Disparate Changes in Kisspeptin and Neurokinin B Expression in the Arcuate Nucleus After Sex Steroid Manipulation Reveal Differential Regulation of the Two KNDy Peptides in Rats

Abstract Kisspeptin, neurokinin B (NKB) and dynorphin A are coexpressed in a population of neurons in the arcuate nucleus (ARC), termed KNDy neurons, which were recently recognized as important elements for the generation of GnRH pulses. However, the topographic distribution of these peptides and their regulated expression by sex steroids are still not well understood. In this study, detailed examination of NKB and kisspeptin immunoreactivity in the rat ARC was carried out, including comparison between sexes, with and without sex steroid replacement. Neurons expressing kisspeptin and NKB were more prominent in the caudal ARC of females, whereas neurons expressing NKB, but not kisspeptin, were the most abundant in the male. Sex steroid manipulation revealed differential regulation of kisspeptin and NKB; although kisspeptin immunoreactive (ir) cells increased in response to gonadectomy, NKB remained unchanged. Furthermore, the number of NKB-ir cells increased upon sex steroid replacement compared with gonadectomy, whereas kisspeptin did not, suggesting that sex steroids differently regulate these peptides. In addition, only in females did the density of kisspeptin- and NKB-ir fibers in the ARC increase upon sex steroid replacement in relation to sham and ovariectomy, respectively, suggesting sex-specific regulation of release. In conclusion, our observations reveal sex differences in the number of kisspeptin- and NKB-ir cells, which are more prominent in the caudal ARC. The divergent regulation of kisspeptin and NKB peptide contents in the ARC as a function of sex and steroid milieu enlarge our understanding on how these neuropeptides are posttranscriptionally regulated in KNDy neurons.

Download Full-text

Neurokinin B and the Control of the Gonadotropic Axis in the Rat: Developmental Changes, Sexual Dimorphism, and Regulation by Gonadal Steroids

Endocrinology ◽

10.1210/en.2012-1287 ◽

2012 ◽

Vol 153 (10) ◽

pp. 4818-4829 ◽

Cited By ~ 45

Author(s):

F. Ruiz-Pino ◽

V. M. Navarro ◽

A. H. Bentsen ◽

D. Garcia-Galiano ◽

M. A. Sanchez-Garrido ◽

...

Keyword(s):

Sexual Dimorphism ◽

Sex Steroids ◽

Developmental Stages ◽

Female Rats ◽

Sex Steroid ◽

Adult Males ◽

Neurokinin B ◽

Male And Female Rats ◽

Gonadotropic Axis ◽

Key Aspects

Abstract Neurokinin B (NKB), encoded by Tac2 in rodents, and its receptor, NK3R, have recently emerged as important regulators of reproduction; NKB has been proposed to stimulate kisspeptin output onto GnRH neurons. Accordingly, NKB has been shown to induce gonadotropin release in several species; yet, null or even inhibitory effects of NKB have been also reported. The basis for these discrepant findings, as well as other key aspects of NKB function, remains unknown. We report here that in the rat, LH responses to the NK3R agonist, senktide, display a salient sexual dimorphism, with persistent stimulation in females, regardless of the stage of postnatal development, and lack of LH responses in males from puberty onward. Such dimorphism was independent of the predominant sex steroid after puberty, because testosterone administration to adult females failed to prevent LH responses to senktide, and LH responsiveness was not restored in adult males treated with estradiol or the nonaromatizable androgen, dihydrotestosterone. Yet, removal of sex steroids by gonadectomy switched senktide effects to inhibitory, both in adult male and female rats. Sexual dimorphism was also evident in the numbers of NKB-positive neurons in the arcuate nucleus (ARC), which were higher in adult female rats. This is likely the result of differences in sex steroid milieu during early periods of brain differentiation, because neonatal exposures to high doses of estrogen decreased ARC NKB neurons at later developmental stages. Likewise, neonatal estrogenization resulted in lower serum LH levels that were normalized by senktide administration. Finally, we document that the ability of estrogen to inhibit hypothalamic Tac2 expression seems region specific, because estrogen administration decreased Tac2 levels in the ARC but increased them in the lateral hypothalamus. Altogether, our data provide a deeper insight into relevant aspects of NKB function as major regulator of the gonadotropic axis in the rat, including maturational changes, sexual dimorphism, and differential regulation by sex steroids.

