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Sighs are long, deep breaths expressing sadness, relief or exhaustion. Sighs also occur spontaneously every few minutes to reinflate alveoli, and sighing increases under hypoxia, stress, and certain psychiatric conditions. Here we use molecular, genetic, and pharmacologic approaches to identify a peptidergic sigh control circuit in murine brain. Small neural subpopulations in a key breathing control centre, the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG), express bombesin-like neuropeptide genes neuromedin B ( Nmb ) or gastrin-releasing peptide ( Grp ). These project to the preBötzinger Complex (preBötC), the respiratory rhythm generator, which expresses NMB and GRP receptors in overlapping subsets of ~200 neurons. Introducing either neuropeptide into preBötC or onto preBötC slices, induced sighing or in vitro sigh activity, whereas elimination or inhibition of either receptor reduced basal sighing, and inhibition of both abolished it. Ablating receptor-expressing neurons eliminated basal and hypoxia-induced sighing, but left breathing otherwise intact initially. We propose that these overlapping peptidergic pathways comprise the core of a sigh control circuit that integrates physiological and perhaps emotional input to transform normal breaths into sighs. The peptidergic neuronal circuit controlling sigh generation has been identified as ~200  Nmb- or Grp -expressing neurons in the RTN/pFRG breathing control centre of the medulla that project to ~200 receptor-expressing neurons in the respiratory rhythm generator, the preBötzinger Complex. Sigh centre neurons identified Although sighs are an integral part of breathing and respiratory physiology, little is known about the neuronal circuits controlling this behaviour. Here, Mark Krasnow and colleagues identify a small subset of genetically defined neurons in the medulla that project to the preBötzinger complex (preBötC), the respiratory rhythm generator, to drive sighing. Inhibition of this connection could completely eliminate sighs, while regular breathing was left intact. The authors propose a mechanism by which specific preBötC neurons may integrate physiological and possibly emotional inputs to turn regular breaths into sighs when appropriate.

The identification of loss-of-function mutations in MKRN3 in patients with central precocious puberty in association with the decrease in MKRN3 expression in the medial basal hypothalamus of mice before the initiation of reproductive maturation suggests that MKRN3 is acting as a brake on gonadotropin-releasing hormone (GnRH) secretion during childhood. In the current study, we investigated the mechanism by which MKRN3 prevents premature manifestation of the pubertal process. We showed that, as in mice, MKRN3 expression is high in the hypothalamus of rats and nonhuman primates early in life, decreases as puberty approaches, and is independent of sex steroid hormones. We demonstrated that Mkrn3 is expressed in Kiss1 neurons of the mouse hypothalamic arcuate nucleus and that MKRN3 repressed promoter activity of human KISS1 and TAC3, 2 key stimulators of GnRH secretion. We further showed that MKRN3 has ubiquitinase activity, that this activity is reduced by MKRN3 mutations affecting the RING finger domain, and that these mutations compromised the ability of MKRN3 to repress KISS1 and TAC3 promoter activity. These results indicate that MKRN3 acts to prevent puberty initiation, at least in part, by repressing KISS1 and TAC3 transcription and that this action may involve an MKRN3-directed ubiquitination-mediated mechanism.

A. Kemal Topaloglu and colleagues report the identification of mutations in the neurokinin B receptor and its ligand in families with severe congenital gonadotropin deficiency and pubertal failure. These findings indicate that neurokinin B is a central regulator of human gonadal function. The timely secretion of gonadal sex steroids is essential for the initiation of puberty, the postpubertal maintenance of secondary sexual characteristics and the normal perinatal development of male external genitalia. Normal gonadal steroid production requires the actions of the pituitary-derived gonadotropins, luteinizing hormone and follicle-stimulating hormone. We report four human pedigrees with severe congenital gonadotropin deficiency and pubertal failure in which all affected individuals are homozygous for loss-of-function mutations in TAC3 (encoding Neurokinin B) or its receptor TACR3 (encoding NK3R). Neurokinin B, a member of the substance P–related tachykinin family, is known to be highly expressed in hypothalamic neurons that also express kisspeptin 1 , a recently identified regulator of gonadotropin-releasing hormone secretion 2 . These findings implicate Neurokinin B as a critical central regulator of human gonadal function and suggest new approaches to the pharmacological control of human reproduction and sex hormone-related diseases.

