KNDy Neurons Research Articles

Selective depletion of kisspeptin neurons in the hypothalamic arcuate nucleus in early juvenile life reduces pubertal LH secretion and delays puberty onset in mice.

Puberty is the critical developmental transition to reproductive capability driven by the activation of gonadotropin-releasing hormone (GnRH) neurons. The complex neural mechanisms underlying pubertal activation of GnRH secretion still remain unknown, yet likely include kisspeptin neurons. However, kisspeptin neurons reside in several hypothalamic areas and the specific kisspeptin population timing pubertal onset remains undetermined. To investigate this, we strategically capitalized on the differential ontological expression of the Kiss1 gene in different hypothalamic nuclei to selectively ablate just arcuate kisspeptin neurons (aka KNDy neurons) during the early juvenile period, well before puberty, while sparing RP3V kisspeptin neurons. Both male and female transgenic mice with a majority of their KNDy neurons ablated (KNDyABL) by diphtheria toxin treatment in juvenile life demonstrated significantly delayed puberty onset and lower peripubertal LH secretion than controls. In adulthood, KNDyABL mice demonstrated normal invivo LH pulse frequency with lower basal and peak LH levels, suggesting that only a small subset of KNDy neurons is sufficient for normal GnRH pulse timing but more KNDy cells are needed to secrete normal LH concentrations. Unlike prior KNDy ablation studies in rats, there was no alteration in the occurrence or magnitude of estradiol-induced LH surges in KNDyABL female mice, indicating that a complete KNDy neuronal population is not essential for normal LH surge generation. This study teases apart the contributions of different kisspeptin neural populations to the control of puberty onset, demonstrating that a majority of KNDy neurons in the arcuate nucleus are necessary for the proper timing of puberty in both sexes.

7441 KNDy Neurons and the Control of the Gonadotropic Axis in Midgestation Fetal Sheep

Abstract Disclosure: R. Amodei: None. S.S. Jonker: None. K. Gribbin: None. E. Lazen: None. C.T. Estill: None. C.E. Roselli: None. Recent evidence suggests that the neuropeptide kisspeptin (Kiss) plays a role to activate and regulate the male reproductive axis to secrete LH and testosterone during a time in gestation critical for brain sexual differentiation. KNDy neurons, an acronym for Kiss neurons that co-express neurokinin B (NKB) and dynorphin (Dyn), regulate pulsatile GnRH/LH secretion and are present in the hypothalamic arcuate nucleus of midgestation sheep fetuses. Notably, KNDy neurons are more abundant in female than in male fetuses. We hypothesize that KNDy-GnRH signaling is established during midgestation and that negative feedback mediated through KNDy neurons serves to regulate levels of testosterone needed for brain masculinization. In the current study, we used immunofluorescence histochemistry to assess the effect of testosterone manipulations on the number of arcuate Kiss neurons in male and female fetuses. In addition, we employed unanesthetized cannulated midgestation fetal sheep to test the effects of KNDy peptides on LH secretion and characterize receptor specificity in a real-time physiological context. We found that biweekly administration of testosterone propionate to the dam from Day 30–58 of gestation significantly increased (P < 0.05) plasma testosterone concentrations, which resulted in a significant decrease (P < 0.05) in both mean plasma LH concentrations and the number of arcuate Kiss neurons in female lamb fetuses. Conversely, chemical castration of midgestation male fetuses by treatment with the GnRH antagonist degarelix from day 60 – 90 of gestation resulted in a significant decrease (P < 0.05) in mean plasma LH and testosterone concentrations that was accompanied by a significant increase (P < 0.05) in the number of arcuate Kiss neurons. In unanesthetized fetuses, we found that bolus administration of both KP-10 (Kiss agonist) and senktide (NKB agonist) elicited a robust release of LH within 15 min. Treatment with the NK3 receptor antagonist SB222200 effectively blocked the senktide response, whereas the Kiss1 receptor antagonist P271 appeared to have an effect in males only. Blocking κ-opiate receptor (KOR) with a bolus injection of the KOR antagonist PF4455242 resulted in a significant increase in LH release and enhanced the stimulatory effect of senktide. These results support the hypothesis that KNDy neurons play a regulatory role in GnRH and hence gonadotropin secretion in sheep fetuses and serve as targets for negative feedback in males for maintaining a stable androgen environment crucial for brain masculinization. Supported by NIH Grant OD11047 to CER. Presentation: 6/1/2024

