Kisspeptin Cells Research Articles

The role of kisspeptin and gonadotropin inhibitory hormone (GnIH) in the seasonality of reproduction in sheep

Sheep are seasonal breeders and reproductive status is controlled by photoperiod. Recent recognition of the significant role for kisspeptin and gonadotropin inhibitory hormone (GnIH) in the regulation of gonadotropin releasing hormone (GnRH) cells has provided a new perspective in the seasonal regulation of reproductive activity. Virtually all kisspeptin cells express estrogen receptors and kisspeptin is a potent stimulator of GnRH secretion. Thus, kisspeptin cells provide a conduit by which changes in estrogen feedback effects may be exerted upon GnRH cells. Changes in the activity of kisspeptin cells with season indicate a major role in the seasonal changes in reproductive activity in the ewe. GnIH is an inhibitor of reproductive function and there is mounting evidence that changing activity of this system is also an important determinant of reproductive status. Reciprocal changes in kisspeptin and GnIH activity explain seasonal changes in the function of GnRH cells.

Neuronal plasticity and seasonal reproduction in sheep

Seasonal reproduction represents a naturally occurring example of functional plasticity in the adult brain as it reflects changes in neuroendocrine pathways controlling gonadotropin-releasing hormone (GnRH) secretion and, in particular, the responsiveness of GnRH neurons to estradiol negative feedback. Structural plasticity within this neural circuitry may, in part, be responsible for seasonal switches in the negative feedback control of GnRH secretion that underlie annual reproductive transitions. We review evidence for structural changes in the circuitry responsible for seasonal inhibition of GnRH secretion in sheep. These include changes in synaptic inputs onto GnRH neurons, as well as onto dopamine neurons in the A15 cell group, a nucleus that plays a key role in estradiol negative feedback. We also present preliminary data suggesting a role for neurotrophins and neurotrophin receptors as an early mechanistic step in the plasticity that accompanies seasonal reproductive transitions in sheep. Finally, we review recent evidence suggesting that kisspeptin cells of the arcuate nucleus constitute a critical intermediary in the control of seasonal reproduction. Although a majority of the data for a role of neuronal plasticity in seasonal reproduction has come from the sheep model, the players and principles are likely to have relevance for reproduction in a wide variety of vertebrates, including humans, and in both health and disease.

Characterization of Kiss1 neurons using transgenic mouse models

Humans and mice with loss-of-function mutations of the genes encoding kisspeptins ( Kiss1) or kisspeptin receptor ( Kiss1r) are infertile due to hypogonadotropic hypogonadism. Within the hypothalamus, Kiss1 mRNA is expressed in the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (Arc). In order to better study the different populations of kisspeptin cells we generated Kiss1-Cre transgenic mice. We obtained one line with Cre activity specifically within Kiss1 neurons (line J2–4), as assessed by generating mice with Cre-dependent expression of green fluorescent protein or β-galactosidase. Also, we demonstrated Kiss1 expression in the cerebral cortex and confirmed previous data showing Kiss1 mRNA in the medial nucleus of amygdala and anterodorsal preoptic nucleus. Kiss1 neurons were more concentrated towards the caudal levels of the Arc and higher leptin-responsivity was observed in the most caudal population of Arc Kiss1 neurons. No evidence for direct action of leptin in AVPV Kiss1 neurons was observed. Melanocortin fibers innervated subsets of Kiss1 neurons of the preoptic area and Arc, and both populations expressed melanocortin receptors type 4 (MC4R). Specifically in the preoptic area, 18–28% of Kiss1 neurons expressed MC4R. In the Arc, 90% of Kiss1 neurons were glutamatergic, 50% of which also were GABAergic. In the AVPV, 20% of Kiss1 neurons were glutamatergic whereas 75% were GABAergic. The differences observed between the Kiss1 neurons in the preoptic area and the Arc likely represent neuronal evidence for their differential roles in metabolism and reproduction.

