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BACKGROUND: Perturbations in the timing of puberty, with potential adverse consequences in later health, are increasingly common. The underlying neurohormonal mechanisms are unfolded, but nutritional alterations are key contributors. Efforts to unveil the basis of normal puberty and its metabolic control have focused on mechanisms controlling expression of Kiss1, the gene encoding the puberty-activating neuropeptide, kisspeptin. However, other regulatory phenomena remain ill-defined. Here, we address the putative role of the G protein-coupled-receptor kinase-2, GRK2, in GnRH neurons, as modulator of pubertal timing via repression of the actions of kisspeptin, in normal maturation and conditions of nutritional deficiency. METHODS: Hypothalamic RNA and protein expression analyses were conducted in maturing female rats. Pharmacological studies involved central administration of GRK2 inhibitor, βARK1-I, and assessment of gonadotropin responses to kisspeptin or phenotypic and hormonal markers of puberty, under normal nutrition or early subnutrition in female rats. In addition, a mouse line with selective ablation of GRK2 in GnRH neurons, aka G-GRKO, was generated, in which hormonal responses to kisspeptin and puberty onset were monitored, in normal conditions and after nutritional deprivation. RESULTS: Hypothalamic GRK2 expression increased along postnatal maturation in female rats, especially in the preoptic area, where most GnRH neurons reside, but decreased during the juvenile-to-pubertal transition. Blockade of GRK2 activity enhanced Ca(+2) responses to kisspeptin in vitro, while central inhibition of GRK2 in vivo augmented gonadotropin responses to kisspeptin and advanced puberty onset. Postnatal undernutrition increased hypothalamic GRK2 expression and delayed puberty onset, the latter being partially reversed by central GRK2 inhibition. Conditional ablation of GRK2 in GnRH neurons enhanced gonadotropin responses to kisspeptin, accelerated puberty onset, and increased LH pulse frequency, while partially prevented the negative impact of subnutrition on pubertal timing and LH pulsatility in mice. CONCLUSIONS: Our data disclose a novel pathway whereby GRK2 negatively regulates kisspeptin actions in GnRH neurons, as major regulatory mechanism for tuning pubertal timing in nutritionally-compromised conditions.

El presente proyecto ha consistido en la implantación de un sistema de gamificación de preguntas y respuestas en clase con el objetivo de mejorar la motivación y el compromiso de los alumnos con el aprendizaje en las asignaturas del área de Fisiología: Fisiología General (Grado de Medicina, 180 alumnos), Fisiología (Grado de Enfermería: 170 alumnos) y Fisiología Especial (Grado de Fisioterapia: 70 alumnos). Como sistema de gamificación se empleó la aplicación web Kahoot!, que permite registrar en tiempo real, mediante el uso de dispositivos remotos individuales (p.ej. teléfonos móviles), la participación de los estudiantes en las diferentes tareas propuestas (p.ej. preguntas tipo test, evaluaciones). Los datos registrados nos permiten concluir que los alumnos han conseguido un grado de motivación significativamente superior al observado en cursos anteriores, en los que este sistema aún no se había implantado. Además, consideramos que dicha motivación viene dada por el papel protagonista que han adquirido los estudiantes tras la implantación de este nuevo sistema. En resumen, estos resultados sugieren que la gamificación es una herramienta muy útil para estimular la motivación de los estudiantes y abre la posibilidad de su implantación en otros contextos educativos, especialmente en aquellos en los que los estudiantes no se sientan motivados.

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 AMPK alpha 1 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.

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.

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.

Background: Kisspeptins, encoded by Kiss1, have emerged as essential regulators of puberty and reproduction by primarily acting on GnRH neurons, via their canonical receptor, Gpr54. Mounting, as yet fragmentary, evidence strongly suggests that kisspeptin signaling may also participate in the control of key aspects of body energy and metabolic homeostasis. However, characterization of such metabolic dimension of kisspeptins remains uncomplete, without an unambiguous discrimination between the primary metabolic actions of kisspeptins vs. those derived from their ability to stimulate the secretion of gonadal hormones, which have distinct metabolic actions on their own. In this work, we aimed to tease apart primary vs. secondary effects of kisspeptins in the control of key aspects of metabolic homeostasis using genetic models of impaired kisspeptin signaling and/or gonadal hormone status. Methods: Body weight (BW) gain and composition, food intake and key metabolic parameters, including glucose tolerance, were comparatively analyzed, in lean and obesogenic conditions, in mice lacking kisspeptin signaling due to global inactivation of Gpr54 (displaying profound hypogonadism; Gpr54−/−) vs. Gpr54 null mice with selective re-introduction of Gpr54 expression only in GnRH cells (Gpr54−/−Tg), where kisspeptin signaling elsewhere than in GnRH neurons is ablated but gonadal function is preserved. Results: In male mice, global elimination of kisspeptin signaling resulted in decreased BW, feeding suppression and increased adiposity, without overt changes in glucose tolerance, whereas Gpr54−/− female mice displayed enhanced BW gain at adulthood, increased adiposity and perturbed glucose tolerance, despite reduced food intake. Gpr54−/−Tg rescued mice showed altered postnatal BW gain in males and mildly perturbed glucose tolerance in females, with intermediate phenotypes between control and global KO animals. Yet, body composition and leptin levels were similar to controls in gonadal-rescued mice. Exposure to obesogenic insults, such as high fat diet (HFD), resulted in exaggerated BW gain and adiposity in global Gpr54−/− mice of both sexes, and worsening of glucose tolerance, especially in females. Yet, while rescued Gpr54−/−Tg males displayed intermediate BW gain and feeding profiles and impaired glucose tolerance, rescued Gpr54−/−Tg females behaved as controls, except for a modest deterioration of glucose tolerance after ovariectomy. Conclusion: Our data support a global role of kisspeptin signaling in the control of body weight and metabolic homeostasis, with a dominant contribution of gonadal hormone-dependent actions. However, our results document also discernible primary effects of kisspeptin signaling in the regulation of body weight gain, feeding and responses to obesogenic insults, which occur in a sexually-dimorphic manner. Summary of translational relevance: Kisspeptins, master regulators of reproduction, may also participate in the control of key aspects of body energy and metabolic homeostasis; yet, the nature of such metabolic actions remains debatable, due in part to the fact that kisspeptins modulate gonadal hormones, which have metabolic actions on their own. By comparing the metabolic profiles of two mouse models with genetic inactivation of kisspeptin signaling but different gonadal status (hypogonadal vs. preserved gonadal function), we provide herein a systematic dissection of gonadal-dependent vs. -independent metabolic actions of kisspeptins. Our data support a global role of kisspeptin signaling in the control of body weight and metabolic homeostasis, with a dominant contribution of gonadal hormone-dependent actions. However, our results document also discernible primary effects of kisspeptin signaling in the regulation of body weight gain, feeding and responses to obesogenic insults, which occur in a sexually-dimorphic manner. These data pave the way for future analyses addressing the eventual contribution of altered kisspeptin signaling in the development of metabolic alterations, especially in conditions linked to reproductive dysfunction.