Mechanisms in the brain controlling secretion of gonadotropin hormones in pigs, particularly luteinizing hormone (LH), are poorly understood. Kisspeptin is a potent LH stimulant that is essential for fertility in many species, including pigs. Neurokinin B (NKB) acting through neurokinin 3 receptor (NK3R) is involved in kisspeptin-stimulated LH release, but organization of NKB and NK3R within the porcine hypothalamus is unknown. Hypothalamic tissue from ovariectomized (OVX) gilts was used to determine the distribution of immunoreactive kisspeptin, NKB, and NK3R cells in the arcuate nucleus (ARC). Almost all kisspeptin neurons coexpressed NKB in the porcine ARC. Immunostaining for NK3R was distributed throughout the preoptic area (POA) and in several hypothalamic areas including the periventricular and retrochiasmatic areas but was not detected within the ARC. There was no colocalization of NK3R with gonadotropin-releasing hormone (GnRH), but NK3R-positive fibers in the POA were in close apposition to GnRH neurons. Treating OVX gilts with the progestin altrenogest decreased LH pulse frequency and reduced mean circulating concentrations of LH compared with OVX control gilts (P < 0.01), but the number of kisspeptin and NKB cells in the ARC did not differ between treatments. The neuroanatomical arrangement of kisspeptin, NKB, and NK3R within the porcine hypothalamus confirms they are positioned to stimulate GnRH and LH secretion in gilts, though differences with other species exist. Altrenogest suppression of LH secretion in the OVX gilt does not appear to involve decreased peptide expression of kisspeptin or NKB. Summary sentence Components of the KNDy system in the pig are characterized for the first time and pig-specific features such as a lack of immunopositive NK3R expression in the ARC and resistance to inhibition of kisspeptin protein expression by a progestin are identified.

Abstract Context Kisspeptin–neurokinin B (NKB)–dynorphin neurons are critical regulators of the hypothalamic–pituitary–gonadal axis. NKB and dynorphin are hypothesized to influence the frequency of GnRH pulses, whereas kisspeptin is hypothesized to be a generator of the GnRH pulse. How these neuropeptides interact remains unclear. Objective To probe the role of NKB in GnRH pulse generation and to determine the interactions between NKB, kisspeptin, and dynorphin in humans and mice with a complete absence of NKB. Design Case/control. Setting Academic medical center. Participants Members of a consanguineous family bearing biallelic loss-of-function mutations in the gene encoding NKB and NKB-deficient mice. Interventions Frequent blood sampling to characterize neuroendocrine profile and administration of kisspeptin, GnRH, and naloxone, a nonspecific opioid receptor antagonist used to block dynorphin. Main Outcome Measures LH pulse characteristics. Results Humans lacking NKB demonstrate slow LH pulse frequency, which can be increased by opioid antagonism. Mice lacking NKB also demonstrate impaired LH secretion, which can be augmented with an identical pharmacologic manipulation. Both mice and humans with NKB deficiency respond to exogenous kisspeptin. Conclusion The preservation of LH pulses in the absence of NKB and dynorphin signaling suggests that both peptides are dispensable for GnRH pulse generation and kisspeptin responsiveness. However, NKB and dynorphin appear to have opposing roles in the modulation of GnRH pulse frequency. This study uses pharmacologic probes to demonstrate that endogenous GnRH-induced LH pulses can be generated in the absence of neurokinin B and dynorphin activity in humans and mice.