7640 The first description of an MC4R variant in a patient with Kallmann syndrome and obesity

Abstract Disclosure: A.A. Aslam: None. S. Lim: None. R. Willemsen: None. K. Koysombat: None. M. Young: None. W.S. Dhillo: None. A. Abbara: None. S. Howard: None. E.F. Gevers: None. Introduction: Pathogenic MC4R gene variants result in hyperphagia and early onset obesity but puberty is not usually affected. We describe an MC4R variant in a patient with Kallmann syndrome and obesity. A 16 year old male with repaired Tetralogy of Fallot, anosmia, autism and anxiety, was referred with obesity and delayed puberty. His birth weight was 3360 g (-0.26 SDS). Height was -1.31 SDS, BMI 30.7 kg/m2. He had high arched palate, normal skin and hair colour, mild hypertelorism and upward slanting palpebral fissures. Pubertal staging was A2P1G1 5ml testes bilaterally. Results: LHRH test showed borderline low peaks (LH 5.0 U/L, FSH 2.6 U/L) and inhibin B 108 pg/mL (25-325 pg/mL). MRI brain showed hypoplastic olfactory bulbs, absent olfactory sulci and normal pituitary anatomy. CGH microarray, karyotype and the UK hypogonadism gene panel were normal. He underwent whole genome sequencing in the Genetic Factors Affecting the Timing of Puberty Study; no abnormalities in 52 known genes associated with GnRH deficiency were found but a previously described heterozygous variant was detected, MC4R c.542G>A, p.Gly181Asp, classified pathogenic and absent in control databases. Management: He was commenced on Testosterone but showed progression of testicular size to 10 mL and testosterone was stopped but required restarting. Repeat investigations off treatment, aged 22 years (height 178 cm, BMI 42 kg/m2, testes 15 mL): inhibin B 66 pg/mL, testosterone 2 nmol/L, baseline LH 2.4 U/L, stimulated peak 24.9 U/L, baseline FSH 1.2 U/L, stimulated peak 4.2 U/L. Cortisol and IGF1 were in the normal range. He also developed autoimmune hypothyroidism with raised TPO antibodies (TSH 24.7 mU/L, FT4 5.6 pmol/L). Discussion: Previous in vitro work has shown complete loss of function of MC4R p.Gly181Asp due to reduced cell surface expression and reduced binding to a-MSH. Heterozygous p.Gly181Asp variants have been described in several children/adults with obesity; but hypogonadism in heterozygous carriers has not. Homozygous MC4R p.Gly181Asp was found in a male with obesity and partial HH thought to be due to abnormal GnRH production but with normal olfactory bulbs. We are the first to describe a heterozygous MC4R p.Gly181Asp variant in a patient with partial hypogonadism and anosmia with hypoplastic bulbs in addition to obesity. Interaction between POMC-MC-leptin circuits and Kisspeptin-GnRH circuits is recognised although not completely understood. Previous work has shown that a subset of kisspeptin neurons express MC4R and that KNDy neurones receive synaptic input from POMC neurons. In addition, inhibition of MC4R/MC3R results in delayed onset of puberty in rats. Our results support a role for MC4R in GnRH secretion and potentially olfactory /GnRH neuronal migration. Assessment of spontaneous gonadotrophin production and response of gonadotrophin production to kisspeptin stimulation is currently in progress. Presentation: 6/1/2024

Absence of sex differences in diabetes-induced suppression of KNDy neurons in rats.

Diabetes mellitus disturbs kisspeptin-neurokinin B-dynorphin A (KNDy) neurons in the arcuate nucleus (ARC), which regulate pulsatile luteinizing hormone (LH) secretion in both sexes. However, it remains unclear whether a sex-specific association with the negative effects of diabetes on KNDy neurons exists. Therefore, we examined mRNA expression in KNDy neurons of diabetic male and female rats 7 weeks after streptozotocin (STZ) injection using histochemistry. In gonad-intact rats, the numbers of Kiss1 and Pdyn mRNA-expressing cells in the ARC decreased in an STZ-dose-dependent manner; moreover, no sex-dependent association with STZ treatment was observed. In males, plasma LH and sex steroid levels decreased in diabetic rats. Conversely, those of females did not vary significantly between the control and diabetic rats. Kiss1 expression in the anteroventral periventricular nucleus was slightly affected in diabetic animals, but did not exhibit sex-dependent differences. In gonadectomized rats, the numbers of KNDy mRNA-expressing cells in the ARC and plasma LH levels decreased in diabetic male and female rats; however, sex-dependent differences did not exist. These results demonstrated that a sex-specific association with the negative effects of diabetes on KNDy neurons did not exist. Therefore, the stage of diabetes that induces the suppression of the hypothalamus may not vary according to sex.