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., The microRNA miR-30 suppresses the expression of Mkrn3 (a known repressor of puberty) in the hypothalamus by binding to a highly conserved region of its 3’ untranslated region, representing a novel mechanism for finely modulating the tempo of puberty.

Background Reproduction is tightly coupled to body energy and metabolic status. GnRH neurons, master elements and final output pathway for the brain control of reproduction, directly or indirectly receive and integrate multiple metabolic cues to regulate reproductive function. Yet, the molecular underpinnings of such phenomenon remain largely unfolded. AMP-activated protein kinase (AMPK), the fundamental cellular sensor that becomes activated in conditions of energy deficit, has been recently shown to participate in the control of Kiss1 neurons, essential gatekeepers of the reproductive axis, by driving an inhibitory valence in situations of energy scarcity at puberty. However, the contribution of AMPK signaling specifically in GnRH neurons to the metabolic control of reproduction remains unknown. Methods Double immunohistochemistry (IHC) was applied to evaluate expression of active (phosphorylated) AMPK in GnRH neurons and a novel mouse line, named GAMKO, with conditional ablation of the AMPK α1 subunit in GnRH neurons, was generated. GAMKO mice of both sexes were subjected to reproductive characterization, with attention to puberty and gonadotropic responses to kisspeptin and metabolic stress. Results A vast majority (>95%) of GnRH neurons co-expressed pAMPK. Female (but not male) GAMKO mice displayed earlier puberty onset and exaggerated LH (as surrogate marker of GnRH) responses to kisspeptin-10 at the prepubertal age. In adulthood, GAMKO females retained increased LH responsiveness to kisspeptin and showed partial resilience to the inhibitory effects of conditions of negative energy balance on the gonadotropic axis. The modulatory role of AMPK in GnRH neurons required preserved ovarian function, since the differences in LH pulsatility detected between GAMKO and control mice subjected to fasting were abolished in ovariectomized animals. Conclusions Altogether, our data document a sex-biased, physiological role of AMPK signaling in GnRH neurons, as molecular conduit of the inhibitory actions of conditions of energy deficit on the female reproductive axis.

Childhood obesity, especially in girls, is frequently bound to earlier puberty, which is linked to higher disease burden later in life. The mechanisms underlying this association remain elusive. Here we show that brain ceramides participate in the control of female puberty and contribute to its alteration in early-onset obesity in rats. Postnatal overweight caused earlier puberty and increased hypothalamic ceramide content, while pharmacological activation of ceramide synthesis mimicked the pubertal advancement caused by obesity, specifically in females. Conversely, central blockade of de novo ceramide synthesis delayed puberty and prevented the effects of the puberty-activating signal, kisspeptin. This phenomenon seemingly involves a circuit encompassing the paraventricular nucleus (PVN) and ovarian sympathetic innervation. Early-onset obesity enhanced PVN expression of SPTLC1, a key enzyme for ceramide synthesis, and advanced the maturation of the ovarian noradrenergic system. In turn, obesity-induced pubertal precocity was reversed by virogenetic suppression of SPTLC1 in the PVN. Our data unveil a pathway, linking kisspeptin, PVN ceramides, and sympathetic ovarian innervation, as key for obesity-induced pubertal precocity.

GH (growth hormone) secretion/action is modulated by alterations in energy homeostasis, such as malnutrition and obesity. Recent data have uncovered the mechanism by which hypothalamic neurons sense nutrient bioavailability, with a relevant contribution of AMPK (AMP-activated protein kinase) and mTOR (mammalian Target of Rapamycin), as sensors of cellular energy status. However, whether central AMPK-mediated lipid signaling and mTOR participate in the regulation of pituitary GH secretion remains unexplored. We provide herein evidence for the involvement of hypothalamic AMPK signaling, but not hypothalamic lipid metabolism or CPT-1 (carnitine palmitoyltransferase I) activity, in the regulation of GH stimulatory responses to the two major elicitors of GH release in vivo, namely GHRH (growth hormone&ndash, releasing hormone) and ghrelin. This effect appeared to be GH-specific, as blocking of hypothalamic AMPK failed to influence GnRH (gonadotropin-releasing hormone)-induced LH (luteinizing hormone) secretion. Additionally, central mTOR inactivation did not alter GH responses to GHRH or ghrelin, nor this blockade affected LH responses to GnRH in vivo. In sum, we document here for the first time the indispensable and specific role of preserved central AMPK, but not mTOR, signaling, through a non-canonical lipid signaling pathway, for proper GH responses to GHRH and ghrelin in vivo.