Kisspeptin and the Preovulatory Gonadotrophin‐Releasing Hormone/Luteinising Hormone Surge in the Ewe: Basic Aspects and Potential Applications in the Control of Ovulation

The identification of the neural mechanisms controlling ovulation in mammals has long been a 'holy grail' over recent decades, although the recent discovery of the kisspeptin systems has totally changed our views on this subject. Kisspeptin cells are the major link between gonadal steroids and gonadotrophin-releasing hormone (GnRH) neurones. In the female rodent, kisspeptin cells of the preoptic area are involved in the positive-feedback action of oestrogen on GnRH secretion, although the picture appears more complicated in the ewe. As in rodents, activation of preoptic kisspeptin neurones accompanies the GnRH surge in the ewe but an active role for arcuate kisspeptin neurones has also been proposed. Experimentally, kisspeptin is able to restore reproductive function when the hypothalamic-hypophyseal ovarian axis is quiescent. For example, i.v. infusion of a low dose of peptide in anoestrous ewes induces an immediate and sustained release of gonadotrophin, which subsides and then provokes a luteinising hormone (LH) surge a few hours later. This pharmacological intervention induces the same hormonal changes normally observed during the follicular phase of the oestrous cycle, including the secretion of oestrogen and its negative- and positive-feedback actions on the secretion of LH and follicle-stimulating hormone. Accordingly, a high percentage of kisspeptin-infused animals ovulated. Although the multiple facets of how the kisspeptin systems modulate GnRH secretion are not totally understood, the demonstration that exogenous kisspeptin administration can induce ovulation in anovulatory animals paves the way for future therapeutic applications aiming to control reproduction.

Kisspeptin Cells in the Ewe Brain Respond to Leptin and Communicate with Neuropeptide Y and Proopiomelanocortin Cells

Kisspeptin stimulates reproduction, and kisspeptin cells in the arcuate nucleus (ARC) express Ob-Rb in the mouse. Herein we report studies in ewes to determine whether kisspeptin cells express Ob-Rb and respond to leptin and whether reciprocal connections exist between kisspeptin cells and proopiomelanocortin (POMC) or neuropeptide Y (NPY) cells to modulate reproduction and metabolic function. Kiss1 mRNA was measured by in situ hybridization in ovariectomized ewes that were normal body weight, lean, or lean with leptin treatment by intracerebroventricular (icv) infusion (4 microg/h, 3 d). Kiss1 expression in the ARC and the preoptic area was lower in hypogonadotropic lean animals than animals of normal weight, and icv infusion of leptin partially restored Kiss1 expression in lean animals. Single-cell laser capture microdissection coupled with real-time PCR showed that Kiss1 cells in the preoptic area and ARC express Ob-Rb. Double-label fluorescent immunohistochemistry showed that reciprocal connections exist between kisspeptin cells and NPY and POMC cells. Accordingly, we treated ovariectomized ewes with kisspeptin (5 microg/h, icv) or vehicle for 20 h and examined POMC and NPY gene expression by in situ hybridization. Kisspeptin treatment reduced POMC and increased NPY gene expression. Thus, kisspeptin neurons respond to leptin and expression of Kiss1 mRNA is affected by leptin status. Kisspeptin cells communicate with NPY and POMC cells, altering expression of the relevant genes in the target cells; reciprocal connections also exist. This network between the three cell types could coordinate brain control of reproduction and metabolic homeostatic systems.

329. IS THERE VARIATION IN THE LEVEL OF INPUT OF KISSPEPTIN TERMINALS ONTO GnRH NEURONS ACROSS THE EQUINE OESTROUS CYCLE?

Kisspeptin (KP) plays a key role in reproductive function including the regulation of gonadotrophin releasing hormone (GnRH) and luteinising hormone (LH) secretion in many species but little is known about its role in the mare. In this study, we examined the location of KP-producing neurons in the brain of the mare, their potential interactions with GnRH neurons, and temporal changes in their expression across the oestrous cycle. Mares (n = 3/group) were killed at oestrus (just prior to ovulation), mid-dioestrus, and late dioestrus and the head was perfusion fixed with paraformaldehyde, and hypothalamus collected. Coronal sections (40 μm) were used for dual-label immuno-stained for KP & GnRH. The majority of KP-immunoreactive (-ir) neurons were located in the arcuate nucleus/median eminence (especially mid and caudal regions), and periventricular nucleus. There was a trend (P = 0.09) towards increasing numbers of KP-ir neurons across the cycle. GnRH-ir neurons, located primarily in the arcuate nucleus (especially mid arcuate), as well as the preoptic area, did not change in number across the cycle. Numerous interactions between KP and GnRH neurons were observed, primarily in the arcuate nucleus; KP fibres interacting with GnRH cell bodies, fibre-fibre interactions between KP and GnRH, and GnRH fibres interacting with KP cell bodies. Overall we found KP inputs to 32% of GnRH-ir cells, but the number of these interactions did not vary across the oestrous cycle. This study has confirmed the reciprocal innervation between KP & GnRH neurons in the mare. Although we did not detect variation in the degree across the oestrous cycle this may reflect the sample size issues inherent to equine research.

