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.

Download Full-text

Neurokinin 3 Receptor Immunoreactivity in the Septal Region, Preoptic Area and Hypothalamus of the Female Sheep: Colocalisation in Neurokinin B Cells of the Arcuate Nucleus but not in Gonadotrophin-Releasing Hormone Neurones

Journal of Neuroendocrinology ◽

10.1111/j.1365-2826.2009.01930.x ◽

2010 ◽

Vol 22 (1) ◽

pp. 1-12 ◽

Cited By ~ 96

Author(s):

M. Amstalden ◽

L. M. Coolen ◽

A. M. Hemmerle ◽

H. J. Billings ◽

J. M. Connors ◽

...

Keyword(s):

B Cells ◽

Arcuate Nucleus ◽

Preoptic Area ◽

Neurokinin B ◽

Releasing Hormone ◽

Septal Region ◽

Gonadotrophin Releasing Hormone ◽

Neurokinin 3 Receptor ◽

Female Sheep

Download Full-text

Evidence of a Role for Kisspeptin and Neurokinin B in Puberty of Female Sheep

Endocrinology ◽

10.1210/en.2011-2009 ◽

2012 ◽

Vol 153 (6) ◽

pp. 2756-2765 ◽

Cited By ~ 60

Author(s):

Casey C Nestor ◽

Amanda M.S. Briscoe ◽

Shay M. Davis ◽

Miro Valent ◽

Robert L. Goodman ◽

...

Keyword(s):

Arcuate Nucleus ◽

Preoptic Area ◽

Age Groups ◽

Neurokinin B ◽

Genetic Studies ◽

Cell Numbers ◽

Puberty Onset ◽

Lh Secretion ◽

Female Sheep ◽

Close Contacts

Puberty onset in female sheep is marked by a decrease in estradiol-negative feedback, allowing for the increase in GnRH and LH pulses that heralds the first ovulation. Based on recent genetic studies in humans, two possible neuropeptides that could promote puberty onset are kisspeptin and neurokinin B (NKB). Our first experiment determined whether the NKB agonist, senktide, could stimulate LH secretion in prepubertal ewes. A second study used prepubertal and postpubertal ewes that were intact or ovariectomized (OVX) to test the hypothesis that expression of kisspeptin and NKB in the arcuate nucleus increased postpubertally. For comparison, kisspeptin and NKB expression in age-matched intact, and castrated males were also examined. In experiment 1, the percentage of ewes showing an LH pulse immediately after injection of senktide (100 μg, 60%; 500 μg, 100%) was greater than that for water-injected controls (experiment 1a, 25%; experiment 1b, 20%). In experiment 2, kisspeptin-positive cell numbers in the arcuate nucleus increased after puberty in intact females and were increased by OVX in prepubertal but not postpubertal ewes. Changes in kisspeptin cell numbers were paralleled by changes in kisspeptin-close contacts onto GnRH neurons in the medial preoptic area. NKB cell numbers did not differ significantly between intact prepubertal and postpubertal ewes but increased with OVX in both age groups. NKB fiber immunoreactivity was greater in postpubertal than in prepubertal intact ewes. In age-matched males, kisspeptin and NKB cell numbers increased with castration, but decreased with age. These results support the hypothesis that kisspeptin is a gatekeeper to female ovine puberty and raise the possibility that NKB may also play a role, albeit through different means.

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

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

Endocrinology ◽

10.1210/en.2014-1200 ◽

2014 ◽

Vol 155 (10) ◽

pp. 3945-3955 ◽

Cited By ~ 24

Author(s):

Agnete Overgaard ◽

Francisco Ruiz-Pino ◽

Juan M. Castellano ◽

Manuel Tena-Sempere ◽

Jens D. Mikkelsen

Keyword(s):

Sex Steroids ◽

Arcuate Nucleus ◽

Detailed Examination ◽

Differential Regulation ◽

Sex Steroid ◽

Neurokinin B ◽

Regulated Expression ◽

Male Sex ◽

Specific Regulation ◽

Kndy Neurons

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

Kisspeptin, Neurokinin B, and Dynorphin Expression during Pubertal Development in Female Sheep

Biology ◽

10.3390/biology10100988 ◽

2021 ◽

Vol 10 (10) ◽

pp. 988

Author(s):

Eliana G. Aerts ◽

KaLynn Harlow ◽

Max J. Griesgraber ◽

Elizabeth C. Bowdridge ◽

Steven L. Hardy ◽

...