Puberty is regulated by epigenetic mechanisms and is highly sensitive to metabolic and nutritional cues. However, the epigenetic pathways mediating the effects of nutrition and obesity on pubertal timing are unknown. Here, we identify Sirtuin 1 (SIRT1), a fuel-sensing deacetylase, as a molecule that restrains female puberty via epigenetic repression of the puberty-activating gene, Kiss1. SIRT1 is expressed in hypothalamic Kiss1 neurons and suppresses Kiss1 expression. SIRT1 interacts with the Polycomb silencing complex to decrease Kiss1 promoter activity. As puberty approaches, SIRT1 is evicted from the Kiss1 promoter facilitating a repressive-to-permissive switch in chromatin landscape. Early-onset overnutrition accelerates these changes, enhances Kiss1 expression and advances puberty. In contrast, undernutrition raises SIRT1 levels, protracts Kiss1 repression and delays puberty. This delay is mimicked by central pharmacological activation of SIRT1 or SIRT1 overexpression, achieved via transgenesis or virogenetic targeting to the ARC. Our results identify SIRT1-mediated inhibition of Kiss1 as key epigenetic mechanism by which nutritional cues and obesity influence mammalian puberty., The onset of mammalian puberty is sensitive to metabolic changes and nutritional status, but the mechanisms underlying this phenomenon are poorly understood. Here the authors show that the epigenetic regulator of transcription, SIRT1, mediates the effects of under and overnutrition on pubertal timing by controlling the expression of Kiss1 in hypothalamic neurons.

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.

Kisspeptin/neurokinin B/dynorphin (KNDy) neurons, which coexpress kisspeptins (Kps), neurokinin B (NKB), and dynorphin (Dyn), regulate gonadotropin secretion. The KNDy model proposes that NKB (a stimulator, through NK3R) and Dyn (an inhibitor, through κ-opioid receptor) shape Kp secretion onto GnRH neurons. However, some aspects of this paradigm remain ill defined. Here we aimed to characterize the following: 1) the effects of NKB signaling on FSH secretion and 2) the role of Dyn in gonadotropin secretion after NK3R activation; 3) additionally, we explored the roles of other tachykinin receptors, NK1R and NK2R, on gonadotropin release. Thus, the effects of the NK3R agonist, senktide, on FSH release were explored across postnatal development in male and female rats; gonadotropin responses to agonists of NK1R substance P and NK2R [neurokinin A (NKA)] were also monitored. Moreover, the effects of senktide on gonadotropin secretion were assessed after antagonizing Dyn actions by nor-binaltorphimine didydrochloride. Before puberty, rats of both sexes showed increased FSH secretion to senktide (and Kp-10). Conversely, adult female rats were irresponsive to senktide in terms of FSH, despite proven LH responses, whereas the adult males did not display FSH or LH responses to senktide, even at high doses. In turn, substance P and NKA stimulated gonadotropin secretion in prepubertal rats, whereas in adults modest gonadotropin responses to NKA were detected. By pretreatment with a Dyn antagonist, adult males became responsive to senktide in terms of LH secretion and displayed elevated basal LH and FSH levels; nor-binaltorphimine didydrochloride treatment uncovered FSH responses to senktide in adult females. Furthermore, the expression of Pdyn and Opkr1 (encoding Dyn and κ-opioid receptor, respectively) in the mediobasal hypothalamus was greater in males than in females at prepubertal ages. Overall, our data contribute to refining our understanding on how the elements of the KNDy node and related factors (ie, other tachykinins) differentially participate in the control of gonadotropins at different stages of rat postnatal maturation.

Tachykinins are comprised of the family of related peptides, substance P (SP), neurokinin A (NKA), and neurokinin B (NKB). NKB has emerged as regulator of kisspeptin release in the arcuate nucleus (ARC), whereas the roles of SP and NKA in reproduction remain unknown. This work explores the roles of SP and NKA in the central regulation of GnRH release. First, central infusion of specific agonists for the receptors of SP (neurokinin receptor 1, NK1R), NKA (NK2R) and NKB (NK3R) each induced gonadotropin release in adult male and ovariectomized, estradiol-replaced female mice, which was absent in Kiss1r-/- mice, indicating a kisspeptin-dependent action. The NK2R agonist, however, decreasedLHrelease in ovariectomized-sham replaced females, as documented forNK3R agonists but in contrast to the NK1R agonist, which further increased LH release. Second, Tac1 (encoding SP and NKA) expression in the ARC and ventromedial nucleus was inhibited by circulating estradiol but did not colocalize with Kiss1 mRNA. Third, about half of isolated ARC Kiss1 neurons expressed Tacr1 (NK1R) and 100% Tacr3 (NK3R); for anteroventral-periventricular Kiss1 neurons andGnRHneurons, approximately one-fourth expressed Tacr1 and one-tenth Tacr3; Tacr2 (NK2R) expression was absent in all cases. Overall, these results identify a potent regulation of gonadotropin release by the SP/NK1R and NKA/NK2R systems in the presence of kisspeptin-Kiss1r signaling, indicating that they may, along with NKB/NK3R, control GnRH release, at least in part through actions on Kiss1 neurons. Copyright © 2015 by the Endocrine Society.

Summary Estrogens play a major role in the modulation of energy balance through central and peripheral actions. Here, we demonstrate that central action of estradiol (E2) inhibits AMP-activated protein kinase (AMPK) through estrogen receptor alpha (ERα) selectively in the ventromedial nucleus of the hypothalamus (VMH), leading to activation of thermogenesis in brown adipose tissue (BAT) through the sympathetic nervous system (SNS) in a feeding-independent manner. Genetic activation of AMPK in the VMH prevented E2-induced increase in BAT-mediated thermogenesis and weight loss. Notably, fluctuations in E2 levels during estrous cycle also modulate this integrated physiological network. Together, these findings demonstrate that E2 regulation of the VMH AMPK-SNS-BAT axis is an important determinant of energy balance and suggest that dysregulation in this axis may account for the common changes in energy homeostasis and obesity linked to dysfunction of the female gonadal axis., Graphical Abstract, Highlights • Central estradiol (E2) promotes negative energy balance • Central E2 increases thermogenic sympathetic nerve activity • Central E2 inhibits AMPK, specifically in the VMH, through ERα • The VMH AMPK-SNS-BAT axis mediates the central actions of E2 on energy balance, Estrogens play a major role in the regulation of body weight. Martínez de Morentin and colleagues show that estradiol (E2), acting through estrogen receptor alpha, selectively inhibits the energy sensor AMPK in the brain hypothalamic ventromedial nucleus, leading to activation of thermogenesis in brown adipose tissue.

