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

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.

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SUN-243 Deletion of KNDy Neuron-Specific KISS1 Disrupts Estrous Cyclicity and LH Pulsatility in Female Mice

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.1253 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Nimisha Nandankar ◽

Ariel L Negron ◽

Jon E Levine ◽

Sally Radovick

Keyword(s):

Feedback Control ◽

Negative Feedback ◽

Vaginal Opening ◽

Pulsatile Release ◽

Female Mice ◽

Lh Pulsatility ◽

Estrous Cyclicity ◽

Lh Release ◽

Negative Feedback Control ◽

Kndy Neurons

Abstract Kisspeptin (encoded by Kiss1), a neuropeptide critically involved in neuroendocrine regulation of reproduction, is primarily synthesized in two discrete hypothalamic nuclei: the anteroventral periventricular area (AVPV) and arcuate nucleus (ARC). AVPV Kiss1 is important for the pre-ovulatory luteinizing hormone (LH) surge unique to females as well as estrogen-induced positive feedback control of GnRH and LH. In contrast, ARC Kiss1 neurons, which largely co-express the neuropeptides NKB and dynorphin (collectively known as KNDy neurons), are major regulators of pulsatile release of GnRH and LH, and mediate estrogen-induced negative feedback control of both GnRH and LH. Previous studies have not fully separated the specific roles for Kiss1 in the AVPV versus KNDy-ARC neurons in the downstream control of GnRH and LH release. Therefore, we generated a Pdyn-Cre/Kiss1fl/fl (KO) mouse model to target Kiss1 in the KNDy neurons to differentiate KNDy neuron-specific function from AVPV Kiss1 function in the maturation and maintenance of the reproductive axis. qRT-PCR data documented a significant reduction of Kiss1 expression in the mediobasal hypothalamus (containing ARC) compared to controls, whereas Kiss1 in the preoptic area (containing AVPV) was similar in both KO and controls. Immunofluorescent IHC confirmed a loss of kisspeptin immunoreactivity in the ARC of KO animals while expression in the AVPV remained intact. Markers of pubertal onset (day of vaginal opening and first estrus in females; day of preputial separation in males) were normal in KO mice, suggesting that AVPV Kiss1 and/or other neural signals may be sufficient for pubertal onset. In addition, body weight throughout pubertal growth was comparable between KO and control animals of both sexes. Interestingly, KO female mice had disrupted estrous cycles presenting with persistent diestrus and a small vaginal opening. In order to test our hypothesis that conditional deletion of Kiss1 in KNDy neurons disrupts or ablates episodic GnRH/LH pulsatile release, we collected serial tail blood samples from mice at diestrus and measured LH. KO female mice exhibited significantly fewer LH pulses in a 3-hour timespan compared to controls, suggesting that KNDy neurons were functionally compromised. These observations indicate the central role of KNDy neurons in the regulation of GnRH/LH pulsatility and estrous cyclicity. The functional effects of disrupted estrous cyclicity and slower LH pulses observed in KO females are currently under study to assess potential abnormalities in ovarian folliculogenesis and fertility. Future experiments will determine whether ARC Kiss1 deletion disrupts the KNDy-driven negative feedback response of LH to gonadectomy, as well as address potential sex differences in ARC Kiss1-mediated negative feedback control of LH release.

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Abnormal Response of the Neuropeptide Y-Deficient Mouse Reproductive Axis to Food Deprivation But Not Lactation

Endocrinology ◽

10.1210/en.2002-221024 ◽

2003 ◽

Vol 144 (5) ◽

pp. 1780-1786 ◽

Cited By ~ 23

Author(s):

Jennifer W. Hill ◽

Jon E. Levine

Keyword(s):

Neuropeptide Y ◽

Food Consumption ◽

Food Deprivation ◽

Normal Female ◽

Estrous Cycles ◽

Energy Deficiency ◽

Steroid Dependent ◽

Reproductive Axis ◽

Estrous Cyclicity ◽

Lh Release

Neuropeptide Y (NPY) plays a key role in both food intake and GnRH secretion. Food deprivation elevates hypothalamic NPY activity and suppresses LH and gonadal steroid secretion. Similarly, lactation up-regulates NPY expression as food consumption increases and estrous cycles cease. These observations suggest that NPY coordinates reproductive suppression in response to energy deficiency; if so, the reproductive axis of NPY knockout (KO) mice should be impervious to lactation and food deprivation. We monitored food consumption, body weight, and estrous cyclicity during lactation in NPY KO mice with large and small litters. NPY KO mice with either litter size resembled wild types (WTs) in weight regulation and food consumption. Large-litter mothers had longer anestrous periods and smaller pups at weaning, but NPY KOs and WTs did not differ in either respect. We also examined the LH response of NPY KO mice to 48 h without food. Basal levels of LH in ovariectomized NPY KO animals decreased in response to fasting, but LH levels in intact and estrogen-treated ovariectomized NPY KO animals did not. In contrast, WTs consistently showed fasting-induced suppression of LH. Our findings suggest that other systems can sustain the hyperphagia of lactation and NPY alone is not responsible for suppressing cyclicity during lactation. Nevertheless, the suppression of basal LH release that accompanies food deprivation in normal female mice appears to require the steroid-dependent actions of NPY.