PurposeThe neurokinin B (NKB)/NK3 receptor (NK3R) and kisspeptin (KISS1)/kisspeptin receptor (KISS1R), two systems essential for reproduction, are present in human granulosa cells (GCs) of healthy women and contribute to the control of fertility, at least partially, by acting on the gonads. However, little is known about the expression of these systems in GCs of women with polycystic ovarian syndrome (PCOS). The aim of this study was to analyze the expression of NKB/NK3R and KISS1/KISS1R in mural granulosa (MGCs) and cumulus cells (CCs) of PCOS women.MethodsA cross-sectional study was performed in 46 healthy women and 43 PCOS women undergoing controlled ovarian stimulation. MGCs and CCs were collected from pre-ovulatory follicles after transvaginal ultrasound-guided oocyte retrieval and the expression of the genes encoding NKB (TAC3), NK3R (TACR3), KISS1, and its receptor (KISS1R) was analyzed using real-time quantitative RT-PCR.ResultsTAC3, TACR3, and KISS1 mRNA levels were decreased in MGCs and CCs of PCOS women. TAC3 positively correlated with KISS1 in MGCs of healthy women and TACR3 was positively associated with KISS1R in CCs from healthy women. These associations were not observed in PCOS women.ConclusionThe NKB/NK3R and KISS1/KISS1R systems are dysregulated in MGCs and CCs of PCOS women. The lower expression of these systems in GCs could contribute to the abnormal follicle development and defective ovulation that characterize the pathogenesis of PCOS.

The gonadotropin-releasing hormone (GnRH) pulse and surge are considered to be generated by arcuate kisspeptin/neurokinin B/dynorphin A (KNDy) neurons and anteroventral periventricular nucleus (AVPV) kisspeptin neurons, respectively, in female rodents. The majority of KNDy and AVPV kisspeptin neurons express κ-opioid receptors (KORs, encoded by Oprk1 ) in female rodents. Thus, this study aimed to investigate the effect of a conditional Oprk1 -dependent Kiss1 deletion in kisspeptin neurons on the luteinizing hormone (LH) pulse/surge and fertility using Kiss1 -floxed/ Oprk1-Cre rats, in which Kiss1 was deleted in cells expressing or once expressed the Oprk1/Cre . The Kiss1 -floxed/ Oprk1-Cre female rats, with Kiss1 deleted in a majority of KNDy neurons, showed normal puberty while having a one-day longer estrous cycle and fewer pups than Kiss1 -floxed controls. Notably, ovariectomized (OVX) Kiss1 -floxed/ Oprk1-Cre rats showed profound disruption of LH pulses in the presence of a diestrous level of estrogen but showed apparent LH pulses without estrogen treatment. Furthermore, Kiss1 -floxed/ Oprk1-Cre rats, with Kiss1 deleted in approximately half of AVPV kisspeptin neurons, showed a lower peak of the estrogen-induced LH surge than controls. These results suggest that arcuate and AVPV kisspeptin neurons expressing or having expressed Oprk1 have a role in maintaining normal GnRH pulse and surge generation, the normal length of the estrous cycle, and the normal offspring number in female rats.

This Review highlights the milestones in the field of kisspeptin research, in particular developments in the putative interplay of kisspeptins and their cotransmitters, such as neurokinin B, in the central control of GnRH neurons; and the possible role of Kiss1 neurons as conduits for the metabolic regulation of puberty and fertility, with emphasis on intermediate regulatory signals and mechanisms involved. The neurohormonal control of reproduction involves a hierarchical network of central and peripheral signals in the hypothalamic–pituitary–gonadal (HPG) axis. Development and function of this neuroendocrine system is the result of a lifelong delicate balance between endogenous regulators and environmental cues, including nutritional and metabolic factors. Kisspeptins are the peptide products of KISS1 , which operate via the G-protein-coupled receptor GPR54 (also known as Kiss1R). These peptides have emerged as essential upstream regulators of neurons secreting gonadotropin-releasing hormone (GnRH), the major hypothalamic node for the stimulatory control of the HPG axis. They are potent elicitors of gonadotropin secretion in various species and physiological settings. Moreover, Kiss1 neurons in the hypothalamus participate in crucial features of reproductive maturation and function, such as brain-level sex differentiation, puberty onset and the neuroendocrine regulation of gonadotropin secretion and ovulation. Cotransmitters of Kiss1 neurons, such as neurokinin B, with roles in controlling the HPG axis have been identified by genetic, neuroanatomical and physiological studies. In addition, a putative role has been proposed for Kiss1 neurons in transmitting metabolic information to GnRH neurons, although the precise mechanisms are as yet unclear. In this Review, we present the major reproductive features of kisspeptins, especially their interplay with neurokinin B and potential roles in the metabolic control of puberty and fertility, and suggest new avenues for research. Key Points Kisspeptins, the ligands of the G-protein-coupled receptor GPR54, are encoded in humans by the KISS1 gene and operate as potent stimulators of gonadotropin-releasing hormone (GnRH) in the reproductive brain Kisspeptin pathways are involved in the control of crucial aspects of reproductive maturation and function, including puberty onset, sex-steroid control of gonadotropin secretion and ovulation Discrete populations of Kiss1 neurons exist in the arcuate nucleus of the hypothalamus in humans and nonprimates and in the rostral periventricular area in rodents; arcuate Kiss1 neurons coexpress neurokinin B Hypothalamic expression of Kiss1 is sensitive to negative energy balance (potentially including obesity) and probably acts as a sensor and transmitter of metabolic information to GnRH neurons The signals and mechanisms underlying metabolic control of Kiss1 neurons are poorly defined; a role for leptin is proposed, but its mechanisms of action are not yet clear Interplay between the Kiss1 system and various molecular mediators and other neuropeptide pathways seems important for energy homeostasis, but data are inconsistent and need to be interpreted with caution