Background: VGF (non-acronymic), a protein expressed in the hypothalamus and pituitary, is involved in the control of metabolism and body weight homeostasis. Different active peptide fragments are generated from VGF, including TLQP-21. Previous studies of our group reported that this molecule participates also in the regulation of reproductive function in male rats, with predominant stimulatory effects. Methods: We report herein a series of studies on the reproductive effects of TLQP-21 in female rats, as evaluated by a combination of in vivo and in vitro analyses. Results: TLQP-21 modestly increased serum LH levels after systemic administration and directly stimulated pituitary LH and FSH secretion in prepubertal female rats, while acute central injection of TLQP-21 was unable to modify LH secretion at this age. Repeated central administration of TLQP-21 during the pubertal transition (between PND-28 and -35) to female rats fed ad libitum advanced the timing of vaginal opening and increased the percentage of animals with signs of ovulation. Moreover, an analogous treatment slightly enhanced ovarian maturation in pubertal female rats subjected to chronic undernutrition, but was unable to rescue the delay of vaginal opening induced by food deprivation. In addition, TLQP-21 oppositely modified LH secretion in adult female rats depending on the stage of the ovarian cycle: it stimulated LH secretion when injected in the morning of diestrus and decreased the magnitude of the preovulatory LH (but not FSH) surge when injected in the afternoon of proestrus. Conclusions: Our data are the first to document the potential involvement of TLQP-21 in the control of reproductive function in female rats.

Kisspeptins (Kp), products of the Kiss1 gene, have emerged as essential elements in the control of GnRH neurons and gonadotropic secretion. However, despite considerable progress in the field, to date limited attention has been paid to elucidate the potential interactions of Kp with other neurotransmitters known to centrally regulate the gonadotropic axis. We characterize herein the impact of manipulations of key aminoacidergic (glutamate and GABA), peptidergic (NKB, Dyn, and MCH), and gaseous [nitric oxide (NO)] neurotransmission on gonadotropin responses to Kp-10 in male rats. Blockade of ionotropic glutamate receptors (of the NMDA and non-NMDA type) variably decreased LH responses to Kp-10, whereas activation of both ionotropic and metabotropic receptors, which enhanced LH and FSH release per se, failed to further increase gonadotropin responses to Kp-10. In fact, coactivation of metabotropic receptors attenuated LH and FSH responses to Kp-10. Selective activation of GABAA receptors decreased Kp-induced gonadotropin secretion, whereas their blockade elicited robust LH and FSH bursts and protracted responses to Kp-10 when combined with GABAB receptor inhibition. Blockade of Dyn signaling (at κ-opioid receptors) enhanced LH responses to Kp-10, whereas activation of Dyn and NKB signaling modestly reduced Kp-induced LH and FSH release. Finally, MCH decreased basal LH secretion and modestly reduced FSH responses to Kp-10, whereas LH responses to Kp-10 were protracted after inhibition of NO synthesis. In summary, we present herein evidence for the putative roles of glutamate, GABA, Dyn, NKB, MCH, and NO in modulating gonadotropic responses to Kp in male rats. Our pharmacological data will help to characterize the central interactions and putative hierarchy of key neuroendocrine pathways involved in the control of the gonadotropic axis.

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.

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., Highlights • Puberty is highly sensitive to metabolic modulation and disturbed by child obesity. • Altered puberty is linked to adverse metabolic health outcomes via unclear mechanisms. • The POMC product, α-MSH, transmit leptin-mediated metabolic regulation of puberty. • A novel α-MSH→kisspeptin→GnRH signaling pathway is involved in the control of puberty • This pathway is important for the metabolic (and pharmacologic) control of puberty.

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.

Kisspeptins, encoded by the Kiss1 gene, and their canonical receptor, GPR54 (also termed Kiss1R), are unanimously recognised as essential regulators of puberty onset and gonadotrophin secretion. These key reproductive functions stem from the capacity of kisspeptins to stimulate gonadotrophin-releasing hormone (GnRH) secretion in the hypothalamus, where discrete populations of Kiss1 neurones have been identified. In rodents, two major groups of hypothalamic Kiss1 neurones exist: one present in the arcuate nucleus (ARC) and the other located in the anteroventral periventricular area (AVPV/RP3V). In recent years, numerous signals have been identified as putative modulators of the hypothalamic Kiss1 system. Among them, the prominent role of sex steroids as being important regulators of Kiss1 neurones has been documented in different species and developmental stages, such as early brain sex differentiation, puberty, adulthood and senescence. These regulatory actions are (mainly) conducted via oestrogen receptor (ER)α, which is expressed in almost all Kiss1 neurones, and likely involve both classical and nonclassical pathways. The regulatory effects of sex steroids are nucleus-specific. Thus, sex steroids inhibit the expression of Kiss1/kisspeptin at the ARC, as a mechanism to conduct their negative-feedback actions on gonadotrophin secretion. By contrast, oestrogens enhance Kiss1 expression at the AVPV/RP3V in rodents, suggesting the involvement of this population in the positive-feedback actions of oestradiol to generate the preovulatory surge of gonadotrophins. In addition, sex steroids have been shown to act post-transcriptionally, modulating GnRH/gonadotrophin responsiveness to kisspeptin. Finally, sex steroids also regulate the expression of co-transmitters of Kiss1 neurones, such as neurokinin B, whose mRNA content in the ARC fluctuates in parallel to that of Kiss1 in response to changes in the circulating levels of sex steroids, therefore suggesting the contribution of this neuropeptide in the feedback control of gonadotrophin secretion. In sum, compelling experimental evidence obtained in different mammalian (and non-mammalian) species, including primates, demonstrates that sex steroids are essential regulators of hypothalamic Kiss1 neurones, which in turn operate as conduits for their effects on GnRH neurones. The physiological relevance of such regulatory phenomena is thoroughly discussed.

