Kisspeptin and its receptor GPR54 have recently been identified as key signaling partners in the neural control of fertility in animal models and humans. The gonadotropin-releasing hormone (GnRH) neurons represent the final output neurons of the neural network controlling fertility and are suspected to be the primary locus of kisspeptin-GPR54 signaling. Using mouse models, the present study addressed whether kisspeptin and GPR54 have a key role in the activation of GnRH neurons to generate the luteinizing hormone (LH) surge responsible for ovulation. Dual-label immunocytochemistry experiments showed that 40 -60% of kisspeptin neurons in the rostral periventricular area of the third ventricle (RP3V) expressed estrogen receptor ␣ and progesterone receptors. Using an ovariectomized, gonadal steroid-replacement regimen, which reliably generates an LH surge, ϳ30% of RP3V kisspeptin neurons were found to express c-FOS in surging mice compared with 0% in nonsurging controls. A strong correlation was found between the percentage of c-FOSpositive kisspeptin neurons and the percentage of c-FOS-positive GnRH neurons. To evaluate whether kisspeptin and/or GPR54 were essential for GnRH neuron activation and the LH surge, Gpr54-and Kiss1-null mice were examined. Whereas wild-type littermates all exhibited LH surges and c-FOS in ϳ50% of their GnRH neurons, none of the mutant mice from either line showed an LH surge or any GnRH neurons with c-FOS. These observations provide the first evidence that kisspeptin-GPR54 signaling is essential for GnRH neuron activation that initiates ovulation. This broadens considerably the potential roles and therapeutic possibilities for kisspeptin and GPR54 in fertility regulation.

Kisspeptin-GPR54 signalling is critical for the proper functioning of the reproductive axis. In humans, loss of function mutations of GPR54 result in failed puberty and infertility (1, 2), whereas gain of function mutations result in precocious puberty (3). Similar phenotypes are observed in Kiss1 and GPR54 knockout mouse lines (4, 5) and laboratory experiments have suggested kisspeptin-GPR54 signalling to be essential for puberty onset (1, 6-9) and the preovulatory LH surge (10-12). A role for kisspeptin and GPR54 has also been suggested in the control of seasonal reproduction (13)(14)(15)(16) and the integration of metabolic cues relevant to fertility (17). Given the importance of kisspeptin in the regulation of the reproductive axis, it is vital to gain a clear understanding of the locations and projection patterns of kisspeptin neurones within the brain.In situ hybridisation (ISH) and immunocytochemical studies indicate remarkable similarities in the neuroanatomical locations of kisspeptin-synthesising cell populations in mammalian species. Foremost, a large population of kisspeptin-expressing neurones has been identified in the arcuate ⁄ infundibular nucleus of mice (18)(19)(20), rats (21, 22), hamsters (23, 24), sheep (16, 25-27), mares (28), primates (29, 30) and humans (31). A second population of kisspeptin-expressing cells exists in the medial preoptic area of mice (18)(19)(20)32), rats (12, 21, 22), hamsters (15, 23), sheep (16, 25-27) and humans (31). The precise position of kisspeptin neurones in the preoptic area varies with species; being immediately adjacent to the ventricle wall in rodents but more lateral in other mammals (25,26,31). In addition to these two main populations of kisspeptin 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 b...

Kisspeptin and G protein-coupled receptor 54 (GPR54) are now acknowledged to play essential roles in the neural regulation of fertility. Using a transgenic Gpr54 LacZ knock-in mouse model, this study aimed to provide 1) a detailed map of cells expressing Gpr54 in the mouse brain and 2) an analysis of Gpr54 expression in GnRH neurons across postnatal development. The highest density of Gpr54-expressing cells in the mouse central nervous system was found in the dentate gyrus of the hippocampus beginning on postnatal d 6 (P6). Abundant Gpr54 expression was also noted in the septum, rostral preoptic area (rPOA), anteroventral nucleus of the thalamus, posterior hypothalamus, periaqueductal grey, supramammillary and pontine nuclei, and dorsal cochlear nucleus. No Gpr54 expression was detected in the arcuate and rostral periventricular nuclei of the hypothalamus. Dual-labeling experiments showed that essentially all Gpr54-expressing cells in the rPOA were GnRH neurons. Analyses of mice at birth, P1, P5, P20, and P30 and as adults revealed a gradual increase in the percentage of GnRH neurons expressing Gpr54 from approximately 40% at birth through to approximately 70% from P20 onward. Whereas GnRH neurons located in the septum displayed a consistent increase across this time, GnRH neurons in the rPOA showed a sharp reduction in Gpr54 expression after birth (to approximately 10% at P5) before increasing to the 70% expression levels by P20. Together these findings provide an anatomical basis for the exploration of Gpr54 actions outside the reproductive axis and reveal a complex temporal and spatial pattern of Gpr54 gene expression in developing GnRH neurons.