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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.

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Mice With Targeted Deletion of ARC Kisspeptin Exhibit Immature Gametogenesis and Impaired Fertility

Journal of the Endocrine Society ◽

10.1210/jendso/bvab048.1107 ◽

2021 ◽

Vol 5 (Supplement_1) ◽

pp. A543-A544

Author(s):

Nimisha Nandankar ◽

Ariel L Negron ◽

Andrew M Wolfe ◽

Jon E Levine ◽

Sally Radovick

Keyword(s):

Antral Follicle ◽

Mature Sperm ◽

Seminiferous Tubules ◽

Corpora Lutea ◽

Knock Out ◽

Targeted Deletion ◽

Female Mice ◽

Lh Pulsatility ◽

Estrous Cyclicity ◽

Fertility Data

Abstract Hypothalamic kisspeptin is primarily synthesized in two discrete nuclei - the anteroventral periventricular (AVPV) and the arcuate (ARC) nuclei. We have previously developed a selective, conditional ARC kisspeptin knock-out (KO) mouse line, namely the Pdyn-Cre/Kissfl/fl KO mice, that exhibited normal puberty onset in both sexes, but impaired estrous cyclicity and LH pulsatility in Pdyn-Cre/Kissfl/fl KO females. To examine the end-organ effect of the lack of ARC kisspeptin, we examined gametogenesis, gonad morphology, and fertility. Hematoxylin and eosin (H&E) staining of serial-sectioned whole ovaries demonstrated that Pdyn-Cre/Kissfl/fl KO female mice lacked corpora lutea - their ovarian folliculogenesis did not progress beyond antral follicle development, suggesting an ovulatory defect in Pdyn-Cre/Kissfl/fl KO females. 75% of the Pdyn-Cre/Kissfl/fl KO male mice had testes exhibiting a striking decrease in mature sperm in the seminiferous tubules. The remaining 25% showed evidence of mature sperm. Further evidence of a hypogonadal phenotype of the Pdyn-Cre/Kissfl/fl KO mice included the significantly low weight and small size of the ovaries, uteri, and testes when compared to control littermates. In a controlled, continuous mating paradigm with proven WT males, 2-4-month-old Pdyn-Cre/Kissfl/fl KO female mice failed to become pregnant or produce any pups, whereas age-matched WT females exhibited normal pregnancies to term. Thus, Pdyn-Cre/Kissfl/fl KO females have complete infertility. Ongoing studies of male fertility data suggest that Pdyn-Cre/Kissfl/fl KO males are subfertile, in accordance with their variable spermatogenesis phenotype - some KO males sired pups when paired with proven, WT females, whereas other KO males are infertile. Future experiments include assessing the capability of Pdyn-Cre/Kissfl/fl KO mice to respond to chronic, exogenous kisspeptin and GnRH administration to rescue abnormal LH pulsatility and estrous cyclicity in females, as well as the impaired fertility in both sexes.

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A role for glial fibrillary acidic protein (GFAP)-expressing cells in the regulation of gonadotropin-releasing hormone (GnRH) but not arcuate kisspeptin neuron output

10.1101/2021.03.10.434805 ◽

2021 ◽

Author(s):

Charlotte Vanacker ◽

R. Anthony DeFazio ◽

Charlene M. Sykes ◽

Suzanne M. Moenter

Keyword(s):

Arcuate Nucleus ◽

Designer Drugs ◽

Neuron Activity ◽

Gnrh Neurons ◽

Hypothalamic Arcuate Nucleus ◽

Lh Release ◽

Main Regulator ◽

Kndy Neurons

AbstractGnRH neurons are the final central neural output regulating fertility. Kisspeptin neurons in the hypothalamic arcuate nucleus (KNDy neurons) are considered the main regulator of GnRH output. GnRH and KNDy neurons are surrounded by astrocytes, which can modulate neuronal activity and communicate over distances. Prostaglandin E2 (PGE2), synthesized primarily by astrocytes, increases GnRH neuron activity and downstream pituitary release of luteinizing hormone (LH). We hypothesized GFAP-expressing astrocytes play a role regulating GnRH and/or KNDy neuron activity and LH release. We used adenoassociated viruses to target designer receptor exclusively activated by designer drugs (DREADDs) to GFAP-expressing cells to activate Gq or Gi-mediated signaling. Activating Gq signaling in the preoptic area, near GnRH neurons, but not in the arcuate, increases LH release in vivo and GnRH firing in vitro via a mechanism in part dependent upon PGE2. These data suggest astrocytes can activate GnRH/LH release in a manner independent of KNDy neurons.