Neurokinin B (NKB) and its cognate receptor neurokinin 3 (NK3R) play a critical role in reproduction. NKB and NK3R are coexpressed with dynorphin ( Dyn) and kisspeptin ( Kiss1) genes in neurons of the arcuate nucleus (Arc). However, the mechanisms of action of NKB as a cotransmitter with kisspeptin and dynorphin remain poorly understood. We explored the role of NKB in the control of LH secretion in the female rat as follows. 1) We examined the effect of an NKB agonist (senktide, 600 pmol, administered into the lateral cerebral ventricle) on luteinizing hormone (LH) secretion. In the presence of physiological levels of estradiol (E2), senktide induced a profound increase in serum levels of LH and a 10-fold increase in the number of Kiss1 neurons expressing c -fos in the Arc ( P < 0.01 for both). 2) We mapped the distribution of NKB and NK3R mRNAs in the central forebrain and found that both are widely expressed, with intense expression in several hypothalamic nuclei that control reproduction, including the Arc. 3) We studied the effect of E2 on the expression of NKB and NK3R mRNAs in the Arc and found that E2 inhibits the expression of both genes ( P < 0.01) and that the expression of NKB and NK3R reaches its nadir on the afternoon of proestrus (when circulating levels of E2 are high). These observations suggest that NKB/NK3R signaling in Kiss1/NKB/Dyn-producing neurons in the Arc has a pivotal role in the control of gonadotropin-releasing hormone (GnRH)/LH secretion and its regulation by E2-dependent negative feedback in the rat.

Kisspeptin (Kiss1) and neurokinin B (NKB) (encoded by the Kiss1 and Tac2 genes, respectively) are indispensable for reproduction. In the female of many species, Kiss1 neurons in the arcuate nucleus (ARC) coexpress dynorphin A and NKB. Such cells have been termed Kiss1/NKB/Dynorphin (KNDy) neurons, which are thought to mediate the negative feedback regulation of GnRH/LH secretion by 17β-estradiol. However, we have less knowledge about the molecular physiology and regulation of Kiss1/Kiss1-expressing neurons in the ARC of the male. Our work focused on the adult male mouse, where we sought evidence for coexpression of these neuropeptides in cells in the ARC, assessed the role of Kiss1 neurons in negative feedback regulation of GnRH/LH secretion by testosterone (T), and investigated the action of NKB on KNDy and GnRH neurons. Results showed that 1) the mRNA encoding Kiss1, NKB, and dynorphin are coexpressed in neurons located in the ARC; 2) Kiss1 and dynorphin A mRNA are regulated by T through estrogen and androgen receptor-dependent pathways; 3) senktide, an agonist for the NKB receptor (neurokinin 3 receptor, encoded by Tacr3), stimulates gonadotropin secretion; 4) KNDy neurons express Tacr3, whereas GnRH neurons do not; and 5) senktide activates KNDy neurons but has no discernable effect on GnRH neurons. These observations corroborate the putative role for KNDy neurons in mediating the negative feedback effects of T on GnRH/LH secretion and provide evidence that NKB released from KNDy neurons is part of an auto-feedback loop that generates the pulsatile secretion of Kiss1 and GnRH in the male.

Kiss1 neurons have recently emerged as a putative conduit for the metabolic gating of reproduction, with leptin being a regulator of hypothalamic Kiss1 expression. Early perturbations of the nutritional status are known to predispose to different metabolic disorders later in life and to alter the timing of puberty; however, the potential underlying mechanisms remain poorly defined. Here we report how changes in the pattern of postnatal feeding affect the onset of puberty and evaluate key hormonal and neuropeptide [Kiss1/kisspeptin (Kp)] alterations linked to these early nutritional manipulations. Female rats were raised in litters of different sizes: small (four pups per dam: overfeeding), normal (12 pups per dam), and large litters (20 pups per litter: underfeeding). Postnatal overfeeding resulted in persistently increased body weight and earlier age of vaginal opening, as an external sign of puberty, together with higher levels of leptin and hypothalamic Kiss1 mRNA. Conversely, postnatal underfeeding caused a persistent reduction in body weight, lower ovarian and uterus weights, and delayed vaginal opening, changes that were paralleled by a decrease in leptin and Kiss1 mRNA levels. Kisspeptin-52 immunoreactivity (Kp-IR) in the hypothalamus displayed similar patterns, with lower numbers of Kp-IR neurons in the arcuate nucleus of postnatally underfed animals, and a trend for increased Kp-positive fibers in the periventricular area of early overfed rats. Yet, gonadotropin responses to Kp at puberty were similar in all groups, except for enhanced responsiveness to low doses of Kp-10 in postnatally underfed rats. In conclusion, our data document that the timing of puberty is sensitive to both overfeeding and subnutrition during early (postnatal) periods and suggest that alterations in hypothalamic expression of Kiss1/kisspeptin may underlie at least part of such programming phenomenon.