Download Full-text

KNDy Neurons Modulate the Magnitude of the Steroid-Induced Luteinizing Hormone Surges in Ovariectomized Rats

Endocrinology ◽

10.1210/en.2015-1070 ◽

2015 ◽

Vol 156 (11) ◽

pp. 4200-4213 ◽

Cited By ~ 23

Author(s):

Cleyde V. Helena ◽

Natalia Toporikova ◽

Bruna Kalil ◽

Andrea M. Stathopoulos ◽

Veronika V. Pogrebna ◽

...

Keyword(s):

Negative Feedback ◽

Arcuate Nucleus ◽

Ovariectomized Rats ◽

Neurokinin B ◽

Lh Surge ◽

Neuronal Populations ◽

Lh Release ◽

Positive And Negative Feedback ◽

Kndy Neurons ◽

Lh Secretion

Kisspeptin is the most potent stimulator of LH release. There are two kisspeptin neuronal populations in the rodent brain: in the anteroventral periventricular nucleus (AVPV) and in the arcuate nucleus. The arcuate neurons coexpress kisspeptin, neurokinin B, and dynorphin and are called KNDy neurons. Because estradiol increases kisspeptin expression in the AVPV whereas it inhibits KNDy neurons, AVPV and KNDy neurons have been postulated to mediate the positive and negative feedback effects of estradiol on LH secretion, respectively. Yet the role of KNDy neurons during the positive feedback is not clear. In this study, ovariectomized rats were microinjected bilaterally into the arcuate nucleus with a saporin-conjugated neurokinin B receptor agonist for targeted ablation of approximately 70% of KNDy neurons. In oil-treated animals, ablation of KNDy neurons impaired the rise in LH after ovariectomy and kisspeptin content in both populations. In estradiol-treated animals, KNDy ablation did not influence the negative feedback of steroids during the morning. Surprisingly, KNDy ablation increased the steroid-induced LH surges, accompanied by an increase of kisspeptin content in the AVPV. This increase seems to be due to lack of dynorphin input from KNDy neurons to the AVPV as the following: 1) microinjections of a dynorphin antagonist into the AVPV significantly increased the LH surge in estradiol-treated rats, similar to KNDy ablation, and 2) intra-AVPV microinjections of dynorphin in KNDy-ablated rats restored LH surge levels. Our results suggest that KNDy neurons provide inhibition to AVPV kisspeptin neurons through dynorphin and thus regulate the amplitude of the steroid-induced LH surges.

Download Full-text

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

Endocrinology ◽

10.1210/en.2014-1609 ◽

2015 ◽

Vol 156 (9) ◽

pp. 3277-3291 ◽

Cited By ~ 25

Author(s):

Maria Cernea ◽

Vasantha Padmanabhan ◽

Robert L. Goodman ◽

Lique M. Coolen ◽

Michael N. Lehman

Keyword(s):

Feedback Control ◽

Arcuate Nucleus ◽

Preoptic Area ◽

Neurokinin B ◽

Medial Basal Hypothalamus ◽

Synaptic Inputs ◽

Prenatal Testosterone ◽

Peptide Expression ◽

Kndy Neurons ◽

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.

Download Full-text

Evidence That Dynorphin Acts Upon KNDy and GnRH Neurons During GnRH Pulse Termination in the Ewe

Endocrinology ◽

10.1210/en.2018-00435 ◽

2018 ◽

Vol 159 (9) ◽

pp. 3187-3199 ◽

Cited By ~ 14

Author(s):

Peyton W Weems ◽

Lique M Coolen ◽

Stanley M Hileman ◽

Steven Hardy ◽

Rick B McCosh ◽

...