The deletion of nuclear progesterone receptors from kisspeptin cells does not impair negative feedback in female mice.

Reproductive function in mammals depends on the ability of progesterone to suppress pulsatile gonadotrophin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion in a homeostatic negative feedback loop. Previous research identified that cells upstream from GnRH neurons expressing the nuclear progesterone receptor (PGR) are required for progesterone-negative feedback. However, the identity of these cells and the mechanism by which they reduce GnRH/LH pulsatile secretion is unknown. We aimed to address the hypothesis that PGR expressed by a neural population in the arcuate nucleus recently identified as the GnRH pulse generator, cells expressing Kisspeptin, Neurokinin B, and Dynorphin (KNDy cells), mediate progesterone negative feedback. To achieve this, we utilized female mice with the PGR gene conditionally deleted from kisspeptin cells (KPRKO mice) and observed a substantial decrease in the percentage of KNDy neurons co-expressing PGR mRNA (11% in KPRKO mice versus 86% in wildtype mice). However, KPRKO mice did not display changes in the frequency or amplitude of LH pulses in diestrus or estrus, nor in the ability of exogenous progesterone to blunt a post-castration rise in LH. Further, mRNA expression of arcuate kisspeptin and dynorphin, which are excitatory and inhibitory to GnRH secretion, respectively, remained unaltered in KPRKO mice compared to wildtype controls. Together, these findings show that the near-complete loss of PGR signaling from KNDy cells does not impact negative feedback regulation of GnRH pulse generation in mice, suggesting that feedback through this receptor can occur via a small number of KNDy cells or a yet unidentified cell population.

Peripheral administration of a κ-opioid receptor agonist nalfurafine inactivates gonadotropin-releasing hormone pulse generator activity in goats

Neurons co-expressing kisspeptin, neurokinin B, and dynorphin A (KNDy neurons), located in the arcuate nucleus (ARC) of the hypothalamus, are indicated to be the gonadotropin-releasing hormone (GnRH) pulse generator. Dynorphin A is reported to suppress GnRH pulse generator activity. Nalfurafine is a selective agonist of the κ-opioid receptor (KOR), a receptor for dynorphin A, clinically used as an anti-pruritic drug. This study aimed to evaluate the effects of nalfurafine on GnRH pulse generator activity and luteinizing hormone (LH) pulses using female goats. Nalfurafine (0, 2, 4, 8, or 16 μg/head) was intravenously injected into ovariectomized Shiba goats. The multiple unit activity (MUA) in the ARC area was recorded, and plasma LH concentrations were measured 2 and 48 h before and after injection, respectively. The MUA volley interval during 0–2 h after injection was significantly increased in the nalfurafine 8 and 16 μg groups compared with the vehicle group. In 0–2 h after injection, the number of LH pulses was significantly decreased in the nalfurafine 8 and 16 μg groups, and the mean and baseline LH were significantly decreased in all nalfurafine-treated groups (2, 4, 8, and 16 μg) compared with the vehicle group. These results suggest that nalfurafine inhibits the activity of the GnRH pulse generator in the ARC, thus suppressing pulsatile LH secretion. Therefore, nalfurafine could be used as a reproductive inhibitor in mammals.

Population bursts in a modular neural network as a mechanism for synchronized activity in KNDy neurons.

The pulsatile activity of gonadotropin-releasing hormone neurons (GnRH neurons) is a key factor in the regulation of reproductive hormones. This pulsatility is orchestrated by a network of neurons that release the neurotransmitters kisspeptin, neurokinin B, and dynorphin (KNDy neurons), and produce episodic bursts of activity driving the GnRH neurons. We show in this computational study that the features of coordinated KNDy neuron activity can be explained by a neural network in which connectivity among neurons is modular. That is, a network structure consisting of clusters of highly-connected neurons with sparse coupling among the clusters. This modular structure, with distinct parameters for intracluster and intercluster coupling, also yields predictions for the differential effects on synchronization of changes in the coupling strength within clusters versus between clusters.

Norepinephrine Neurons in the Nucleus of the Solitary Tract Suppress Luteinizing Hormone Secretion in Female Mice.