The Kisspeptin/Neurokinin B/Dynorphin (KNDy) Cell Population of the Arcuate Nucleus: Sex Differences and Effects of Prenatal Testosterone in Sheep

Recent work in sheep has identified a neuronal subpopulation in the arcuate nucleus that coexpresses kisspeptin, neurokinin B, and dynorphin (referred to here as KNDy cells) and that mediate the negative feedback influence of progesterone on GnRH secretion. We hypothesized that sex differences in progesterone negative feedback are due to sexual dimorphism of KNDy cells and compared neuropeptide and progesterone receptor immunoreactivity in this subpopulation between male and female sheep. In addition, because sex differences in progesterone negative feedback and neurokinin B are due to the influence of testosterone (T) during fetal life, we determined whether prenatal T exposure would mimic sex differences in KNDy cells. Adult rams had nearly half the number of kisspeptin, neurokinin B, dynorphin, and progesterone receptor-positive cells in the arcuate nucleus as did females, but the percentage of KNDy cells colocalizing progesterone receptors remained high in both sexes. Prenatal T treatment also reduced the number of dynorphin, neurokinin B, and progesterone receptor-positive cells in the female arcuate nucleus; however, the number of kisspeptin cells remained high and at levels comparable to control females. Thus, sex differences in kisspeptin in the arcuate nucleus, unlike that of dynorphin and neurokinin B, are not due solely to exposure to prenatal T, suggesting the existence of different critical periods for multiple peptides coexpressed within the same neuron. In addition, the imbalance between inhibitory (dynorphin) and stimulatory (kisspeptin) neuropeptides in this subpopulation provides a potential explanation for the decreased ability of progesterone to inhibit GnRH neurons in prenatal T-treated ewes.

Melanocortins May Stimulate Reproduction by Activating Orexin Neurons in the Dorsomedial Hypothalamus and Kisspeptin Neurons in the Preoptic Area of the Ewe

To further test the hypothesis that melanocortins stimulate the reproductive axis, we treated ewes with melanocortin agonist (MTII) in the luteal phase of the estrous cycle and during seasonal anestrus. Lateral ventricular infusion of MTII (10 microg/h) during the luteal phase increased LH secretion. Retrograde neuronal tracing in the brain showed few proopiomelanocortin or kisspeptin cells in the arcuate nucleus, but more than 70% of kisspeptin cells in the dorsolateral preoptic area (POA), projecting to the ventromedial POA in which GnRH cells are located. MTII infusion (20 h) was repeated in luteal phase ewes and brains were harvested to measure gene expression of preproorexin and kisspeptin. Expression of orexin in the dorsomedial hypothalamus and kisspeptin in the POA was up-regulated by MTII treatment and Kiss1 in the arcuate nucleus was down-regulated. Seasonally anestrous ewes were progesterone primed and then treated (lateral ventricular) with MTII (10 microg/h) or vehicle for 30 h, and blood samples were collected every 2 h from 4 h before infusion until 6 h afterward to monitor acute response in terms of LH levels. A rise in basal LH levels was seen, but samples collected around the time of the predicted LH surge did not indicate that an ovulatory event occurred. We conclude that melanocortins are positive regulators of the reproductive neuroendocrine system, but treatment with melanocortins does not fully overcome seasonal acyclicity. The stimulatory effect of melanocortin in the luteal phase of the estrous cycle may be via the activation of kisspeptin cells in the POA and/or orexin cells in the dorsomedial hypothalamus.

