Hypothalamic miR-30 regulates puberty onset via repression of the puberty-suppressing factor, Mkrn3.

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.

Metabolic regulation of female puberty via hypothalamic AMPK-kisspeptin signaling.

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.

An alternative transcription start site yields estrogen-unresponsive Kiss1 mRNA transcripts in the hypothalamus of prepubertal female rats.

The importance of the Kiss1 gene in the control of reproductive development is well documented. However, much less is known about the transcriptional regulation of Kiss1 expression in the hypothalamus. Critical for these studies is an accurate identification of the site(s) where Kiss1 transcription is initiated. Employing 5'-RACE PCR, we detected a transcription start site (TSS1) used by the hypothalamus of rats, mice, nonhuman primates and humans to initiate Kiss1 transcription. In rodents, an exon 1 encoding 5'-untranslated sequences is followed by an alternatively spliced second exon, which encodes 5'-untranslated regions of two different lengths and contains the translation initiation codon (ATG). In nonhuman primates and humans, exon 2 is not alternatively spliced. Surprisingly, in rat mediobasal hypothalamus (MBH), but not preoptic area (POA), an additional TSS (TSS2) located upstream from TSS1 generates an exon 1 longer (377 bp) than the TSS1-derived exon 1 (98 bp). The content of TSS1-derived transcripts increased at puberty in the POA and MBH of female rats. It also increased in the MBH after ovariectomy, and this change was prevented by estrogen. In contrast, no such changes in TSS2-derived transcript abundance were detected. Promoter assays showed that the proximal TSS1 promoter is much more active than the putative TSS2 promoter, and that only the TSS1 promoter is regulated by estrogen. These differences appear to be related to the presence of a TATA box and binding sites for transcription factors activating transcription and interacting with estrogen receptor-α in the TSS1, but not TSS2, promoter.

Role of neurokinin B in the control of female puberty and its modulation by metabolic status.

Human genetic studies have revealed that neurokinin B (NKB) and its receptor, neurokinin-3 receptor (NK3R), are essential elements for normal reproduction; however, the precise role of NKB-NK3R signaling in the initiation of puberty remains unknown. We investigated here the regulation of Tac2 and Tacr3 mRNAs (encoding NKB and NK3R, respectively) in female rats and demonstrated that their hypothalamic expression is increased along postnatal maturation. At puberty, both genes were widely expressed throughout the brain, including the lateral hypothalamic area and the arcuate nucleus (ARC)/medial basal hypothalamus, where the expression of Tacr3 increased across pubertal transition. We showed that central administration of senktide (NK3R agonist) induced luteinizing hormone (LH) secretion in prepubertal and peripubertal females. Conversely, chronic infusion of an NK3R antagonist during puberty moderately delayed the timing of vaginal opening (VO) and tended to decrease LH levels. The expression of NKB and its receptor was sensitive to changes in metabolic status during puberty, as reflected by a reduction in Tacr3 (and, to a lesser extent, Tac2) expression in the ARC after a 48 h fast. Yet, acute LH responses to senktide in pubertal females were preserved, if not augmented, under fasting conditions, suggesting sensitization of the NKB-NK3R-gonadotropin-releasing hormone signaling pathway under metabolic distress. Moreover, repeated administration of senktide to female rats with pubertal arrest due to chronic undernutrition rescued VO (in ∼50% of animals) and potently elicited LH release. Altogether, our observations suggest that NKB-NK3R signaling plays a role in pubertal maturation and that its alterations may contribute to pubertal disorders linked to metabolic stress and negative energy balance.

A system biology approach to identify regulatory pathways underlying the neuroendocrine control of female puberty in rats and nonhuman primates.

This article is part of a Special Issue "Puberty and Adolescence". Puberty is a major developmental milestone controlled by the interaction of genetic factors and environmental cues of mostly metabolic and circadian nature. An increased pulsatile release of the decapeptide gonadotropin releasing hormone (GnRH) from hypothalamic neurosecretory neurons is required for both the initiation and progression of the pubertal process. This increase is brought about by coordinated changes that occur in neuronal and glial networks associated with GnRH neurons. These changes ultimately result in increased neuronal and glial stimulatory inputs to the GnRH neuronal network and a reduction of transsynaptic inhibitory influences. While some of the major players controlling pubertal GnRH secretion have been identified using gene-centric approaches, much less is known about the system-wide control of the overall process. Because the pubertal activation of GnRH release involves a diversity of cellular phenotypes, and a myriad of intracellular and cell-to-cell signaling molecules, it appears that the overall process is controlled by a highly coordinated and interactive regulatory system involving hundreds, if not thousands, of gene products. In this article we will discuss emerging evidence suggesting that these genes are arranged as functionally connected networks organized, both internally and across sub-networks, in a hierarchical fashion. According to this concept, the core of these networks is composed of transcriptional regulators that, by directing expression of downstream subordinate genes, provide both stability and coordination to the cellular networks involved in initiating the pubertal process. The integrative response of these gene networks to external inputs is postulated to be coordinated by epigenetic mechanisms.

Metabolic regulation of kisspeptin.

Body energy balance and metabolic signals are important modulators of puberty and reproductive function, so that perturbations of metabolism and energy reserves (ranging from persistent energy insufficiency to morbid obesity) are frequently linked to reproductive disorders. The mechanisms for the tight association between body metabolic state and reproduction are multifaceted, and likely involve numerous peripheral hormones and central transmitters. In recent years, a prominent role of kisspeptins in the central pathways responsible for conveying metabolic information into the brain centers responsible for reproductive control, and specifically GnRH neurons, has been proposed on the basis of a wealth of expression and functional data. In this chapter, we will summarize such evidence, with special attention to the potential (direct and/or indirect) interaction of leptin and kisspeptin pathways. In addition, other potential metabolic modulators of kisspeptin signaling, as well as some of the putative molecular mechanisms for the metabolic regulation of Kiss1 will be briefly reviewed. Conflictive data, including those questioning an essential role of Kiss1 neurons in mediating leptin effects on the reproductive axis, will be also discussed. All in all, we aim to provide an integral and balanced view of the physiological relevance and potential mechanisms for the metabolic control of the kisspeptin system, as important pathway for the integral regulation of energy balance, puberty onset, and fertility.