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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.

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The Gonadotropin-Releasing Hormone Pulse Generator

Endocrinology ◽

10.1210/en.2018-00653 ◽

2018 ◽

Vol 159 (11) ◽

pp. 3723-3736 ◽

Cited By ~ 51

Author(s):

Allan E Herbison

Keyword(s):

Arcuate Nucleus ◽

Pulse Generator ◽

Pulse Generation ◽

Sufficient Evidence ◽

Gnrh Neuron ◽

Lh Pulsatility ◽

Intrinsic Mechanism ◽

Lh Release ◽

Lh Secretion ◽

Present Understanding

Abstract The pulsatile release of GnRH and LH secretion is essential for fertility in all mammals. Pulses of LH occur approximately every hour in follicular-phase females and every 2 to 3 hours in luteal-phase females and males. Many studies over the last 50 years have sought to identify the nature and mechanism of the “GnRH pulse generator” responsible for pulsatile LH release. This review examines the characteristics of pulsatile hormone release and summarizes investigations that have led to our present understanding of the GnRH pulse generator. There is presently little compelling evidence for an intrinsic mechanism of pulse generation involving interactions between GnRH neuron cell bodies. Rather, data support the presence of an extrinsic pulse generator located within the arcuate nucleus, and attention has focused on the kisspeptin neurons and their projections to GnRH neuron dendrons concentrated around the median eminence. Sufficient evidence has been gathered in rodents to conclude that a subpopulation of arcuate kisspeptin neurons is, indeed, the GnRH pulse generator. Findings in other species are generally compatible with this view and suggest that arcuate/infundibular kisspeptin neurons represent the mammalian GnRH pulse generator. With hindsight, it is likely that past arcuate nucleus multiunit activity recordings have been from kisspeptin neurons. Despite advances in identifying the cells forming the pulse generator, almost nothing is known about their mechanisms of synchronicity and the afferent hormonal and transmitter modulation required to establish the normal patterns of LH pulsatility in mammals.

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Ablation of KNDy Neurons Results in Hypogonadotropic Hypogonadism and Amplifies the Steroid-Induced LH Surge in Female Rats

Endocrinology ◽

10.1210/en.2015-1740 ◽

2016 ◽

Vol 157 (5) ◽

pp. 2015-2027 ◽

Cited By ~ 24

Author(s):

Melinda A. Mittelman-Smith ◽

Sally J. Krajewski-Hall ◽

Nathaniel T. McMullen ◽

Naomi E. Rance

Keyword(s):

Arcuate Nucleus ◽

Hypogonadotropic Hypogonadism ◽

Reproductive Function ◽

Female Rats ◽

Lh Surge ◽

Serum Lh ◽

Estrous Cyclicity ◽

17Β Estradiol ◽

Neurokinin 3 Receptor ◽

Kndy Neurons

Abstract In the human infundibular (arcuate) nucleus, a subpopulation of neurons coexpress kisspeptin and neurokinin B (NKB), 2 peptides required for normal reproductive function. A homologous group of neurons exists in the arcuate nucleus of rodents, termed KNDy neurons based on the coexpression of kisspeptin, NKB, and dynorphin. To study their function, we recently developed a method to selectively ablate KNDy neurons using NK3-SAP, a neurokinin 3 receptor agonist conjugated to saporin (SAP). Here, we ablated KNDy neurons in female rats to determine whether these neurons are required for estrous cyclicity and the steroid induced LH surge. NK3-SAP or Blank-SAP (control) was microinjected into the arcuate nucleus using stereotaxic surgery. After monitoring vaginal smears for 3–4 weeks, rats were ovariectomized and given 17β-estradiol and progesterone in a regimen that induced an afternoon LH surge. Rats were killed at the time of peak LH levels, and brains were harvested for NKB and dual labeled GnRH/Fos immunohistochemistry. In ovary-intact rats, ablation of KNDy neurons resulted in hypogonadotropic hypogonadism, characterized by low levels of serum LH, constant diestrus, ovarian atrophy with increased follicular atresia, and uterine atrophy. Surprisingly, the 17β-estradiol and progesterone-induced LH surge was 3 times higher in KNDy-ablated rats. Despite the marked increase in the magnitude of the LH surge, the number of GnRH or anterior ventral periventricular nucleus neurons expressing Fos was not significantly different between groups. Our studies show that KNDy neurons are essential for tonic levels of serum LH and estrous cyclicity and may play a role in limiting the magnitude of the LH surge.