The G protein-coupled receptor GPR54 (AXOR12, OT7T175) is central to acquisition of reproductive competency in mammals. Peptide ligands (kisspeptins) for this receptor are encoded by the Kiss1 gene, and administration of exogenous kisspeptins stimulates hypothalamic gonadotropin-releasing hormone (GnRH) release in several species, including humans. To establish that kisspeptins are the authentic agonists of GPR54 in vivo and to determine whether these ligands have additional physiological functions we have generated mice with a targeted disruption of the Kiss1 gene. Kiss1-null mice are viable and healthy with no apparent abnormalities but fail to undergo sexual maturation. Mutant female mice do not progress through the estrous cycle, have thread-like uteri and small ovaries, and do not produce mature Graffian follicles. Mutant males have small testes, and spermatogenesis arrests mainly at the early haploid spermatid stage. Both sexes have low circulating gonadotropin (luteinizing hormone and follicle-stimulating hormone) and sex steroid (␤-estradiol or testosterone) hormone levels. Migration of GnRH neurons into the hypothalamus appears normal with appropriate axonal connections to the median eminence and total GnRH content. The hypothalamicpituitary axis is functional in these mice as shown by robust luteinizing hormone secretion after peripheral administration of kisspeptin. The virtually identical phenotype of Gpr54-and Kiss1-null mice provides direct proof that kisspeptins are the true physiological ligand for the GPR54 receptor in vivo. Kiss1 also does not seem to play a vital role in any other physiological processes other than activation of the hypothalamic-pituitary-gonadal axis, and loss of Kiss1 cannot be overcome by compensatory mechanisms.Gpr54 ͉ kisspeptin ͉ mouse ͉ puberty N euroendocrine events within the hypothalamus control sexual maturation and seasonal breeding in mammals (1). The gonadotropin-releasing hormone (GnRH)-induced secretion of the gonadotropic hormones luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary is essential to invoke puberty and maintain reproductive function. The G protein-coupled receptor GPR54 (2) is a key protein involved in the pubertal activation of the hypothalamic pituitary gonadal axis because mice and humans with mutations in this receptor are sterile with hypogonadotropic hypogonadism (3-7).A series of overlapping peptide ligands (kisspeptins) for the GPR54 receptor are produced by the Kiss1 gene (8-10). Kiss1 mRNA is expressed in hypothalamic regions that regulate gonadotropin secretion including the anteroventral periventricular nucleus (AVPV), the periventricular nucleus, and the arcuate nucleus (ARC) (11). Kiss1 expression increases at puberty in rodents (12-14) and primates (15) and fluctuates during the rat estrous cycle (12,16). Kiss1 expression is also subject to differential regulation by sex steroids, providing a plausible mechanism for the feedback control of gonadotropin secretion by these hormones (17, 18). Ad...

The anteroventral periventricular nucleus (AVPV) is thought to play a key role in regulating the excitability of gonadotropin-releasing hormone (GnRH) neurons that control fertility. Using an angled, parahorizontal brain slice preparation we have undertaken a series of electrophysiological experiments to examine how the AVPV controls GnRH neurons in adult male and female mice. More than half (59%) of GnRH neurons located in the rostral preoptic area were found to receive monosynaptic inputs from the AVPV in a sex-dependent manner. AVPV stimulation frequencies Ͻ1 Hz generated short-latency action potentials in GnRH neurons with GABA and glutamate mediating Ͼ90% of the evoked fast synaptic currents. The AVPV GABA input was dominant and found to excite or inhibit GnRH neurons in a cell-dependent manner. Increasing the AVPV stimulation frequency to 5-10 Hz resulted in the appearance of additional poststimulus inhibitory as well as delayed excitatory responses in GnRH neurons that were independent of ionotropic amino acid receptors. The inhibition observed immediately following the end of the stimulation period was mediated partly by GABA B receptors, while the delayed activation was mediated by the neuropeptide kisspeptin. The latter response was essentially absent in Gpr54 knock-out mice and abolished by a Gpr54 antagonist. Together, these studies show that AVPV neurons provide direct amino acid and neuropeptidergic inputs to GnRH neurons. Low-frequency activation generates predominant GABA/glutamate release with higher frequency activation recruiting release of kisspeptin. This frequency-dependent release of amino acid and neuropeptide neurotransmitters greatly expands the range of AVPV control of GnRH neuron excitability.