Stress impairs fertility, at least in part, via inhibition of gonadotropin secretion. Luteinizing hormone (LH) is an important gonadotropin that is released in a pulsatile pattern in males and in females throughout the majority of the ovarian cycle. Several models of stress, including acute metabolic stress, suppress LH pulses via inhibition of neurons in the arcuate nucleus of the hypothalamus that coexpress kisspeptin, neurokinin B, and dynorphin (termed KNDy cells) which form the pulse generator. The mechanism for inhibition of KNDy neurons during stress, however, remains a significant outstanding question. Here, we investigated a population of catecholamine neurons in the nucleus of the solitary tract (NTS), marked by expression of the enzyme dopamine beta-hydroxylase (DBH), in female mice. First, we found that a subpopulation of DBH neurons in the NTS is activated (express c-Fos) during metabolic stress. Then, using chemogenetics, we determined that activation of these cells is sufficient to suppress LH pulses, augment corticosterone secretion, and induce sickness-like behavior. In subsequent studies, we identified evidence for suppression of KNDy cells (rather than downstream signaling pathways) and determined that the suppression of LH pulses was not dependent on the acute rise in glucocorticoids. Together these data support the hypothesis that DBH cells in the NTS are important for regulation of neuroendocrine and behavioral responses to stress.

Malnutrition alters protein expression of KNDy neuropeptides in the arcuate nucleus of mature ewes.

The neuropeptides kisspeptin, neurokinin B, and dynorphin A are imperative for the pulsatile secretion of gonadotropin-releasing hormone and luteinizing hormone to ultimately regulate reproductive cyclicity. A population of neurons co-expressing these neuropeptides, KNDy neurons, within the arcuate nucleus of the hypothalamus (ARC) are positioned to integrate energy status from afferent neuronal and glial cells. We hypothesized that KNDy-expressing neurons in the ARC of mature ewes are influenced by energy balance. To test this hypothesis, ovary-intact, mature ewes were fed to lose, maintain, or gain body weight and hypothalamic tissue harvested during the luteal phase of the estrous cycle. Fluorescent, multiplex immunohistochemistry with direct antibody conjugation was employed to identify and quantify neurons expressing a single neuropeptide, as well as for the first time report co-expression of kisspeptin, neurokinin B, and dynorphin A protein in the ARC. Previous reports using this population of ewes demonstrated that concentrations of insulin and leptin differed between ewes fed to achieve different body weights and that ewes fed to gain body weight had increased concentrations of progesterone. Moreover, within this population of ewes tanycyte density and cellular penetration into the ARC was increased in ewes fed to gain body weight. Within the current report we have revealed that the number of neurons in the ARC expressing kisspeptin, neurokinin B, and dynorphin A protein was increased in ewes fed to gain body weight. Moreover, the number of KNDy neurons in the ARC expressing all three neuropeptides within a single neuron was decreased in ewes fed to lose body weight and increased in ewes fed to gain body weight when compared to ewes fed to maintain body weight. The cumulative findings of this experimental model suggest that expression of kisspeptin, neurokinin B, and dynorphin A protein in the ARC during the luteal phase of the estrous cycle are influenced by energy balance-induced alterations in circulating concentrations of progesterone that drive changes in morphology and density of tanycytes to ultimately regulate central perception of global energy status. Moreover, these results demonstrate that changes in KNDy neurons within the ARC occur as an adaptation to energy balance, potentially regulated divergently by metabolic milieu via proopiomelanocortin afferents.

Estrogen promotes gonadotropin-releasing hormone expression by regulating tachykinin 3 and prodynorphin systems in chicken

The "KNDy neurons" located in the hypothalamic arcuate nucleus (ARC) of mammals are known to co-express kisspeptin, neurokinin B (NKB), and dynorphin (DYN), and have been identified as key mediators of the feedback regulation of steroid hormones on gonadotropin-releasing hormone (GnRH). However, in birds, the genes encoding kisspeptin and its receptor GPR54 are genomic lost, leaving unclear mechanisms for feedback regulation of GnRH by steroid hormones. Here, the genes tachykinin 3 (TAC3) and prodynorphin (PDYN) encoding chicken NKB and DYN neuropeptides were successfully cloned. Temporal expression profiling indicated that TAC3, PDYN and their receptor genes (TACR3, OPRK1) were mainly expressed in the hypothalamus, with significantly higher expression at 30W than at 15W. Furthermore, overexpression or interference of TAC3 and PDYN can regulate the GnRH mRNA expression. In addition, in vivo and in vitro assays showed that estrogen (E2) could promote the mRNA expression of TAC3, PDYN, and GnRH, as well as the secretion of GnRH/LH. Mechanistically, E2 could dimerize the nuclear estrogen receptor 1 (ESR1) to regulate the expression of TAC3 and PDYN, which promoted the mRNA and protein expression of GnRH gene as well as the secretion of GnRH. In conclusion, these results revealed that E2 could regulate the GnRH expression through TAC3 and PDYN systems, providing novel insights for reproductive regulation in chickens.