International audience; Puberty, as the end-point of a complex series of maturational events affecting the components of the hypothalamic-pituitary-gonadal (HPG) axis, is gated by the state of body energy reserves and sensitive to different metabolic cues; conditions of severe metabolic stress and energy unbalance (from anorexia to morbid obesity) being commonly linked to perturbation of the onset of puberty. In the last two decades, the neuroendocrine mechanisms responsible for the tight coupling between energy homeostasis and puberty onset have begun to be deciphered. These seemingly involve a plethora of metabolic hormones and neuropeptides, which impinge and integrate (mostly) at the hypothalamic centers governing reproduction. Yet, characterization of the mechanisms of action of such regulators (and even their nature and physiological relevance) still remains incomplete. In this review, we will summarize some recent developments in our knowledge of the effects and mechanisms of action of two key metabolic hormones, leptin and ghrelin, in the control of puberty onset. In addition, the roles of the hypothalamic Kiss1 system in the metabolic gating of puberty will be reviewed, with special attention to its regulation by leptin and the recent identification of the putative roles of Crtc1 and mTOR signaling as molecular conduits for the metabolic control of Kiss1 expression. Elucidation of these novel players and regulatory mechanisms will help for a better understanding of the determinants of the timing of puberty, and its eventual alterations in adverse metabolic conditions.

Severe inflammatory challenges are frequently coupled to decreased food intake and disruption of reproductive function, the latter via deregulation of different signaling pathways that impinge onto GnRH neurons. Recently, the hypothalamic Kiss1 system, a major gatekeeper of GnRH function, was suggested as potential target for transmitting immune-mediated repression of the gonadotropic axis during acute inflammation, and yet key facets of such a phenomenon remain ill defined. Using lipopolysaccharide S (LPS)-treated male rats as model of inflammation, we document herein the pattern of hypothalamic kisspeptin immunoreactivity (IR) and hormonal responses to kisspeptin during the acute inflammatory phase. LPS injections induced a dramatic but transient drop of serum LH and testosterone levels. Suppression of gonadotropic function was associated with a significant decrease in kisspeptin-IR in the arcuate nucleus (ARC) that was not observed under conditions of metabolic stress induced by 48-h fasting. In addition, absolute responses to kisspeptin-10 (Kp-10), in terms of LH and testosterone secretion, were significantly attenuated in LPS-treated males that also displayed a decrease in food intake and body weight. Yet pair-fed males did not show similar alterations in LH and testosterone secretory responses to Kp-10, whose magnitude was preserved, if not augmented, during food restriction. In summary, our data document the impact of acute inflammation on kisspeptin content at the ARC as key center for the neuroendocrine control of reproduction. Our results also suggest that suppressed gonadotropic function following inflammatory challenges might involve a reduction in absolute responsiveness to kisspeptin that is independent of the anorectic effects of inflammation.

The hypothalamic peptide, nesfatin-1, derived from the precursor NEFA/nucleobindin 2 (NUCB2), was recently identified as anorexigenic signal, acting in a leptin-independent manner. Yet its participation in the regulation of other biological functions gated by body energy status remains unexplored. We show herein that NUCB2/nesfatin-1 is involved in the control of female puberty. NUCB2/nesfatin mRNA and protein were detected at the hypothalamus of pubertal female rats, with prominent signals at lateral hypothalamus (LHA), paraventricular (PVN), and supraoptic (SON) nuclei. Hypothalamic NUCB2 expression raised along pubertal transition, with detectable elevations of its mRNA levels at LHA, PVN, and SON, and threefold increase of its total protein content between late-infantile and peripubertal periods. Conditions of negative energy balance, such as 48 h fasting or sustained subnutrition, decreased hypothalamic NUCB2 mRNA and/or protein levels in pubertal females. At this age, central administration of nesfatin-1 induced modest but significant elevations of circulating gonadotropins, whose magnitude was notably augmented in conditions of food deprivation. Continuous intracerebroventricular infusion of antisense morpholino oligonucleotides (as-MONs) against NUCB2 along pubertal maturation, which markedly reduced hypothalamic NUCB2 protein content, delayed vaginal opening and decreased ovarian weights and serum luteinizing hormone (LH) levels. In contrast, in adult female rats, intracerebroventricular injection of nesfatin did not stimulate LH or follicle-stimulating hormone secretion; neither did central as-MON infusion alter preovulatory gonadotropin surges, despite suppression of hypothalamic NUCB2. In sum, our data are the first to disclose the indispensable role of NUCB2/nesfatin-1 in the central networks driving puberty onset, a function that may contribute to its functional coupling to energy homeostasis.

Kisspeptins (Kp) have recently emerged as master regulators of the reproductive axis and among the most potent elicitors of GnRH-gonadotropin secretion. Despite their paramount importance in reproductive physiology and their potential therapeutic implications, development of Kp antagonists has remained elusive, and only recently has the first compound with the ability to block Kp actions in vitro and in vivo, namely p234, been reported. However, previous in vivo studies all used acute central injections, whereas characterization of the effects of the antagonist after continuous or systemic administration, which poses pharmacological challenges, is still pending. We report herein a comprehensive series of analyses on the impact of continuous intracerebroventricular infusion of p234 on puberty onset and the preovulatory surge of gonadotropins in the female rat. In addition, the effects of systemic (ip) administration of a tagged p234-penetratin, with a predicted higher permeability at the blood-brain barrier, on Kp-10 induced gonadotropin secretion were evaluated. Central infusion of p234 to pubertal females delayed vaginal opening and decreased uterine and ovarian weights at the expected time of puberty, without affecting body weight. Likewise, chronic intracerebroventricular administration of p234 for 4 d prevented the preovulatory surges of LH and FSH. In addition, systemic (ip) administration of p234-penetratin significantly attenuated acute LH and FSH responses to Kp-10, either after intracerebroventricular or ip injection of Kp. Our data document the validity of p234 for antagonizing Kp actions in vivo and provide direct experimental evidence for the important role of Kp signaling in the key events of female reproduction, such as puberty onset and the preovulatory surge of gonadotropins.

