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At the present time, no viable treatment exists for cognitive and olfactory deficits in Down syndrome (DS). We show in a DS model (Ts65Dn mice) that these progressive nonreproductive neurological symptoms closely parallel a postpubertal decrease in hypothalamic as well as extrahypothalamic expression of a master molecule that controls reproduction-gonadotropin-releasing hormone (GnRH)-and appear related to an imbalance in a microRNA-gene network known to regulate GnRH neuron maturation together with altered hippocampal synaptic transmission. Epigenetic, cellular, chemogenetic, and pharmacological interventions that restore physiological GnRH levels abolish olfactory and cognitive defects in Ts65Dn mice, whereas pulsatile GnRH therapy improves cognition and brain connectivity in adult DS patients. GnRH thus plays a crucial role in olfaction and cognition, and pulsatile GnRH therapy holds promise to improve cognitive deficits in DS.

Neurons that produce gonadotropin-releasing hormone (GnRH), which control fertility, complete their nose-to-brain migration by birth. However, their function depends on integration within a complex neuroglial network during postnatal development. Here, we show that rodent GnRH neurons use a prostaglandin D 2 receptor DP1 signaling mechanism during infancy to recruit newborn astrocytes that 'escort' them into adulthood, and that the impairment of postnatal hypothalamic gliogenesis markedly alters sexual maturation by preventing this recruitment, a process mimicked by the endocrine disruptor bisphenol A. Inhibition of DP1 signaling in the infantile preoptic region, where GnRH cell bodies reside, disrupts the correct wiring and firing of GnRH neurons, alters minipuberty or the first activation of the hypothalamic-pituitary-gonadal axis during infancy, and delays the timely acquisition of reproductive capacity. These findings uncover a previously unknown neuron-to-neural-progenitor communication pathway and demonstrate that postnatal astrogenesis is a basic component of a complex set of mechanisms used by the neuroendocrine brain to control sexual maturation., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Mkrn3, the maternally imprinted gene encoding the makorin RING-finger protein-3, has recently emerged as putative pubertal repressor, as evidenced by central precocity caused by MKRN3 mutations in humans; yet, the molecular underpinnings of this key regulatory action remain largely unexplored. We report herein that the microRNA, miR-30, with three binding sites in a highly conserved region of its 3' UTR, operates as repressor of Mkrn3 to control pubertal onset. Hypothalamic miR-30b expression increased, while Mkrn3 mRNA and protein content decreased, during rat postnatal maturation. Neonatal estrogen exposure, causing pubertal alterations, enhanced hypothalamic Mkrn3 and suppressed miR-30b expression in female rats. Functional in vitro analyses demonstrated a strong repressive action of miR-30b on Mkrn3 3' UTR. Moreover, central infusion during the juvenile period of target site blockers, tailored to prevent miR-30 binding to Mkrn3 3' UTR, reversed the prepubertal down-regulation of hypothalamic Mkrn3 protein and delayed female puberty. Collectively, our data unveil a novel hypothalamic miRNA pathway, involving miR-30, with a prominent role in the control of puberty via Mkrn3 repression. These findings expand our current understanding of the molecular basis of puberty and its disease states., Competing Interests: The authors have declared that no competing interests exist.