Keyword(s):

Arcuate Nucleus ◽

Third Ventricle ◽

Pulse Generation ◽

G Protein Coupled Receptors ◽

Neurokinin B ◽

Gnrh Secretion ◽

Pulsatile Gnrh ◽

Gnrh Neurons ◽

Kndy Neurons ◽

G Protein Coupled

Abstract A subpopulation of neurons located within the arcuate nucleus, colocalizing kisspeptin, neurokinin B, and dynorphin (Dyn; termed KNDy neurons), represents key mediators of pulsatile GnRH secretion. The KNDy model of GnRH pulse generation proposes that Dyn terminates each pulse. However, it is unknown where and when during a pulse that Dyn is released to inhibit GnRH secretion. Dyn acts via the κ opioid receptor (KOR), and KOR is present in KNDy and GnRH neurons in sheep. KOR, similar to other G protein–coupled receptors, are internalized after exposure to ligand, and thus internalization can be used as a marker of endogenous Dyn release. Thus, we hypothesized that KOR will be internalized at pulse termination in both KNDy and GnRH neurons. To test this hypothesis, GnRH pulses were induced in gonad-intact anestrous ewes by injection of neurokinin B (NKB) into the third ventricle and animals were euthanized at times of either pulse onset or termination. NKB injections produced increased internalization of KOR within KNDy neurons during both pulse onset and termination. In contrast, KOR internalization into GnRH neurons was seen only during pulse termination, and only in GnRH neurons within the mediobasal hypothalamus (MBH). Overall, our results indicate that Dyn is released onto KNDy cells at the time of pulse onset, and continues to be released during the duration of the pulse. In contrast, Dyn is released onto MBH GnRH neurons only at pulse termination and thus actions of Dyn upon KNDy and GnRH cell bodies may be critical for pulse termination.

Download Full-text

Increased Neurokinin B (Tac2) Expression in the Mouse Arcuate Nucleus Is an Early Marker of Pubertal Onset with Differential Sensitivity to Sex Steroid-Negative Feedback than Kiss1

Endocrinology ◽

10.1210/en.2012-1529 ◽

2012 ◽

Vol 153 (10) ◽

pp. 4883-4893 ◽

Cited By ~ 41

Author(s):

John C. Gill ◽

Víctor M. Navarro ◽

Cecilia Kwong ◽

Sekoni D. Noel ◽

Cecilia Martin ◽

...

Keyword(s):

Negative Feedback ◽

Arcuate Nucleus ◽

Pubertal Development ◽

Differential Sensitivity ◽

Sex Steroid ◽

Wild Type ◽

Neurokinin B ◽

Pubertal Onset ◽

Steroid Dependent

Abstract At puberty, neurokinin B (NKB) and kisspeptin (Kiss1) may help to amplify GnRH secretion, but their precise roles remain ambiguous. We tested the hypothesis that NKB and Kiss1 are induced as a function of pubertal development, independently of the prevailing sex steroid milieu. We found that levels of Kiss1 mRNA in the arcuate nucleus (ARC) are increased prior to the age of puberty in GnRH/sex steroid-deficient hpg mice, yet levels of Kiss1 mRNA in wild-type mice remained constant, suggesting that sex steroids exert a negative feedback effect on Kiss1 expression early in development and across puberty. In contrast, levels of Tac2 mRNA, encoding NKB, and its receptor (NK3R; encoded by Tacr3) increased as a function of puberty in both wild-type and hpg mice, suggesting that during development Tac2 is less sensitive to sex steroid-dependent negative feedback than Kiss1. To compare the relative responsiveness of Tac2 and Kiss1 to the negative feedback effects of gonadal steroids, we examined the effect of estradiol (E2) on Tac2 and Kiss1 mRNA and found that Kiss1 gene expression was more sensitive than Tac2 to E2-induced inhibition at both juvenile and adult ages. This differential estrogen sensitivity was tested in vivo by the administration of E2. Low levels of E2 significantly suppressed Kiss1 expression in the ARC, whereas Tac2 suppression required higher E2 levels, supporting differential sensitivity to E2. Finally, to determine whether inhibition of NKB/NK3R signaling would block the onset of puberty, we administered an NK3R antagonist to prepubertal (before postnatal d 30) females and found no effect on markers of pubertal onset in either WT or hpg mice. These results indicate that the expression of Tac2 and Tacr3 in the ARC are markers of pubertal activation but that increased NKB/NK3R signaling alone is insufficient to trigger the onset of puberty in the mouse.

