tested this hypothesis by determining the actions, if any, of four neurotransmitters found in KNDy neurons (kisspeptin, NKB, dynorphin, and glutamate) on episodic LH secretion using local administration of agonists and antagonists to receptors for these transmitters in ovariectomized ewes. We also obtained evidence that GnRH-containing afferents contact KNDy neurons, so we tested the role of two components of these afferents: GnRH and orphanin-FQ. Microimplants of a Kiss1r antagonist briefly inhibited LH pulses and microinjections of 2 nmol of this antagonist produced a modest transitory decrease in LH pulse frequency. An antagonist to the NKB receptor also decreased LH pulse frequency, whereas NKB and an antagonist to the receptor for dynorphin both increased pulse frequency. In contrast, antagonists to GnRH receptors, orphanin-FQ receptors, and the N-methyl-D-aspartate glutamate receptor had no effect on episodic LH secretion. We thus conclude that the KNDy neuropeptides act in the arcuate nucleus to control episodic GnRH secretion in the ewe, but afferent input from GnRH neurons to this area does not. These data support the proposed roles for NKB and dynorphin within the KNDy neural network and raise the possibility that kisspeptin contributes to the control of GnRH pulse frequency in addition to its established role as an output signal from KNDy neurons that drives GnRH pulses.