Conditions of metabolic distress, from malnutrition to obesity, impact, via as yet ill-defined mechanisms, the timing of puberty, whose alterations can hamper later cardiometabolic health and even life expectancy. AMP-activated protein kinase (AMPK), the master cellular energy sensor activated in conditions of energy insufficiency, has a major central role in whole-body energy homeostasis. However, whether brain AMPK metabolically modulates puberty onset remains unknown. We report here that central AMPK interplays with the puberty-activating gene, Kiss1 , to control puberty onset. Pubertal subnutrition, which delayed puberty, enhanced hypothalamic pAMPK levels, while activation of brain AMPK in immature female rats substantially deferred puberty. Virogenetic overexpression of a constitutively active form of AMPK, selectively in the hypothalamic arcuate nucleus (ARC), which holds a key population of Kiss1 neurons, partially delayed puberty onset and reduced luteinizing hormone levels. ARC Kiss1 neurons were found to express pAMPK, and activation of AMPK reduced ARC Kiss1 expression. The physiological relevance of this pathway was attested by conditional ablation of the AMPKα1 subunit in Kiss1 cells, which largely prevented the delay in puberty onset caused by chronic subnutrition. Our data demonstrate that hypothalamic AMPK signaling plays a key role in the metabolic control of puberty, acting via a repressive modulation of ARC Kiss1 neurons in conditions of negative energy balance., Competing Interests: The authors declare no conflict of interest.

Background: RF-amide-related peptide-3 (RFRP-3), the mammalian ortholog of gonadotropin-inhibiting hormone, operates as inhibitory signal for the reproductive axis. Recently, RFRP-3 has been also suggested to stimulate feeding, and therefore might contribute to the control of body weight and its alterations. Yet, characterization of the metabolic actions of RFRP-3 has been so far superficial and mostly pharmacological. Here, we aim to investigate the physiological roles of RFRP-3 signaling in the control of feeding and metabolic homeostasis using a novel mouse model of genetic ablation of its canonical receptor, NPFF1R., Methods: Food intake, body weight gain and composition, and key metabolic parameters, including glucose tolerance and insulin sensitivity, were monitored in mice with constitutive inactivation of NPFF1R., Results: Congenital elimination of NPFF1R in male mice resulted in changes in feeding patterns, with a decrease in spontaneous food intake and altered responses to leptin and ghrelin: leptin-induced feeding suppression was exaggerated in NPFF1R null mice, whereas orexigenic responses to ghrelin were partially blunted. Concordant with this pro-anorectic phenotype, hypothalamic expression of Pomc was increased in NPFF1R null mice. In contrast, spontaneous feeding and neuropeptide expression remained unaltered in NPFF1R KO female mice. Despite propensity for reduced feeding, ablation of NPFF1R signaling in male mice did not cause overt alterations in body weight (BW) gain or composition, neither it affected BW responses to high fat diet (HFD), total energy expenditure or RQ ratios. Yet, NPFF1R KO males showed a decrease in locomotor activity. Conversely, NPFF1R null female mice tended to be heavier and displayed exaggerated BW increases in response to obesogenic insults, such as HFD or ovariectomy. These were associated to increased fat mass, decreased total energy expenditure in HFD, and unaltered RQ ratios or spontaneous locomotor activity. Finally, lack of NPFF1R signaling worsened the metabolic impact of HFD on glycemic homeostasis in males, as revealed by impaired glucose tolerance and insulin sensitivity, while female mice remained unaffected., Conclusion: Our data support a discernible orexigenic role of NPFF1R signaling selectively in males, which might modulate the effects of leptin and ghrelin on food intake. In addition, our study is the first to disclose the sex-biased, deleterious impact of the lack of NPFF1R signaling on body weight and fat composition, energy expenditure, locomotor activity and glucose balance, which exaggerates some of the metabolic consequences of concurrent obesogenic insults, such as HFD, in a sexually dimorphic manner., Summary of Translational Relevance: Our data are the first to document the nature and magnitude of the regulatory actions of RFRP-3/NPFF1R signaling in the control of feeding and metabolic homeostasis in a physiological setting. Our results not only suggest an orexigenic action of endogenous RFRP-3, specifically in males, but reveal also the detrimental impact of ablation of NPFF1R signaling on body composition, energy expenditure, locomotor activity or glucose balance, especially when concurrent with other obesogenic insults, as HFD, thereby providing the first evidence for additional metabolic effects of RFRP-3, other that the mere control of feeding. Interestingly, alterations