Kisspeptin Neurons in the Ovine Arcuate Nucleus and Preoptic Area Are Involved in the Preovulatory Luteinizing Hormone Surge

Kisspeptin is the product of the Kiss1 gene that regulates GnRH secretion. In sheep, Kiss1 mRNA-expressing cells are found in the preoptic area (POA) and arcuate nucleus (ARC), and expression is up-regulated in the caudal ARC during the periovulatory period. We hypothesized that kisspeptin neurons in the ARC are activated by estradiol-17beta prior to the preovulatory LH surge. Ovariectomized ewes were treated as follows: 1) estradiol-17beta implants (sc 2 wk) to cause tonic negative feedback; 2) vehicle (no estrogen negative or positive feedback); or 3) positive feedback/GnRH surge-inducing injection of estradiol-17beta (50 microg iv). For groups 2 and 3, brains were collected 1 h after treatment and kisspeptin/Fos immunoreactivity was examined. In the caudal and mid-ARC, the percentage of kisspeptin cells that were Fos immunoreactive increased after acute estradiol treatment (group 3) over that seen in the other two groups. Kisspeptin/Fos colocalization was also quantified in ewes during the luteal and late-follicular phase of the estrous cycle, showing a trend toward an increase in colocalization in the late-follicular phase. Kisspeptin/Fos colocalization was similar in the POA across groups in both experiments. Analysis of Kiss1 mRNA by in situ hybridization revealed an increase in expression during the late-follicular phase in the caudal ARC and POA. These data suggest kisspeptin neurons located in the caudal extent of the ARC are involved in generating the positive feedback preovulatory GnRH/LH surge in the ewe, but there may also be a role for Kiss1-expressing cells in the POA.

Studies of the localisation of kisspeptin within the pituitary of the rhesus monkey (Macaca mulatta) and the effect of kisspeptin on the release of non-gonadotropic pituitary hormones.

Kisspeptin neurones in the arcuate nucleus play a pivotal role in the regulation of hypothalamic gonadotrophin-releasing hormone (GnRH) secretion in higher primates. To examine whether kisspeptin also influences the function of the primate pituitary directly, two experiments were performed in adult male rhesus monkeys. First, the distribution of kisspeptin-containing cells in the pituitary was described using fluorescence immunohistochemistry. Second, the secretion of non-gonadotrophin adenohypophysial hormones [growth hormone (GH), prolactin and thyroid-stimulating hormone (TSH)] and cortisol in response to i.v. kisspeptin administration was examined. Eight animals were deeply anaesthetised and transcardially perfused with 4% paraformaldehyde. Fluorescence immunohistochemistry was performed on 25-microm thick free-floating pituitary sections to localise immunopositive kisspeptin cells and to examine their relationship with immunostaining for luteinising hormone (LH), follicle-stimulating hormone, GH, prolactin, alpha-melanocyte-stimulating hormone (MSH), adrenocorticotrophic hormone (ACTH) and GnRH. Kisspeptin cells were found in the intermediate lobe of all animals and, in four monkeys, this neuropeptide was also observed in cells scattered in the periphery of the anterior lobe. Kisspeptin colocalised with alpha-MSH-immunopositive cells in the intermediate lobe and, in 50% of the monkeys, with ACTH-immuunopositive cells in the periphery of the adenohypophysis. There was no evidence for colocalisation of kisspeptin with gonadotrophs, somatotrophs or lactotrophs. Beaded kisspeptin axons were observed in the neural lobe. In addition, assay of plasma samples that had been collected for a previous study documenting kisspeptin-10-induced LH release in male monkeys revealed that kisspeptin administration failed to influence circulating concentrations of GH, prolactin, TSH and cortisol. Release of all four of these non-gonadotrophic hormones, however, was stimulated markedly by NMDA, which is considered to act centrally. Although the morphological findings obtained in the present study are consistent with the notion that kisspeptin may act directly at the level of the pituitary, the nature of such an action remains to be defined.