Raphe glucose-sensing serotonergic neurons stimulate KNDy neurons to enhance LH pulses via 5HT2CR: rat and goat studies

Dysfunction of central serotonergic neurons is known to cause depressive disorders in humans, who often show reproductive and/or glucose metabolism disorders. This study examined whether dorsal raphe (DR) serotonergic neurons sense high glucose availability to upregulate reproductive function via activating hypothalamic arcuate (ARC) kisspeptin neurons (= KNDy neurons), a dominant stimulator of gonadotropin-releasing hormone (GnRH)/gonadotropin pulses, using female rats and goats. RNA-seq and histological analysis revealed that stimulatory serotonin-2C receptor (5HT2CR) was mainly expressed in the KNDy neurons in female rats. The serotonergic reuptake inhibitor administration into the mediobasal hypothalamus (MBH), including the ARC, significantly blocked glucoprivic suppression of luteinizing hormone (LH) pulses and hyperglycemia induced by intravenous 2-deoxy-D-glucose (2DG) administration in female rats. A local infusion of glucose into the DR significantly increased in vivo serotonin release in the MBH and partly restored LH pulses and hyperglycemia in the 2DG-treated female rats. Furthermore, central administration of serotonin or a 5HT2CR agonist immediately evoked GnRH pulse generator activity, and central 5HT2CR antagonism blocked the serotonin-induced facilitation of GnRH pulse generator activity in ovariectomized goats. These results suggest that DR serotonergic neurons sense high glucose availability to reduce gluconeogenesis and upregulate reproductive function by activating GnRH/LH pulse generator activity in mammals.

Mechanism of elevated LH/FSH ratio in lean PCOS revisited: a path analysis

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder affecting 5–20% of reproductive-age women. However, the treatment of PCOS is mainly based on symptoms and not on its pathophysiology. Neuroendocrine disturbance, as shown by an elevated LH/FSH ratio in PCOS patients, was thought to be the central mechanism of the syndrome, especially in lean PCOS. LH and FSH secretion are influenced by GnRH pulsatility of GnRH neurons in the hypothalamus. Kisspeptin is the main regulator of GnRH secretion, whereas neurokinin B (NKB) and dynorphin regulate kisspeptin secretion in KNDy neurons. This study aims to deepen the understanding of the neuroendocrine disorder in lean PCOS patients and its potential pathophysiology-based therapy. A cross-sectional study was performed at Dr. Cipto Mangunkusumo Kencana Hospital and the IMERI UI HRIFP cluster with 110 lean PCOS patients as subjects. LH, FSH, LH/FSH ratio, kisspeptin, NKB, dynorphin, leptin, adiponectin, AMH, fasting blood glucose, fasting insulin, HOMA-IR, testosterone, and SHBG were measured. Bivariate and path analyses were performed to determine the relationship between variables. There was a negative association between dynorphin and kisspeptin, while NKB levels were not associated with kisspeptin. There was no direct association between kisspeptin and the LH/FSH ratio; interestingly, dynorphin was positively associated with the LH/FSH ratio in both bivariate and pathway analyses. AMH was positively correlated with the LH/FSH ratio in both analyses. Path analysis showed an association between dynorphin and kisspeptin levels in lean PCOS, while NKB was not correlated with kisspeptin. Furthermore, there was a correlation between AMH and the LH/FSH ratio, but kisspeptin levels did not show a direct significant relationship with the LH/FSH ratio. HOMA-IR was negatively associated with adiponectin levels and positively associated with leptin and FAI levels. In conclusion, AMH positively correlates with FAI levels and is directly associated with the LH/FSH ratio, showing its important role in neuroendocrinology in lean PCOS. From the path analysis, AMH was also an intermediary variable between HOMA-IR and FAI with the LH/FSH ratio. Interestingly, this study found a direct positive correlation between dynorphin and the LH/FSH ratio, while no association between kisspeptin and the LH/FSH ratio was found. Further research is needed to investigate AMH and dynorphin as potential therapeutic targets in the management of lean PCOS patients.

Puberty in beef heifers: effects of prenatal and postnatal nutrition on the development of the neuroendocrine axis.