of such key metabolic parameters occurred in a sex-biased manner, with males being more sensitive to deregulation of locomotor activity and glycemic control, while females displayed clearer obesogenic responses and deregulated energy expenditure. While our study cannot discard the possibility of RFRP-3 actions via alternative pathways, such as NPFF2R, our data pave the way for future analyses addressing the eventual contribution of altered RFRP-3/NPFF1R signaling in the development of metabolic alterations (including obesity and its comorbidities), especially in conditions associated to reproductive dysfunction., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Obesity and its comorbidities are reaching epidemic proportions worldwide. Maternal obesity is known to predispose the offspring to metabolic disorders, independently of genetic inheritance. This intergenerational transmission has also been suggested for paternal obesity, with a potential negative impact on the metabolic and, eventually, reproductive health of the offspring, likely via epigenetic changes in spermatozoa. However, the neuroendocrine component of such phenomenon and whether paternal obesity sensitizes the offspring to the disturbances induced by high-fat diet (HFD) remain poorly defined. We report in this work the metabolic and reproductive impact of HFD in the offspring from obese fathers, with attention to potential sex differences and alterations of hypothalamic Kiss1 system. Lean and obese male rats were mated with lean virgin female rats; male and female offspring were fed HFD from weaning onward and analyzed at adulthood. The increases in body weight and leptin levels, but not glucose intolerance, induced by HFD were significantly augmented in the male, but not female, offspring from obese fathers. Paternal obesity caused a decrease in luteinizing hormone (LH) levels and exacerbated the drop in circulating testosterone and gene expression of its key biosynthetic enzymes caused by HFD in the male offspring. LH responses to central kisspeptin-10 administration were also suppressed in HFD males from obese fathers. In contrast, paternal obesity did not significantly alter gonadotropin levels in the female offspring fed HFD, although these females displayed reduced LH responses to kisspeptin-10. Our findings suggest that HFD-induced metabolic and reproductive disturbances are exacerbated by paternal obesity preferentially in males, whereas kisspeptin effects are affected in both sexes., (Copyright © 2018 Endocrine Society.)

Objective: Puberty is a key developmental phenomenon highly sensitive to metabolic modulation. Worrying trends of changes in the timing of puberty have been reported in humans. These might be linked to the escalating prevalence of childhood obesity and could have deleterious impacts on later (cardio-metabolic) health, but their underlying mechanisms remain unsolved. The neuropeptide α-MSH, made by POMC neurons, plays a key role in energy homeostasis by mediating the actions of leptin and likely participates in the control of reproduction. However, its role in the metabolic regulation of puberty and interplay with kisspeptin, an essential puberty-regulating neuropeptide encoded by Kiss1, remain largely unknown. We aim here to unveil the potential contribution of central α-MSH signaling in the metabolic control of puberty by addressing its role in mediating the pubertal effects of leptin and its potential interaction with kisspeptin., Methods: Using wild type and genetically modified rodent models, we implemented pharmacological studies, expression analyses, electrophysiological recordings, and virogenetic approaches involving DREADD technology to selectively inhibit Kiss1 neurons, in order to interrogate the physiological role of a putative leptin→α-MSH→kisspeptin pathway in the metabolic control of puberty., Results: Stimulation of central α-MSH signaling robustly activated the reproductive axis in pubertal rats, whereas chronic inhibition of melanocortin receptors MC3/4R, delayed puberty, and prevented the permissive effect of leptin on puberty onset. Central blockade of MC3/4R or genetic elimination of kisspeptin receptors from POMC neurons did not affect kisspeptin effects. Conversely, congenital ablation of kisspeptin receptors or inducible, DREADD-mediated inhibition of arcuate nucleus (ARC) Kiss1 neurons resulted in markedly attenuated gonadotropic responses to MC3/4R activation. Furthermore, close appositions were observed between POMC fibers and ARC Kiss1 neurons while blockade of α-MSH signaling suppressed Kiss1 expression in the ARC of pubertal rats., Conclusions: Our physiological, virogenetic, and functional genomic studies document a novel α-MSH→kisspeptin→GnRH neuronal signaling pathway involved in transmitting the permissive effects of leptin on pubertal maturation, which is relevant for the metabolic (and, eventually, pharmacological) regulation of puberty onset.