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that operates as sensor of cellular energy status and effector for its coupling to cell growth and proliferation. At the hypothalamic arcuate nucleus, mTOR signaling has been recently proposed as transducer for leptin effects on energy homeostasis and food intake. However, whether central mTOR also participates in metabolic regulation of fertility remains unexplored. We provide herein evidence for the involvement of mTOR in the control of puberty onset and LH secretion, likely via modulation of hypothalamic expression of Kiss1. Acute activation of mTOR by l-leucine stimulated LH secretion in pubertal female rats, whereas chronic l-leucine infusion partially rescued the state of hypogonadotropism induced by food restriction. Conversely, blockade of central mTOR signaling by rapamycin caused inhibition of the gonadotropic axis at puberty, with significantly delayed vaginal opening, decreased LH and estradiol levels, and ovarian and uterine atrophy. Inactivation of mTOR also blunted the positive effects of leptin on puberty onset in food-restricted females. Yet the GnRH/LH system retained their ability to respond to ovariectomy and kisspeptin-10 after sustained blockade of mTOR, ruling out the possibility of unspecific disruption of GnRH function by rapamycin. Finally, mTOR inactivation evoked a significant decrease of Kiss1 expression at the hypothalamus, with dramatic suppression of Kiss1 mRNA levels at the arcuate nucleus. Altogether our results unveil the role of central mTOR signaling in the control of puberty onset and gonadotropin secretion, a phenomenon that involves the regulation of Kiss1 and may contribute to the functional coupling between energy balance and gonadal activation and function.

Summary. The role of N-methyl-D-aspartate (NMDA) and non-NMDA receptors in the control of FSH and LH secretion was analysed in prepubertal male rats. In the first experiment 4, 8, 12, 16, 20 and 30-day-old males were decapitated 15 min after vehicle, NMDA or kainic acid (KA) administration. In the second experiment, 23-day-old males were sham-orchidectomized or orchidectomized. Orchidectomized males were or were not implanted with silastic capsules containing testosterone and were sacrificed on day 30 after injection with vehicle, NMDA (15 mg kg−1), LHRH (100 ng rat−1), NMDA plus LHRH, or MK801, a non-competitive NMDA antagonist, (1 mg kg−1). In the third experiment, 30-day-old intact males pre-treated with Nw-nitro-L-arginine methyl ester (NAME) or NG-methyl-L-arginine (MA), blockers of nitric oxide (NO) synthase, were sacrificed after NMDA or KA administration. In the fourth experiment, the effects of NMDA, KA, LHRH and NAME were analysed in monolayer cultures of dispersed adenohypophyseal cells. We found that: (i) NMDA and KA stimulated LH and FSH secretion in intact males; (ii) the NMDA effect on LH secretion remained after orchidectomy; (iii) FSH and LH responses to LHRH were not affected by simultaneous NMDA administration; (iv) antagonization of NMDA receptors with MK801 reduced the LH secretion in intact and orchidectomized males and blunted the FSH response to orchidectomy; (v) the stimulatory effect of NMDA or KA on LH secretion was not affected by pre-treatment with blockers of NO synthase; (vi) the in vitro LH secretion remained unchanged after administration of NMDA, KA or NAME. We conclude that NMDA and non-NMDA receptors play a physiological role in the control of the basal and post-orchidectomy secretion of gonadotropins, and that this effect is independent of changes in NO generation and does not include changes in pituitary responsiveness to LHRH. FSH—

Attainment of reproductive capacity at puberty relies on a complex series of maturational events that include sexual differentiation of the brain; a hormonally driven phenomenon that takes place at early stages of development (critical period). Alterations of sex steroid milieu during such critical period disrupt pubertal maturation and gonadotropic function later in life, through mechanisms that remain partially unknown. Kisspeptins, products of the KiSS-1 gene acting via G protein-coupled receptor 54, have recently emerged as essential gatekeepers of puberty onset and reproductive function. By using rat models of neonatal administration of estrogenic compounds, we provide herein compelling evidence for the functional impairment of the hypothalamic KiSS-1 system at the time preceding puberty after early inappropriate exposures during brain sex differentiation. Neonatal injection of estradiol benzoate to male and female rats resulted in a dose-dependent decrease in hypothalamic KiSS-1 mRNA levels at the prepubertal stage, linked to lowering of serum LH concentrations. Yet, despite persistently decreased basal gonadotropin levels in estrogenized animals, intracerebral injection of kisspeptin evoked potent LH and FSH secretory responses, similar in magnitude to those of control animals. Estrogenized rats also showed defective levels of hypothalamic KiSS-1 mRNA and circulating gonadotropins in response to gonadectomy, whereas exogenous kisspeptin was capable to enhance further LH and FSH secretion in this model. Finally, protocols of neonatal exposure to high doses of an environmentally relevant estrogen, bisphenol-A, mimicked the effects of estradiol benzoate in terms of hypothalamic expression of KiSS-1 gene at the prepubertal period. Altogether, our data document the sensitivity of the hypothalamic KiSS-1 system to alterations in sex steroid milieu during critical periods of brain sex differentiation, and suggest that lowering of endogenous kisspeptin tone induced by early exposures to xeno-estrogens might be mechanistically relevant for disruption of gonadotropin secretion and puberty onset later in life.

The existence of a close relationship between energy status and reproductive function is well-documented, especially in females, but its underlying mechanisms remain to be fully unfolded. This study aimed to examine the effects of restriction of daily calorie intake, as well as chronic treatments with the metabolic hormones leptin and ghrelin, on the secretion of different reproductive hormones, namely pituitary gonadotropins and prolactin, as well as testosterone, in male rats. Restriction (50%) in daily food intake for 20 days significantly reduced body weight as well as plasma PRL and T levels, without affecting basal LH and FSH concentrations and testicular weight. Chronic administration of leptin to rats fed ad libitum increased plasma PRL levels and decreased circulating T, while it did not alter other hormonal parameters under analysis. In contrast, in rats subjected to 50% calorie restriction, leptin administration increased plasma T levels and reduced testis weight. Conversely, ghrelin failed to induce major hormonal changes but tended to increase testicular weight in fed animals, while repeated ghrelin injections in food-restricted males dramatically decreased plasma LH and T concentrations and reduced testis weight. In sum, we document herein the isolated and combined effects of metabolic stress (50% food restriction) and leptin or ghrelin treatments on several reproductive hormones in adult male rats. Overall, our results further stress the impact and complex way of action of different metabolic cues, such as energy status and key hormones, in reproductive function also in the male.