Reproductive maturation is a complex physiological process controlled by the neuroendocrine system and is characterized by an increase in gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) pulsatile secretion. Nutrition during early development is a key factor regulating puberty onset, which is defined as first ovulation in females. In heifers, nutrient restriction after weaning delays puberty, whereas elevated levels of nutrition and energy reserves advance reproductive maturation. Recent studies in cattle and other animal models have shown that the dam's nutrition during gestation can also program the neuroendocrine system in the developing fetus and has the potential to alter timing of puberty in the offspring. Among the metabolic signals that modulate brain development and control timing of puberty is leptin, a hormone produced primarily by adipocytes that communicates energy status to the brain. Leptin acts within the arcuate nucleus of the hypothalamus to regulate GnRH secretion via an upstream network of neurons that includes neurons that express neuropeptide Y (NPY), an orexigenic peptide with inhibitory effects on GnRH secretion, and alpha melanocyte-stimulating hormone (αMSH), an anorexigenic peptide with excitatory effects on GnRH neurons. Another important population of neurons are KNDy neurons, neurons in the arcuate nucleus that co-express the neuropeptides kisspeptin, neurokinin B, and dynorphin and have strong stimulatory effects on GnRH secretion. Our studies in beef heifers indicate that increased nutrition between 4 to 8 months of age advances puberty by diminishing NPY inhibitory tone and by increasing excitatory inputs of αMSH and kisspeptin, which collectively lead to increased GnRH/LH pulsatility. Our ongoing studies indicate that different planes of nutrition during gestation can alter maternal leptin concentrations and promote changes in the fetal brain. Nonetheless, at least in Bos indicus-influenced heifers, deficits programmed prenatally can be overcome by adequate postnatal nutrition without negatively impacting age at puberty or subsequent fertility.

Engineering hfCas12Max for improved gene editing efficiency

<p>Hormone studies are a trove of captivating narratives, showcasing dedication, adversity, serendipity, and human triumph. This article delves into riveting stories of hormone discovery, characterization, clinical application, and the scientists themselves. Our journey begins with Arnold Berthod��s pivotal rooster castration experiments, laying the groundwork for understanding testosterone, and Ernest Starling��s 1902 revelation that secretin wasn��t neuron-released. We��ll explore the origins of kisspeptin and its role in GnRH pulsatile secretion by KNDY neurons. Discussions will include GH therapy��s surprising link to Creutzfeldt-Jakob disease and the isolation of GHRH from pancreatic tumors. We��ll also delve into drug screenings for GH release stimulants and the design of long-acting FSH for infertility, alongside discoveries of inhibin and activin. Further, we��ll uncover the genetic basis of chemokines and relaxin-related ligands, highlighting scientists�� perseverance and ingenuity. From collecting Mexican yam tubers for progestin synthesis to using beard shavings to study testosterone regulation, their creativity knows no bounds. We��ll touch on parabiotic animal experiments leading to leptin��s discovery and polarized ligand-receptor expression. Anecdotes will include a Nobel laureate displaying a rejection letter during an award ceremony and a researcher pursuing receptor structure despite funding setbacks, both eventually rewarded. With dedicated scientists and serendipitous findings, we��ve unlocked the secrets of over a hundred hormones, understanding their pivotal role in maintaining bodily homeostasis.</p>

The role of KNDy neurons in human reproductive health.

In the early 2000s, metastin, an endogenous ligand for G protein-coupled receptor 54 (GPR54), was discovered in human placental extracts. In 2003, GPR54 receptor mutations were found in a family with congenital hypogonadotropic hypogonadism. Metastin was subsequently renamed kisspeptin after its coding gene, Kiss1. Since then, studies in mice and other animals have revealed that kisspeptin is located at the apex of the hypothalamic-pituitary-gonadal axis and regulates reproductive functions by modulating gonadotropin-releasing hormone (GnRH). In rodents, kisspeptin (Kiss1) neurons localize to two regions, the hypothalamic arcuate nucleus (ARC) and the anteroventral periventricular nucleus (AVPV). ARC Kiss1 neurons co-express neurokinin B (NKB) and dynorphin and are thus termed KNDy neurons. Kiss1 neurons in humans are concentrated in the infundibular nucleus (equivalent to the ARC), with few Kiss1 neurons localized to the preoptic area (equivalent to the AVPV), and the mechanisms underlying GnRH surge secretion in humans are poorly understood. However, peripheral administration of kisspeptin to humans promotes gonadotropin secretion, and administration of kisspeptin to patients with hypothalamic amenorrhea or congenital hypogonadotropic hypogonadism restores the pulsatile secretion of GnRH/luteinizing hormone. Thus, kisspeptin undoubtedly plays an important role in reproductive function in humans. Studies are currently underway to develop kisspeptin receptor agonists or antagonists for clinical application. Modification of KNDy neurons by NKB agonists/antagonists is also being attempted to develop therapeutic agents for various menstrual abnormalities, including polycystic ovary syndrome and menopausal hot flashes. Here, we review the role of kisspeptin in humans and its clinical applications.