Kisspeptins, ligands of the receptor, Gpr54, are potent stimulators of puberty and fertility. Yet, whether direct kisspeptin actions on GnRH neurons are sufficient for the whole repertoire of their reproductive effects remains debatable. To dissect out direct vs. indirect effects of kisspeptins on GnRH neurons in vivo, we report herein the detailed reproductive/gonadotropic characterization of a Gpr54 null mouse line with selective re-introduction of Gpr54 expression only in GnRH cells (Gpr54(-/-)Tg; rescued). Despite preserved fertility, adult rescued mice displayed abnormalities in gonadal microstructure, with signs of precocious ageing in females and elevated LH levels with normal-to-low testosterone secretion in males. Gpr54(-/-)Tg rescued mice showed also altered gonadotropin responses to negative feedback withdrawal, while luteinizing hormone responses to various gonadotropic regulators were variably affected, with partially blunted relative (but not absolute) responses to kisspeptin-10, NMDA and the agonist of tachykinin receptors, NK2R. Our data confirm that direct effects of kisspeptins on GnRH cells are sufficient to attain fertility. Yet, such direct actions appear to be insufficient to completely preserve proper functionality of gonadotropic axis, suggesting a role of kisspeptin signaling outside GnRH cells.

The reproductive impact of persistent energy excess in the female remains incompletely defined, yet the escalating prevalence of obesity calls for better understanding of this phenomenon. Also along this line, the influence of ovarian hormones on the pathophysiology of obesity and its comorbidities merits further investigation. We study here the metabolic and gonadotropic impact of sequential obesogenic insults, namely postnatal overnutrition [by rearing in small litters (SL)] and high-fat diet (HFD) after weaning, in gonadal-intact and ovariectomized (OVX) female rats. In young (4 mo) females, SL or HFD similarly increased body weight, yet only a HFD evoked additional metabolic perturbations, some of which were worsened by precedent SL. In addition, HFD concomitantly decreased LH and estradiol levels and, when combined with SL, suppressed Kiss1 expression in the hypothalamic arcuate nucleus in 4-month females, whereas HFD up to 10-month also reduced LH responses to kisspeptin-10. OVX caused rapid deterioration of the metabolic profile, with overweight, increased energy intake, and deregulation of leptin and glucose/insulin levels, effects whose magnitude was similar to, if not higher than, HFD. Summation of previous obesogenic insults maximally increased body weight, basal leptin, insulin and glucose levels, and glucose intolerance. Yet OVX obliterated the inhibitory effects of overweight/HFD on gonadotropin levels and arcuate nucleus Kiss1 expression. Our study documents the deleterious consequences of sequential obesogenic insults on the female gonadotropin axis, which involve central impairment of the Kiss1 system. In addition, our work delineates the dramatic impact of the loss of ovarian secretions, as the menopausal model, on the metabolic profile of female rats, especially when combined with preceding obesogenic challenges.