Distribution of Kisspeptin Neurones in the Adult Female Mouse Brain

Kisspeptin-GPR54 signalling is essential for normal reproductive functioning. However, the distribution of kisspeptin neuronal cell bodies and their projections is not well established. The present study aimed to provide a detailed account of kisspeptin neuroanatomy in the mouse brain. Using a polyclonal rabbit antibody AC566, directed towards the final ten C-terminal amino acids of murine kisspeptin, three populations of kisspeptin-expressing cell bodies were identified in the adult female mouse brain. One exists as a dense periventricular continuum of cells within the rostral part of the third ventricle, another is found within the arcuate nucleus, and another is identified as a low-density group of scattered cells within the dorsomedial nucleus and posterior hypothalamus. Kisspeptin-immunoreactive fibres were abundant within the ventral aspect of the lateral septum and within the hypothalamus running in periventricular and ventral retrochiasmatic pathways. Notable exclusions from the kisspeptin fibre innervation were the suprachiasmatic and ventromedial nuclei. Outside of the hypothalamus, a small number of kisspeptin fibres were identified in the bed nucleus of the stria terminalis, subfornical organ, medial amygdala, paraventricular thalamus, periaqueductal grey and locus coerulus. All kisspeptin cell body and fibre immunoreactivity was absent in brain tissue from Kiss1 knockout mice. These observations provide a map of kisspeptin neurones in the mouse brain and indicate that a limited number of mostly medial hypothalamic and lateral septal brain regions are innervated by the three hypothalamic kisspeptin cell populations; the functions of these projections remain to be established.

Kisspeptin cell models from the hypothalamus

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Photoperiod and Testosterone Interact to Drive Seasonal Changes in Kisspeptin Expression in Siberian Hamsters (Phodopus sungorus)

Kisspeptin, a neuropeptide product of the KiSS-1 gene, has recently been implicated in the regulation of seasonal breeding in a number of species, including Siberian hamsters. In this species, kisspeptin expression is reduced in the anteroventral periventricular nucleus (AVPV) following exposure to inhibitory day lengths, and exogenous kisspeptin activates the reproductive neuroendocrine axis of reproductively quiescent animals. Because sex steroids can impact kisspeptin expression, it is unclear whether changes in kisspeptin occur in direct response to photoperiodic cues or secondarily in response to changes in sex steroid concentrations resulting from the transition to reproductive quiescence. The present study aimed to assess the relative contributions of photoperiod and testosterone in regulating kisspeptin expression in Siberian hamsters. Animals housed in long or short day lengths for 8 weeks were either castrated or received sham surgeries. Half of the hamsters in each photoperiod were given testosterone to mimic long-day sex steroid concentrations. The results obtained indicate that kisspeptin neurones in the AVPV and arcuate nuclei were influenced by both photoperiod and testosterone. In the AVPV, removal of testosterone or exposure to inhibitory day lengths led to a marked reduction in kisspeptin-immunoreactive cells, and testosterone treatment increased cell numbers across conditions. Importantly, long-day castrates exhibited significantly more kisspeptin cells than short-day castrates or intact short-day animals with empty capsules, suggesting the influences of photoperiod, independent of gonadal steroids. In general, the opposite pattern emerged for the arcuate nuclei. Collectively, these data suggest a role for both gonadal-dependent and independent (i.e. photoperiodic) mechanisms regulating seasonal changes in kisspeptin expression in Siberian hamsters.

Basic Aspects of the Control of GnRH and LH Secretions by Kisspeptin: Potential Applications for Better Control of Fertility in Females

The neuronal control of fertility and sterility has been a subject of research for years. However, nowadays, in spite of considerable literature about GnRH during the last few decades, the precise cellular and molecular mechanisms whereby gonadal steroids and other peripheral signals converge in the brain to achieve the fine regulation of GnRH secretion remains partially unknown. In this scenario, a major breakthrough in our understanding of the neuronal signals governing reproduction took place in 2003 with the discovery of metastin/kisspeptin as a major player in the control of GnRH secretion. This molecule, first described as having a crucial role in triggering the onset of puberty, is involved in all phases of reproductive life and hence has attracted the interest of many reproductive neuroendocrinologists. Administered either centrally or peripherally, kisspeptin strongly induces the secretion of gonadotropin in many species, mainly through stimulation of GnRH secretion. Kisspeptin cells involved in the control of GnRH secretion are located in two regions of the brain: the preoptic area and the arcuate nucleus. Carrying oestradiol receptor alpha, kisspeptin cells of these regions appear to be the main integration centres for the expression of both the positive and negative feedback of steroid on GnRH secretion. More recently, this molecule has been shown to be able to synchronize preovulatory surges in cyclic ewes and cause ovulation in seasonally acyclic ewes. This review summarizes the most relevant aspects of the role of kisspeptin in GnRH/LH release and the potential application of this molecule in new strategies for controlling female fertility.