Conditional Oprk1-dependent Kiss1 deletion in kisspeptin neurons caused estrogen-dependent LH pulse disruption and LH surge attenuation in female rats

The gonadotropin-releasing hormone (GnRH) pulse and surge are considered to be generated by arcuate kisspeptin/neurokinin B/dynorphin A (KNDy) neurons and anteroventral periventricular nucleus (AVPV) kisspeptin neurons, respectively, in female rodents. The majority of KNDy and AVPV kisspeptin neurons express κ-opioid receptors (KORs, encoded by Oprk1) in female rodents. Thus, this study aimed to investigate the effect of a conditional Oprk1-dependent Kiss1 deletion in kisspeptin neurons on the luteinizing hormone (LH) pulse/surge and fertility using Kiss1-floxed/Oprk1-Cre rats, in which Kiss1 was deleted in cells expressing or once expressed the Oprk1/Cre. The Kiss1-floxed/Oprk1-Cre female rats, with Kiss1 deleted in a majority of KNDy neurons, showed normal puberty while having a one-day longer estrous cycle and fewer pups than Kiss1-floxed controls. Notably, ovariectomized (OVX) Kiss1-floxed/Oprk1-Cre rats showed profound disruption of LH pulses in the presence of a diestrous level of estrogen but showed apparent LH pulses without estrogen treatment. Furthermore, Kiss1-floxed/Oprk1-Cre rats, with Kiss1 deleted in approximately half of AVPV kisspeptin neurons, showed a lower peak of the estrogen-induced LH surge than controls. These results suggest that arcuate and AVPV kisspeptin neurons expressing or having expressed Oprk1 have a role in maintaining normal GnRH pulse and surge generation, the normal length of the estrous cycle, and the normal offspring number in female rats.

Neuroendocrine features of the pathogenesis of polycystic ovary syndrome (literature review)

Polycystic ovary syndrome (PCOS) is one of the most pressing problems in endocrine gynecology. The main signs of the disease are hyperandrogenism, menstrual and/or ovulatory dysfunction, and polycystic ovarian structure according to ultrasound. Women with PCOS are at risk for developing metabolic syndrome, type 2 diabetes, cardiovascular disease, and endometrial cancer. In this connection, the pathogenetic mechanisms of the occurrence of this syndrome are continuously studied and new methods of treatment are being sought. PCOS is characterized by a wide range of various disorders of the neuroendocrine regulation of the reproductive system. The main focus of the review is aimed at summarizing information about the etiological role of neuropeptides and neurotransmitters, such as phoenixin, galanins, orexins, GABA, in the pathophysiology of PCOS and about the possibility of their use for diagnostic and therapeutic purposes. In recent decades, the interest of scientists has been focused on the study of KNDy neurons, because it is the kisspeptin synthesized by them that is one of the main regulators of the hypothalamic-pituitary-ovarian axis. This article discusses data on the significance of KNDy neurons in the pathogenesis of the syndrome. Information is provided on the effect of elevated levels of androgens and anti-Müllerian hormone on GnRH neurons. Also analyzed are studies on functional and structural disorders in the hypothalamus in PCOS. Literature search was carried out in national (eLibrary, CyberLeninka.ru) and international (PubMed, Cochrane Library) databases in Russian and English. The priority was free access to the full text of articles. The choice of sources was prioritized for the period from 2018 to 2023.However, taking into account the insufficient knowledge of the chosen topic, the choice of sources dates back to 1998.

25 Role of Kndy and Arcuate Kiss1r-Containing Neurons in the Preovulatory Luteinizing Hormone Surge and Puberty Onset of Female sheep