Premature ovarian failure (POF) affects 1% of women in reproductive age, but its etiology remains uncertain. Whereas kisspeptins, the products of Kiss1 that act via Kiss1r (aka, Gpr54), are known to operate at the hypothalamus to control GnRH/gonadotropin secretion, additional actions at other reproductive organs, including the ovary, have been proposed. Yet, their physiological relevance is still unclear. We present here a series of studies in Kiss1r haplo-insufficient and null mice suggesting a direct role of kisspeptin signaling in the ovary, the defect of which precipitates a state of primary POF. Kiss1r hypomorph mice displayed a premature decline in ovulatory rate, followed by progressive loss of antral follicles, oocyte loss, and a reduction in all categories of preantral follicles. These alterations were accompanied by reduced fertility. Because of this precocious ovarian ageing, mice more than 48 weeks of age showed atrophic ovaries, lacking growing follicles and corpora lutea. This phenomenon was associated with a drop in ovarian Kiss1r mRNA expression, but took place in the absence of a decrease in circulating gonadotropins. In fact, FSH levels increased in aged hypomorph animals, reflecting loss of follicular function. In turn, Kiss1r-null mice, which do not spontaneously ovulate and have arrested follicular development, failed to show normal ovulatory responses to standard gonadotropin priming and required GnRH prestimulation during 1 week in order to display gonadotropin-induced ovulation. Yet, the magnitude of such ovulatory responses was approximately half of that seen in control immature wild-type animals. Altogether, our data are the first to demonstrate that Kiss1r haplo-insufficiency induces a state of POF, which is not attributable to defective gonadotropin secretion. We also show that the failure of follicular development and ovulation linked to the absence of Kiss1r cannot be fully rescued by (even extended) gonadotropin replacement. These findings suggest a direct ovarian role of kisspeptin signaling, the perturbation of which may contribute to the pathogenesis of POF.

RF-amide-related peptide-3 (RFRP-3), the mammalian ortholog of the avian gonadotropin-inhibiting hormone (GnIH), operates via the NPFF1 receptor (NPFF1R) to repress the reproductive axis, therefore acting as counterpart of the excitatory RF-amide peptide, kisspeptin (ligand of Gpr54). In addition, RFRP-3 modulates feeding and might contribute to the integrative control of energy homeostasis and reproduction. Yet, the experimental evidence supporting these putative functions is mostly indirect, and the physiological roles of RFRP-3 remain debatable and obscured by the lack of proper analytical tools and models. To circumvent these limitations, we characterize herein the first mouse line with constitutive inactivation of NPFF1R. Ablation of NPFF1R did not compromise fertility; rather, litters from NPFF1R null mice were larger than those from wild-type animals. Pubertal timing was not altered in NPFF1R deficient mice; yet, pre-pubertal knockout (KO) males displayed elevated LH levels, which normalized after puberty. Adult NPFF1R null male mice showed increased Kiss1 expression in the hypothalamic arcuate nucleus, higher serum FSH levels, and enhanced LH responses to GnRH. However, genetic elimination of NPFF1R was unable to reverse the state of hypogonadism caused by the lack of kisspeptin signaling, as revealed by double NPFF1R/Gpr54 KO mice. NPFF1R null mice displayed altered feedback responses to gonadal hormone withdrawal. In addition, metabolic challenges causing gonadotropin suppression, such as short-term fasting and high-fat diet, were less effective in dampening LH secretion in NPFF1R-deficient male mice, suggesting that absence of this inhibitory pathway partially prevented gonadotropin suppression by metabolic stress. Our data are the first to document the impact of elimination of GnIH signaling on reproductive parameters and their modulation by metabolic challenges. Whereas, in keeping with its inhibitory role, the NPFF1R pathway seems dispensable for preserved puberty and fertility, our results surface different alterations due to the lack of GnIH signaling that prominently include changes in the sensitivity to fasting- and obesity-associated hypogonadotropism.