Adiponectin is an adipocyte hormone, with relevant roles in lipid metabolism and glucose homeostasis, recently involved in the control of different endocrine organs, such as the placenta, pituitary and, likely, the ovary. However, whether as described previously for other adipokines, such as leptin and resistin, adiponectin is expressed and/or conducts biological actions in the male gonad remains unexplored. In this study, we provide compelling evidence for the expression, putative hormonal regulation, and direct effects of adiponectin in the rat testis. Testicular expression of adiponectin was demonstrated along postnatal development, with a distinctive pattern of RNA transcripts and discernible protein levels that appeared mostly located at interstitial Leydig cells. Testicular levels of adiponectin mRNA were marginally regulated by pituitary gonadotropins but overtly modulated by metabolic signals, such as glucocorticoids, thyroxine, and peroxisome proliferator-activated receptor-γ, whose effects were partially different from those on circulating levels of adiponectin. In addition, expression of the genes encoding adiponectin receptor (AdipoR)-1 and AdipoR2 was detected in the rat testis, with developmental changes and gonadotropin regulation for AdipoR2 mRNA, and prominent levels of AdipoR1 in seminiferous tubules. Moreover, recombinant adiponectin significantly inhibited basal and human choriogonadotropin-stimulated testosterone secretion ex vivo, whereas it failed to change relative levels of several Sertoli cell-expressed mRNAs, such as stem cell factor and anti-Müllerian hormone. In summary, our data are the first to document the expression, regulation and functional role of adiponectin in the rat testis. Taken together with its recently reported expression in the ovary and its effects on LH secretion and ovarian steroidogenesis, these results further substantiate a multifaceted role of adiponectin in the control of the reproductive axis, which might operate as endocrine integrator linking metabolism and gonadal function.

Background Ghrelin is a pleiotropic hormone, involved in the control of growth and metabolism, whose circulating levels fluctuate in relation to food intake and body mass index. Ghrelin has been detected in the decidualized endometrium, as well as in human and rat placenta. Methods A total of 106 patients undergoing IVF procedures were prospectively recruited. On Days 16 and 23 after oocyte retrieval, the patients were subjected to blood sampling after overnight fasting, for determination of serum ghrelin, hCGbeta and progesterone levels. In addition, ghrelin levels were assayed in these groups, 2 h after ingestion of a fixed-calorie meal. Results The subjects were divided according to whether they achieved an ongoing pregnancy. On Days 16 and 23 after oocyte retrieval, pre-prandial serum ghrelin levels were not statistically different, although a general trend toward a decrease in circulating ghrelin by Day 23 was detected in pregnant groups. Although in non-conceiving subjects, maternal ghrelin levels showed an expected 15% decline after meal ingestion, such a post-prandial decrease was not statistically significant in pregnant women, selectively on Day 16 after oocyte retrieval. Conclusions Maternal ghrelin levels at early gestational age do not appear to pose diagnostic (as marker) or prognostic value for pregnancy outcome in IVF procedures.

Identification, in late 2003, of inactivating mutations of the G protein-coupled receptor GPR54 as causative factor for absence of puberty and hypogonadotropic hypogonadism in humans and mice was a major breakthrough in modern Neuroendocrinology, and drew considerable interest on the characterization of the roles of this receptor and its ligands (kisspeptins, encoded by the KiSS-1 gene) in the physiological control of essential facets of reproduction. After 3 years of intense research activity, kisspeptins are universally recognized as essential activators of the gonadotropic axis, with key roles in puberty onset and the control of gonadotropin secretion. While these fundamental functions are now well settled, novel aspects of kisspeptin/GPR54 physiology have emerged, including their involvement in the neuroendocrine control of ovulation and the metabolic gating of reproductive function. In addition, the 'comparative endocrinology' of this system has begun to be explored recently. These facets of kisspeptin/GPR54 function, as fundamental gatekeepers of reproduction, will be comprehensively reviewed herein.

Different signals with key roles in energy homeostasis regulate the reproductive axis. These include neuropeptide Y and polypeptide YY3-36, whose type Y2 receptor is the most abundant of this family in the brain. We evaluated herein the putative roles of Y2 receptors in the control of gonadotropin secretion by means of central administration of PYY13-36 (agonist of Y2 receptors) and BIIE 0246 (antagonist of Y2 receptors) to intact and orchidectomized male rats. In addition, the ability of PYY13-36 to elicit GnRH and gonadotropin secretion in vitro and the impact of fasting on LH responses to PYY13-36 in vivo were also monitored. Central administration of PYY13-36 significantly decreased the circulating levels of both gonadotropins, an effect that was observed in prepubertal and adult rats. Yet a dual action of Y2 receptors in the control of male gonadotropic axis was evidenced as their activation induced 1) stimulation of gonadotropin responses to GnRH at the pituitary but 2) inhibition of GnRH secretion at the hypothalamus. Antagonization of Y2 receptors failed to modify basal LH secretion in intact males either after being fed ad libitum or after being fasted. In contrast, their central blockade in orchidectomized rats evoked a significant increase in circulating LH and FSH level, suggesting the constitutive activation of Y2 receptor in such stimulated conditions. In summary, our data evidence a complex mode of action of Y2 receptors in the control of gonadotropic axis, with stimulatory and inhibitory actions at different levels of the system that are sensitive to the gonadal status.