Structural Interactions between Kisspeptin and GnRH Neurons in the Mediobasal Hypothalamus of the Male Rhesus Monkey (Macaca mulatta) as Revealed by Double Immunofluorescence and Confocal Microscopy

Kisspeptin is recognized to play a critical role in eliciting the pubertal resurgence of pulsatile GnRH release, the proximal trigger of puberty in higher primates. Expression of the kisspeptin receptor (GPR54) by GnRH neurons indicates a direct action of kisspeptin on the GnRH neuronal network. The purpose of the present study was to examine the distribution of kisspeptin cell bodies in the monkey hypothalamus and to assess the structural basis for the stimulatory action of kisspeptin on the GnRH neuronal network. Three castrated male rhesus monkeys, 39-51 months of age, were deeply anesthetized and their brains perfused transcardially with 4% paraformaldehyde in PBS. Serial 25-microm coronal sections throughout the hypothalamus were prepared, and immunopositive neurons identified using a cocktail of specific primary antibodies (sheep anti-kisspeptin at 1:120,000, and rabbit anti-GnRH at 1:100,000) detected with fluorescently tagged secondary antibodies (antisheep, Alexa Fluor 488; antirabbit, Cy3) in combination with confocal microscopy. Kisspeptin perikarya were found only in the mediobasal hypothalamus (MBH) almost exclusively in the posterior two-thirds of the arcuate nucleus. Surprisingly, kisspeptin-beaded axons made only infrequent contacts with GnRH neurons (kisspeptin and GnRH profiles abutting in a 0.5- to 1.0-mum optical section) in the MBH. In the median eminence, kisspeptin and GnRH axons were found in extensive and intimate association. GnRH contacts on kisspeptin perikarya and dendrites were observed. These findings indicate that nonsynaptic pathways of communication in the median eminence should be considered as a possible mechanism of kisspeptin regulation of GnRH release, and provide an anatomical basis for reciprocal control of kisspeptin neuronal activity by GnRH.

Localization of Kisspeptin Cells and Axonal Fibers in the Pituitary of the Male Rhesus Monkey (Macaca mulatta).

Localization of Kisspeptin Cells and Axonal Fibers in the Pituitary of the Male Rhesus Monkey (Macaca mulatta).

Environmental Control of Kisspeptin: Implications for Seasonal Reproduction

The KiSS-1 gene encodes the peptide hormone kisspeptin, which acts as a principal positive regulator of the reproductive axis by directly stimulating GnRH neuron activity. To gain insight into a potential role for kisspeptin in integrating and relaying reproductively relevant stimuli to the GnRH system, we investigated changes in kisspeptin peptide expression associated with photoperiodic changes in reproductive state as well as pituitary and gonadal responses to peripheral kisspeptin injections. Seasonally breeding rodents undergo pronounced fluctuations in reproductive state in response to changing day lengths. In common with other rodent species, a majority of male Siberian hamsters (Phodopus sungorus) exhibit reproductive decline after exposure to short-day lengths. A subset of individuals fails to respond to day length information, however, and maintains their reproductive function. We exploited these individual differences to examine whether kisspeptin may act at the interface between external stimuli and the reproductive system. After extended exposure to short days, animals with a quiescent reproductive axis displayed a marked reduction in kisspeptin cell labeling in the anteroventral periventricular nucleus but robust kisspeptin-immunoreactive staining in the arcuate nucleus. In contrast, animals with functional reproductive systems displayed high numbers of kisspeptin-immunoreactive neurons in the anteroventral periventricular nucleus but a paucity of expression in the arcuate nucleus. Kisspeptin injections significantly elevated LH over preinjection levels regardless of photoperiod or reproductive state. Collectively, these findings suggest an important role for kisspeptin in coordinating and relaying environmentally relevant information to the reproductive axis as well as a role for this peptide in regulating seasonal changes in reproductive function.