Pulsatile gonadotropin-releasing hormone (GnRH) is crucial to normal reproductive function and abnormalities in pulse frequency give rise to reproductive dysfunction. Kisspeptin and neurokinin B (NKB), neuropeptides secreted by the same neuronal population in the ventral hypothalamus, have emerged recently as critical central regulators of GnRH and thus gonadotropin secretion. Patients with mutations resulting in loss of signaling by either of these neuroendocrine peptides fail to advance through puberty but the mechanisms mediating this remain unresolved. We report here that continuous kisspeptin infusion restores gonadotropin pulsatility in patients with loss-of-function mutations in NKB (TAC3) or its receptor (TAC3R), indicating that kisspeptin on its own is sufficient to stimulate pulsatile GnRH secretion. Moreover, our findings suggest that NKB action is proximal to kisspeptin in the reproductive neuroendocrine cascade regulating GnRH secretion, and may act as an autocrine modulator of kisspeptin secretion. The ability of continuous kisspeptin infusion to induce pulsatile gonadotropin secretion further indicates that GnRH neurons are able to set up pulsatile secretion in the absence of pulsatile exogenous kisspeptin.

G protein-coupled receptors (GPCRs) play an important role in physiology and disease and represent the most productive drug targets. Orphan GPCRs, with their endogenous ligands unknown, were considered a source of drug targets and consequently attract great interest to identify their endogenous cognate ligands for deorphanization. However, a contrary view to the ubiquitous existence of endogenous ligands for every GPCR is that there might be a significant overlooked fraction of orphan GPCRs that function constitutively in a ligand-independent manner only. Here, we investigated the evolution of the bombesin receptor-ligand family in vertebrates in which one member-bombesin receptor subtype-3 (BRS3)-is a potential orphan GPCR. With analysis of 17 vertebrate BRS3 structures and 10 vertebrate BRS3 functional data, our results demonstrated that nonplacental vertebrate BRS3 still connects to the original ligands-neuromedin B (NMB) and gastrin-releasing peptide (GRP)-because of adaptive evolution, with significantly changed protein structure, especially in three altered key residues (Q127R, P205S, and R294H) originally involved in ligand binding/activation, whereas the placental mammalian BRS3 lost the binding affinity to NMB/GRP and constitutively activates Gs/Gq/G12 signaling in a ligand-independent manner. Moreover, the N terminus of placental mammalian BRS3 underwent positive selection, exhibiting significant structural differences compared to nonplacental vertebrate BRS3, and this domain plays an important role in constitutive activity of placental mammalian BRS3. In conclusion, constitutively active BRS3 is a genuinely orphan GPCR in placental mammals, including human. To our knowledge, this study identified the first example that might represent a new group of genuinely orphan GPCRs that will never be deorphanized by the discovery of a natural ligand and provided new perspectives in addition to the current ligand-driven GPCR deorphanization.