Download Full-text

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

Endocrinology ◽

10.1210/en.2013-1331 ◽

2013 ◽

Vol 154 (11) ◽

pp. 4259-4269 ◽

Cited By ~ 101

Author(s):

Robert L. Goodman ◽

Stanley M. Hileman ◽

Casey C Nestor ◽

Katrina L. Porter ◽

John M. Connors ◽

...

Keyword(s):

Arcuate Nucleus ◽

Pulse Generator ◽

Pulse Frequency ◽

Afferent Input ◽

Pulse Generation ◽

Orphanin Fq ◽

Neurokinin B ◽

Control Activity ◽

Kndy Neurons ◽

Lh Secretion

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.

Download Full-text

Neurokinin B activates synchronized intracellular Ca2+ oscillations in KNDy neurons obtained from the hypothalamic arcuate nucleus of Kiss1-GFP transgenic mice

Reproduction Abstracts ◽

10.1530/repabs.1.p335 ◽

2014 ◽

Author(s):

Kana Ikegami ◽

Shiori Minabe ◽

Nahoko Ieda ◽

Teppei Goto ◽

Hitomi Abe ◽

...

Keyword(s):

Transgenic Mice ◽

Arcuate Nucleus ◽

Neurokinin B ◽

Hypothalamic Arcuate Nucleus ◽

Kndy Neurons

Download Full-text

Arcuate Kisspeptin/Neurokinin B/Dynorphin (KNDy) Neurons Mediate the Estrogen Suppression of Gonadotropin Secretion and Body Weight

Endocrinology ◽

10.1210/en.2012-1045 ◽

2012 ◽

Vol 153 (6) ◽

pp. 2800-2812 ◽

Cited By ~ 97

Author(s):

Melinda A. Mittelman-Smith ◽

Hemalini Williams ◽

Sally J. Krajewski-Hall ◽

Josephine Lai ◽

Philippe Ciofi ◽

...

Keyword(s):

Body Weight ◽

Arcuate Nucleus ◽

Estrogen Receptor Α ◽

Neurokinin B ◽

Abdominal Girth ◽

Gonadotropin Secretion ◽

Serum Lh ◽

17Β Estradiol ◽

Neurokinin 3 Receptor ◽

Kndy Neurons

Estrogen withdrawal increases gonadotropin secretion and body weight, but the critical cell populations mediating these effects are not well understood. Recent studies have focused on a subpopulation of hypothalamic arcuate neurons that coexpress estrogen receptor α, neurokinin 3 receptor (NK3R), kisspeptin, neurokinin B, and dynorphin for the regulation of reproduction. To investigate the function of kisspeptin/neurokinin B/dynorphin (KNDy) neurons, a novel method was developed to ablate these cells using a selective NK3R agonist conjugated to the ribosome-inactivating toxin, saporin (NK3-SAP). Stereotaxic injections of NK3-SAP in the arcuate nucleus ablated KNDy neurons, as demonstrated by the near-complete loss of NK3R, NKB, and kisspeptin-immunoreactive (ir) neurons and depletion of the majority of arcuate dynorphin-ir neurons. Selectivity was demonstrated by the preservation of proopiomelanocortin, neuropeptide Y, and GnRH-ir elements in the arcuate nucleus and median eminence. In control rats, ovariectomy (OVX) markedly increased serum LH, FSH, and body weight, and these parameters were subsequently decreased by treatment with 17β-estradiol. KNDy neuron ablation prevented the rise in serum LH after OVX and attenuated the rise in serum FSH. KNDy neuron ablation did not completely block the suppressive effects of E2 on gonadotropin secretion, a finding consistent with redundant pathways for estrogen negative feedback. However, regardless of estrogen status, KNDy-ablated rats had lower levels of serum gonadotropins compared with controls. Surprisingly, KNDy neuron ablation prevented the dramatic effects of OVX and 17β-estradiol (E2) replacement on body weight and abdominal girth. These data provide evidence that arcuate KNDy neurons are essential for tonic gonadotropin secretion, the rise in LH after removal of E2, and the E2 modulation of body weight.

Download Full-text