The hypothalamus plays a crucial role in body weight homeostasis through an intricate network of neuronal circuits that are under the precise regulation of peripheral hormones and central transmitters. Although deregulated function of such circuits might be a major contributing factor in obesity, the molecular mechanisms responsible for the hypothalamic control of energy balance remain partially unknown. MicroRNAs (miRNAs) have been recognized as key regulators of different biological processes, including insulin sensitivity and glucose metabolism. However, the roles of miRNA pathways in the control of metabolism have been mostly addressed in peripheral tissues, whereas the potential deregulation of miRNA expression in the hypothalamus in conditions of metabolic distress remains as yet unexplored. In this work, we used high-throughput screening to define to what extent the hypothalamic profiles of miRNA expression are perturbed in two extreme conditions of nutritional stress in male rats, namely chronic caloric restriction and high-fat diet-induced obesity. Our analyses allowed the identification of sets of miRNAs, including let-7a, mir-9*, mir-30e, mir-132, mir-145, mir-200a, and mir-218, whose expression patterns in the hypothalamus were jointly altered by caloric restriction and/or a high-fat diet. The predicted targets of these miRNAs include several elements of key inflammatory and metabolic pathways, including insulin and leptin. Our study is the first to disclose the impact of nutritional challenges on the hypothalamic miRNA expression profiles. These data will help to characterize the molecular miRNA signature of the hypothalamus in extreme metabolic conditions and pave the way for targeted mechanistic analyses of the involvement of deregulated central miRNAs pathways in the pathogenesis of obesity and related disorders.

Objective: To assess the presence and numbers of multi-oocyte follicles (MOFs) in the rat ovary at different stages of postnatal maturation., Design: Animal (rat) study., Setting: Research laboratory., Animal(s): Female Wistar rats., Intervention(s): Histologic/morphometric analyses in ovaries from infantile, juvenile, pubertal, and adult female rats., Main Outcome Measure(s): Numbers and characteristics of MOFs in rat ovaries at different stages of postnatal maturation., Result(s): Female rats displayed low numbers (<5/ovary) of MOFs in the infantile period (postnatal day [PND] 15). The occurrence of MOFs increased sharply by PND-21 and remained at high values (>15/ovary) up to PND-60, to decline thereafter by PND-90. The presence of irregularly shaped and connected adjacent follicles, together with the identification of ruptures at the follicle surface and the occasional invasion of the ovarian stroma by granulosa cells, strongly suggests that the majority of MOFs in peripubertal rats are generated by fusion of adjacent growing follicles., Conclusion(s): A new mechanism for the generation of MOFs linked to the potential invasive capacity of granulosa cells is proposed. The basis for the upsurge in the generation of MOFs during the peripubertal period and whether, as predictable, this phenomenon is applicable to other mammalian species warrant further investigation., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Reproduction is sensitive to insufficient body energy reserves, especially in females. Metabolic regulation of the male reproductive axis is less obvious, and the impact of conditions of persistent energy excess has received moderate attention. Yet, the escalating prevalence of obesity and the clinical evidence of its deleterious effects on male fertility have raised considerable concerns. We report here phenotypic and mechanistic studies of the reproductive impact of postnatal nutritional manipulations (mainly overnutrition) coupled to a high-fat diet (HFD) after weaning. Metabolic and hormonal analyses in young (4 months old) and middle-aged (10 months old) animals revealed that HFD caused profound metabolic perturbations, including glucose intolerance, which were worsened by precedent postnatal overfeeding; these were detectable already in young males but aggravated in 10-month-old rats. Impairment of reproductive parameters took place progressively, and HFD alone was sufficient to explain most of these alterations, regardless of postnatal under- or overnutrition. In young males, testosterone (T) levels and steroidogenic enzyme expression were suppressed by HFD, without compensatory increases of LH levels, which were in fact partially inhibited in heavier males. In addition, obese males displayed suppressed hypothalamic Kiss1 expression despite low T, and HFD inhibited LH responses to kisspeptin. Overweight anticipated some of the neuroendocrine effects of aging, such as the suppression of hypothalamic Kiss1 expression and the decline in serum T and LH levels. Nonetheless, HFD per se caused a detectable worsening of key reproductive indices in middle-aged males, such as basal LH and FSH levels as well as LH responses to kisspeptin. Our study demonstrates that nutritional stress, especially HFD, has a profound deleterious impact on metabolic and gonadotropic function as well as on the Kiss1 system and precipitates neuroendocrine reproductive senescence in the male.