Keyword(s):

Mrna Expression ◽

Pubertal Development ◽

Pulse Frequency ◽

Neurokinin B ◽

Immunopositive Cell ◽

Hypothalamic Tissue ◽

Puberty Onset ◽

Kndy Neurons ◽

Lh Secretion ◽

Female Sheep

The neural mechanisms underlying increases in gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion that drive puberty onset are unknown. Neurons coexpressing kisspeptin, neurokinin B (NKB), and dynorphin, i.e., KNDy neurons, are important as kisspeptin and NKB are stimulatory, and dynorphin inhibitory, to GnRH secretion. Given this, we hypothesized that kisspeptin and NKB expression would increase, but that dynorphin expression would decrease, with puberty. We collected blood and hypothalamic tissue from ovariectomized lambs implanted with estradiol at five, six, seven, eight (puberty), and ten months of age. Mean LH values and LH pulse frequency were the lowest at five to seven months, intermediate at eight months, and highest at ten months. Kisspeptin and NKB immunopositive cell numbers did not change with age. Numbers of cells expressing mRNA for kisspeptin, NKB, or dynorphin were similar at five, eight, and ten months of age. Age did not affect mRNA expression per cell for kisspeptin or NKB, but dynorphin mRNA expression per cell was elevated at ten months versus five months. Thus, neither KNDy protein nor mRNA expression changed in a predictable manner during pubertal development. These data raise the possibility that KNDy neurons, while critical, may await other inputs for the initiation of puberty.

Download Full-text

Deficiency of Arcuate Nucleus Kisspeptin Results in Post-Pubertal Central Hypogonadism

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00088.2021 ◽

2021 ◽

Author(s):

Nimisha Nandankar ◽

Ariel L. Negron ◽

Andrew Wolfe ◽

Jon E Levine ◽

Sally Radovick

Keyword(s):

Feedback Control ◽

Arcuate Nucleus ◽

Critical Role ◽

Knock Out ◽

Protein Levels ◽

Reproductive Axis ◽

Lh Pulsatility ◽

Estrous Cyclicity ◽

Lh Release ◽

Kndy Neurons

Kisspeptin (encoded by Kiss1), a neuropeptide critically involved in neuroendocrine regulation of reproduction, is primarily synthesized in two hypothalamic nuclei: the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC). AVPV kisspeptin is thought to regulate the estrogen-induced positive feedback control of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH), and the pre-ovulatory LH surge in females. In contrast, ARC kisspeptin neurons, which largely co-express neurokinin B and dynorphin A (collectively named KNDy neurons), are thought to mediate estrogen-induced negative feedback control of GnRH/LH and be the major regulators of pulsatile GnRH/LH release. However, definitive data to delineate the specific roles of AVPV versus ARC kisspeptin neurons in the control of GnRH/LH release is lacking. Therefore, we generated a novel mouse model targeting deletion of Kiss1 to the ARC nucleus (Pdyn-Cre/Kiss1fl/fl KO) to determine the functional differences between ARC and AVPV kisspeptin neurons on the reproductive axis. The efficacy of the knock-out was confirmed at both the mRNA and protein levels. Adult female Pdyn-Cre/Kiss1fl/fl KO mice exhibited persistent diestrus and significantly fewer LH pulses when compared to controls, resulting in arrested folliculogenesis, hypogonadism, and infertility. Pdyn-Cre/Kiss1fl/fl KO males also exhibited disrupted LH pulsatility, hypogonadism, and variable, defective spermatogenesis and subfertility. The timing of pubertal onset in males and females was equivalent to controls. These findings add to the current body of evidence for the critical role of kisspeptin in ARC KNDy neurons in GnRH/LH pulsatility in both sexes, while directly establishing ARC kisspeptin's role in regulating estrous cyclicity in female mice, and gametogenesis in both sexes, and culminating in disrupted fertility. The Pdyn-Cre/Kiss1fl/fl KO mice present a novel mammalian model of post-pubertal central hypogonadism.