The G protein-coupled receptor GPR54, and its peptide ligand kisspeptin (Kp), are crucial for the induction and maintenance of mammalian reproductive function. GPR54 is expressed by GnRH neurons and is directly activated by Kp to stimulate GnRH release. We hypothesized that Kp may be able to act at the GnRH nerve terminals located in the mediobasal hypothalamus (MBH) region. To test this hypothesis, we used organotypic culture of MBH explants challenged with Kp, followed by RIA to detect GnRH released into the cultured medium. Kp stimulation for 1 h induced GnRH release from wild-type male MBH in a dose-dependent manner, whereas this did not occur in MBH explants isolated from Gpr54 null mice. Continuous Kp stimulation caused a sustained GnRH release for 4 h, followed by a decrease of GnRH release, suggesting a desensitization of GPR54 activity. Tetrodotoxin did not alter the Kp-induced GnRH release, indicating that Kp can act directly at the GnRH nerve terminals. To localize Gpr54 expression within the MBH, we used transgenic mice, in which Gpr54 expression is tagged with an IRES-LacZ reporter gene and can be visualized by beta-galactosidase staining. Gpr54 expression was detected outside of the median eminence, in the pars tuberalis. In conclusion, our results provide evidence for a potent stimulating effect of Kp at GnRH nerve terminals in the MBH of the mouse. This study suggests a new point at which Kp can act on GnRH neurons.

Reproduction is controlled in the brain by a neural network that drives the secretion of gonadotropin-releasing hormone (GnRH).Various permissive homeostatic signals must be integrated to achieve ovulation in mammals. However, the neural events controlling the timely activation of GnRH neurons are not completely understood. Here we show that kisspeptin, a potent activator of GnRH neuronal activity, directly communicates with neurons that synthesize the gaseous transmitter nitric oxide (NO) in the preoptic region to coordinate the progression of the ovarian cycle. Using a transgenic Gpr54-null IRES-LacZ knock-in mouse model, we demonstrate that neurons containing neuronal NO synthase (nNOS), which are morphologically associated with kisspeptin fibers, express the kisspeptin receptor GPR54 in the preoptic region, but not in the tuberal region of the hypothalamus. The activation of kisspeptin signaling in preoptic neurons promotes the activation of nNOS through its phosphorylation on serine 1412 via the AKT pathway and mimics the positive feedback effects of estrogens. Finally, we show that while NO release restrains the reproductive axis at stages of the ovarian cycle during which estrogens exert their inhibitory feedback, it is required for the kisspeptin-dependent preovulatory activation of GnRH neurons. Thus, interactions between kisspeptin and nNOS neurons may play a central role in regulating the hypothalamic-pituitary-gonadal axis in vivo.

In the ever-changing physiological context of the neuroendocrine brain, the mechanisms by which cellular events involving neurons, astroglia and vascular cells are coordinated to bring forth the appropriate neuronal signaling is not yet known but is amenable to examination. In the median eminence of the hypothalamus, endothelial cells are key players in the plasticity of tanycytes – specialized astroglia-- and neuroendocrine synapse efficacy. Here we report that estradiol acts on both purified endothelial cells and isolated tanycytes to trigger endothelial-to-glial communication that leads to a sudden and massive retraction of tanycyte processes. The blockade of endothelial nitric oxide (NO) synthase (eNOS) by in vitro adenoviral-mediated gene transfer of a dominant negative form of eNOS abrogates the estradiol-induced tanycyte plasticity mediated by endothelial cells. In parallel, increases in prostaglandin-E2 (PGE2) due to changes in cyclooxygenase-1 (COX-1) and COX-2 expression induced by the exposure of tanycytes to estradiol, promote acute tanycyte plasticity. We also demonstrate by electron microscopy that the administration of PGE2 to median eminence explants induces rapid neuroglial plasticity at the neurovascular junction of neurons that release gonadotropin releasing hormone (GnRH) (the neuropeptide controlling reproduction). Conversely, preventing local PGE2 synthesis in the median eminence of adult female rats with the COX inhibitor indomethacin impairs the ovarian cycle, a process that requires a pulsatile, coordinated delivery of GnRH into the hypothalamo-hypophyseal portal system. Taken together, our findings show that estradiol controls the dialogue between endothelial cells and astroglia to regulate neuroglial plasticity in the neuroendocrine brain.