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Food restriction during lactation suppresses Kiss1 mRNA expression and kisspeptin-stimulated LH release in rats

Reproduction ◽

10.1530/rep-13-0426 ◽

2014 ◽

Vol 147 (5) ◽

pp. 743-751 ◽

Cited By ~ 9

Author(s):

Sharon R Ladyman ◽

Barbara Woodside

Keyword(s):

Food Intake ◽

Mrna Expression ◽

Food Restriction ◽

Arcuate Nucleus ◽

Immunoreactive Cell ◽

Physiological Changes ◽

Cell Counts ◽

Reproductive Axis ◽

Lh Release ◽

Ad Libitum

Among the numerous physiological changes that accompany lactation is the suppression of the reproductive axis. The aim of this study was to investigate a possible role for the kisspeptin system in the restoration of the hypothalamic–pituitary–gonadal axis during late lactation in rats using a food restriction model that allows manipulation of the duration of lactational anovulation. Kiss1 mRNA expression and kisspeptin-immunoreactive cell counts were examined in both food-restricted dams and ad libitum (AL)-fed dams across late lactation when LH concentrations begin to increase. In the arcuate nucleus, Kiss1 mRNA expression and kisspeptin-positive cell counts were suppressed during late lactation. In the anteroventral periventricular (AVPV), day 15 food-restricted dams had significantly lower AVPV Kiss1 mRNA expression and a decreased LH response to exogenous kisspeptin compared with the AL-fed dams. Following 5 days of ad libitum food intake, these values were restored to levels similar to those in dams that had been fed ad libitum throughout lactation. In conclusion, this study shows that delayed restoration of the reproductive axis due to food restriction is associated with a decrease in kisspeptin sensitivity and low AVPV Kiss1 mRNA in late lactation.

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Estrogen-Negative Feedback and Estrous Cyclicity Are Critically Dependent Upon Estrogen Receptor-α Expression in the Arcuate Nucleus of Adult Female Mice

Endocrinology ◽

10.1210/en.2014-1128 ◽

2014 ◽

Vol 155 (8) ◽

pp. 2986-2995 ◽

Cited By ~ 34

Author(s):

Shel-Hwa Yeo ◽

Allan E. Herbison

Keyword(s):

Estrogen Receptor ◽

Negative Feedback ◽

Arcuate Nucleus ◽

Critical Role ◽

Estrogen Receptor Α ◽

Feedback Mechanism ◽

Neuron Activity ◽

Female Mice ◽

Negative Feedback Mechanism ◽

Estrous Cyclicity

The location and characteristics of cells within the brain that suppress GnRH neuron activity to contribute to the estrogen-negative feedback mechanism are poorly understood. Using adeno-associated virus (AAV)-mediated Cre-LoxP recombination in estrogen receptor-α (ERα) floxed mice (ERαflox/flox), we aimed to examine the role of ERα-expressing neurons located in the arcuate nucleus (ARN) in the estrogen-negative feedback mechanism. Bilateral injection of AAV-Cre into the ARN of ERαflox/flox mice (n = 14) resulted in the time-dependent ablation of up to 99% of ERα-immunoreactive cell numbers throughout the rostrocaudal length of the ARN. These mice were all acyclic by 5 weeks after AAV-Cre injections with most mice in constant estrous. Control wild-type mice injected with AAV-Cre (n = 13) were normal. Body weight was not altered in ERαflox/flox mice. After ovariectomy, a significant increment in LH secretion was observed in all genotypes, although its magnitude was reduced in ERαflox/flox mice. Acute and chronic estrogen-negative feedback were assessed by administering 17β-estradiol to mice as a bolus (LH measured 3 h later) or SILASTIC brand capsule implant (LH measured 5 d later). This demonstrated that chronic estrogen feedback was absent in ERαflox/flox mice, whereas the acute feedback was normal. These results reveal a critical role for ERα-expressing cells within the ARN in both estrous cyclicity and the chronic estrogen negative feedback mechanism in female mice. This suggests that ARN cells provide a key indirect, transsynpatic route through which estradiol suppresses the activity of GnRH neurons.

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