Abstract Neurons within the arcuate nucleus (ARC) of the hypothalamus containing kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons) have an important role in regulating the pulsatile secretion of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH). In sheep, kisspeptin neurons also contribute to the LH surge, as kisspeptin receptor (Kiss1r) antagonist administration reduces surge amplitude by 50% and KNDy neurons are likely involved, based on increased Fos expression at the time of the surge. However, the extent to which kisspeptin acts within the ARC regulate the GnRH/LH surge remains unclear. Thus, herein we tested the hypothesis that deletion of KNDy or ARC Kiss1r-containing neurons would impair the LH surge. Adult female sheep received bilateral injections targeting the ARC of NKB-saporin (NKB-SAP, n = 8), kisspeptin-saporin (Kiss-SAP, n = 10), or blank-saporin (Blank-SAP, n = 7) as a control. In other work, NKB-SAP lesioned over 90% of ovine KNDy neurons, while Kiss-SAP lesioned 67% of Kiss1r-containing cells without affecting KNDy or GnRH cell number. Ewes were also ovariectomized and a subcutaneous silastic estradiol (E2) implant was inserted at the time of neurosurgery. Two artificial luteal phases were simulated with progesterone-containing CIDRs, immediately followed by E2 treatment via implants to induce an LH surge. Blood samples were collected every two to four hours over two days and analyzed for LH via radioimmunoassay. LH surge amplitude in six of eight NKB-SAP ewes (49.5 ± 11.7 ng/mL) was significantly reduced compared with Blank-SAP control ewes (156.7 ± 20.2 ng/mL, p = 0.0001), a reduction similar to that produced by treatment with a Kiss1r antagonist. Nine of ten Kiss-SAP treated ewes displayed little to no increase of LH at the time of the expected surge (16.6 ± 5.3 ng/mL, p < 0.0001). Lesion effectiveness is currently being assessed by RNAscope, however all Kiss-SAP animals examined to date have significantly reduced ARC Kiss1r cell numbers except a single ewe which exhibited a normal LH surge. Based on these data, we propose that in ewes, KNDy neurons contribute to, but are not required for, the LH surge. In contrast, ARC Kiss1r-containing cells are essential for a functional LH surge. Given these results, we are currently assessing the role of ARC Kiss1r neurons in ovine puberty using a similar approach. Our data to date shows that time to puberty onset is similar for Kiss-SAP, Blank-SAP, and non-surgical control animals as measured by an increase in progesterone (p = 0.35). Blood samples to detect LH pulses and the LH surge are currently being analyzed, as are ARC Kiss1r cell numbers.

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.

26 The Kisspeptin Knockout Pig: a New Model for Dissecting Mechanisms Controlling Reproduction

Abstract The biological role of kisspeptin in the reproduction of swine has been far less studied than in other species. In the porcine hypothalamus, kisspeptin is coexpressed with neurokinin B and dynorphin in KNDy neurons. Although kisspeptin-stimulated secretion of gonadotropin hormones in gilts was established early, increased expression of hypothalamic KISS1 was only recently confirmed to be associated with puberty and cyclicity in pigs. Gene knockout (KO) models to study kisspeptin in livestock did not exist until TALEN gene editing technology was used to knockout KISS1R in pigs, resulting in a hypogonadotropic-hypogonadal (HH) phenotype that caused pubertal failure. Little is known about the reproductive physiology of KISS1R KO pigs, except that exogenous follicle-stimulating hormone and gonadotropin-releasing hormone only partially restored gonadal development in boars. To further explore the biological function of kisspeptin and its receptor in swine, we recently used CRISPR/cas9-ribonucleoproteins to knockout KISS1 in pigs (10.3389/fgene.2022.1078991). A total of 684 zygotes were injected resulting in 69 piglets with 58% bearing KISS1 KO alleles (KISS1-/-). The proportion of KISS1-/- alleles did not affect BW or ADG in these mosaic founder pigs. Two boars and 2 gilts with complete KISS1 KO exhibited HH and failed to become pubertal. Germ and Sertoli cells were disorganized in small seminiferous tubules. Mating mosaic F0 pigs (frequency of KISS1-/-, 35 to 88%) resulted in 170 F1 piglets with KISS1-/- alleles observed in a Mendelian frequency. These KISS1-/- pigs displayed normal BW growth during development but were also HH. Ovarian development of KISS1-/- gilts was arrested at a juvenile state, although antral follicles were present. The KISS1-/- boars lacked testicular development and had dramatically reduced concentrations of androgens and skatole, suggesting the lack of boar taint in their meat. Importantly, KISS1 and KISS1R are expressed in ovarian, uterine and testicular tissue; however, the direct role of kisspeptin in these reproductive organs remains unknown in livestock. These KISS1-/- pigs provide a unique animal model that opens new avenues for understanding the role of kisspeptin and gonadotropin hormones in regulation of reproductive development and function in pigs as well as livestock in general. These animals facilitate our ability to explore questions such as: 1) how KNDy neurons function and control gonadotropin pulses or 2) what role does KISS1 play in the development and function of the ovary, uterus and testis in domestic animals. Some of these questions have heretofore only been addressable with difficult surgical modifications, and therefore, KISS1-/- pigs represent an alternative to painful procedures in livestock research. Pursuing these fundamental studies is expected to lead to methods that restore fertility in KISS1-/- pigs. This, in turn, could provide new possibilities for the elimination of surgical castration from pork production. The USDA is an equal opportunity provider and employer.