Download Full-text

Arcuate nucleus kisspeptin response to increased nutrition in rams

Reproduction Fertility and Development ◽

10.1071/rd19063 ◽

2019 ◽

Vol 31 (11) ◽

pp. 1682 ◽

Cited By ~ 1

Author(s):

S. E. Rietema ◽

P. A. R. Hawken ◽

C. J. Scott ◽

M. N. Lehman ◽

G. B. Martin ◽

...

Keyword(s):

Arcuate Nucleus ◽

Preoptic Area ◽

Pulse Generator ◽

Pulse Frequency ◽

Nutritional Supplementation ◽

Environmental Cues ◽

Dorsomedial Hypothalamus ◽

Gonadotrophin Releasing Hormone ◽

Kndy Neurons

Rams respond to acute nutritional supplementation by increasing the frequency of gonadotrophin-releasing hormone (GnRH) pulses. Kisspeptin neurons may mediate the effect of environmental cues on GnRH secretion, so we tested whether the ram response to nutrition involves activation of kisspeptin neurons in the arcuate nucleus (ARC), namely kisspeptin, neurokin B, dynorphin (KNDy) neurons. Rams were given extra lupin grain with their normal ration. Blood was sampled before feeding, and continued until animals were killed for collection of brain tissue at 2 or 11h after supplementation. In supplemented rams, LH pulse frequency increased after feeding, whereas control animals showed no change. Within the caudal ARC, there were more kisspeptin neurons in supplemented rams than in controls and a higher proportion of kisspeptin cells coexpressed Fos, regardless of the time the rams were killed. There were more Fos cells in the mid-ARC and mid-dorsomedial hypothalamus of the supplemented compared with control rams. No effect of nutrition was found on kisspeptin expression in the rostral or mid-ARC, or on GnRH expression in the preoptic area. Kisspeptin neurons in the caudal ARC appear to mediate the increase in GnRH and LH production due to acute nutritional supplementation, supporting the hypothesised role of the KNDy neurons as the pulse generator for GnRH.

Download Full-text

Patterns of preoptic–hypothalamic neuronal activation and LH secretion in female sheep following the introduction and withdrawal of novel males

Reproduction Fertility and Development ◽

10.1071/rd19079 ◽

2019 ◽

Vol 31 (11) ◽

pp. 1674

Author(s):

Penny A. R. Hawken ◽

Jeremy T. Smith ◽

Trina Jorre de St Jorre ◽

Tammi Esmaili ◽

Christopher J. Scott ◽

...

Keyword(s):

Neural Activity ◽

Arcuate Nucleus ◽

Preoptic Area ◽

Ventromedial Nucleus ◽

Neural Activation ◽

Lamina Terminalis ◽

Neuronal Activation ◽

Neuroendocrine Response ◽

Lh Secretion ◽

Female Sheep

The neuroendocrine response of female sheep to a novel male involves neural activation in the hypothalamus. However, if males are removed, the gonadotrophic signal declines, so the neural activity is likely to change. We examined Fos-immunoreactive (IR) cells in hypothalamic tissues from seasonally anovulatory female sheep exposed to males for 2 or 6h, or for 2h followed by 4h isolation from males. Control females were killed in the absence of male exposure. Male introduction increased LH secretion in all females; male removal was associated with a reduction only in mean and basal LH concentrations. Females exposed to males for 2h had more Fos-IR cells in the arcuate nucleus (ARC), ventromedial nucleus of the hypothalamus (VMH) and organum vasculosum of the lamina terminalis (OVLT) than control females. Fos-IR cells in the preoptic area (POA) were only greater than in control females after 6h exposure to a male. Removal of males decreased the number of Fos-IR cells in the ARC, VMH and OVLT, but not in the POA. Thus, hypothalamic neural activation and LH secretion in female sheep are stimulated by males and decline after male removal. However, activation in the POA persists after removal and may explain the incomplete decline in the LH response.

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