Lin28 (also termed Lin28a) and Lin28b are related RNA-binding proteins, involved in the control of microRNA synthesis, especially of the let-7 family, with putative functions in early (embryo) development. However, their roles during postnatal maturation remain ill defined. Despite the general assumption that Lin28 and Lin28b share similar targets and functions, conclusive demonstration of such redundancy is still missing. In addition, recent observations suggest a role of Lin28 proteins in mammalian reproduction, which is yet to be defined. We document herein the patterns of RNA expression and protein distribution of Lin28 and Lin28b in mouse testis during postnatal development and in a model of hypogonadotropic hypogonadism as a result of inactivation of the kisspeptin receptor, Gpr54. Lin28 and Lin28b mRNAs were expressed in mouse testis across postnatal maturation, but their levels disparately varied between neonatal and pubertal periods, with peak Lin28 levels in infantile testes and sustained elevation of Lin28b mRNA in young adult male gonads, where relative levels of let-7a and let-7b miRNAs were significantly suppressed. In addition, Lin28 peptides displayed totally different patterns of cellular distribution in mouse testis: Lin28 was located in undifferentiated and type-A1 spermatogonia, whereas Lin28b was confined to spermatids and interstitial Leydig cells. These profiles were perturbed in Gpr54 null mouse testis, which showed preserved but irregular Lin28 signal and absence of Lin28b peptide, which was rescued by administration of gonadotropins, mainly hCG (as super-agonist of LH). In addition, increased relative levels of Lin28, but not Lin28b, mRNA and of let-7a/let-7b miRNAs were observed in Gpr54 KO mouse testes. Altogether, our data are the first to document the divergent patterns of cellular distribution and mRNA expression of Lin28 and Lin28b in the mouse testis along postnatal maturation and their alteration in a model of congenital hypogonadotropic hypogonadism. Our findings suggest distinct functional roles of these two related, but not overlapping, miRNA-binding proteins in the male gonad.

Kisspeptins (Kp), products of the Kiss1 gene that act via Gpr54 to potently stimulate GnRH secretion, operate as mediators of other regulatory signals of the gonadotropic axis. Mouse models of Gpr54 and/or Kiss1 inactivation have been used to address the contribution of Kp in the central control of gonadotropin secretion; yet, phenotypic and hormonal differences have been detected among the transgenic lines available. We report here a series of neuroendocrine analyses in male mice of a novel Gpr54 knockout (KO) model, generated by heterozygous crossing of a loxP-Gpr54/Protamine-Cre double mutant line. Gpr54-null males showed severe hypogonadotropic hypogonadism but retained robust responsiveness to GnRH. Gonadotropic responses to the agonist of ionotropic glutamate receptors, N-methyl-d-aspartate, were attenuated, but persisted, in Gpr54-null mice. In contrast, LH secretion after activation of metabotropic glutamate receptors was totally preserved in the absence of Gpr54 signaling. Detectable, albeit reduced, LH responses were also observed in Gpr54 KO mice after intracerebroventricular administration of galanin-like peptide or RF9, putative antagonist of neuropeptide FF receptors for the mammalian ortholog of gonadotropin-inhibiting hormone. In contrast, the stimulatory effect of senktide, agonist of neurokinin B (NKB; cotransmitter of Kiss1 neurons), was totally abrogated in Gpr54 KO males. Lack of Kp signaling also eliminated feedback LH responses to testosterone withdrawal. However, residual but sustained increases of FSH were detected in gonadectomized Gpr54 KO males, in which testosterone replacement failed to fully suppress circulating FSH levels. In sum, our study provides novel evidence for the relative importance of Kp-dependent vs. -independent actions of several key regulators of GnRH secretion, such as glutamate, galanin-like peptide, and testosterone. In addition, our data document for the first time the indispensable role of Kp signaling in mediating the stimulatory effects of NKB on LH secretion, thus supporting the hypothesis that NKB actions on GnRH neurons are indirectly mediated via its ability to regulate Kiss1 neuronal output.