First-in-human evaluation of a biological regenerative vascular conduit for hemodialysis access.

INTRODUCTION: The long-term survival and low complication rate of autogenous fistulas for hemodialysis access is often offset by early thrombosis and slow or failed maturation leading to the use of central venous catheters. A regenerative material may have the potential to overcome these limitations. A completely biological acellular vascular conduit was investigated in this first-in-human clinical study. METHODS: With approval of the ethics board and patients' informed consent, five subjects were enrolled based on predetermined inclusion criteria. Five patients underwent implant of a novel acellular, biological tissue conduit (TRUE AVC™) in the upper arm in a curved configuration between brachial artery and axillary vein. After maturation, standard dialysis was commenced through the new access. Patients were followed up to 26 weeks with ultrasound and physical exam. Serum samples were evaluated for an immune response to the novel allogeneic human tissue implant. RESULTS: This new tissue conduit handled well surgically, with properties similar to that of native human vein. Post procedure conduit flow was excellent in all cases, averaging 1098 ± 388 ml/min at week 4 and remaining stable through 1248 ± 355 ml/min at 26 weeks. Surgical site healing was normal with no edema or erythema by week 4. Six-month primary assisted patency was 80% and secondary patency was 100%. Prescribed dialysis was successfully delivered without infection, and there was no significant change in conduit diameter. Serum testing showed no increase in PRA or IgG specific to the TRUE AVC. One implant required intervention at 5 months with thrombectomy and covered stent procedure. CONCLUSION: This first-in-human 6-month study with favorable patency and low complication rate establishes the initial safety and feasibility of this novel biological tissue conduit for dialysis access in patients with end-stage kidney disease. Its mechanical durability and lack of immune response establishes TRUE AVC as a potential regenerative material for clinical use.

Differential modulation of gonadotropin responses to kisspeptin by aminoacidergic, peptidergic, and nitric oxide neurotransmission.

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 GABA(A) receptors decreased Kp-induced gonadotropin secretion, whereas their blockade elicited robust LH and FSH bursts and protracted responses to Kp-10 when combined with GABA(B) 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.

The anorexigenic neuropeptide, nesfatin-1, is indispensable for normal puberty onset in the female rat.

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.

Interactions between kisspeptin and neurokinin B in the control of GnRH secretion in the female rat.

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 (E(2)), 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 E(2) on the expression of NKB and NK3R mRNAs in the Arc and found that E(2) 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 E(2) 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 E(2)-dependent negative feedback in the rat.

Central Ceramide Signaling Mediates Obesity-Induced Precocious Puberty.

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.

Environmentally Relevant Perinatal Exposures to Bisphenol A Disrupt Postnatal Kiss1/NKB Neuronal Maturation and Puberty Onset in Female Mice.

BACKGROUND: The timing of puberty is highly sensitive to environmental factors, including endocrine disruptors. Among them, bisphenol A (BPA) has been previously analyzed as potential modifier of puberty. Yet, disparate results have been reported, with BPA advancing, delaying, or being neutral in its effects on puberty onset. Likewise, mechanistic analyses addressing the central and peripheral actions/targets of BPA at puberty remain incomplete and conflictive. OBJECTIVE: We aimed to provide a comprehensive characterization of the impact of early BPA exposures, especially at low, real-life doses, on the postnatal development of hypothalamic Kiss1/NKB neurons, and its functional consequences on female pubertal maturation. METHODS: Pregnant CD1 female mice were orally administered BPA at 5, 10, or 40µg/kg body weight (BW)/d from gestational day 11 to postnatal day 8 (PND8). Vaginal opening, as an external marker of puberty onset, was monitored daily from PND19 to PND30 in the female offspring. Blood and brain samples were collected at PND12, 15, 18, 21, and 30 for measuring circulating levels of gonadotropins and analyzing the hypothalamic expression of Kiss1/kisspeptin and NKB. RESULTS: Perinatal exposure to BPA, in a range of doses largely below the no observed adverse effect level (NOAEL; 5mg/kg BW/d, according to the FDA), was associated with pubertal differences in the female progeny compared with those exposed to vehicle alone, with an earlier age of vaginal opening but consistently lower levels of circulating luteinizing hormone. Mice treated with BPA exhibited a persistent, but divergent, impairment of Kiss1 neuronal maturation, with more kisspeptin cells in the rostral (RP3V) hypothalamus but consistently fewer kisspeptin neurons in the arcuate nucleus (ARC). Detailed quantitative analysis of the ARC population, essential for pubertal development, revealed that mice treated with BPA had persistently lower Kiss1 expression during (pre)pubertal maturation, which was associated with lower Tac2 (encoding NKB) levels, even at low doses (5µg/kg BW/d), in the range of the tolerable daily intake (TDI), recently updated by the European Food Safety Authority. CONCLUSIONS: Our data attest to the consistent, but divergent, effects of gestational exposures to low concentrations of BPA, via the oral route, on phenotypic and neuroendocrine markers of puberty in female mice, with an unambiguous impact on the developmental maturation not only of Kiss1, but also of the NKB system, both essential regulators of puberty onset. https://doi.org/10.1289/EHP5570.

Thermoneutrality improves skeletal impairment in adult Prader-Willi syndrome mice.

Human Prader-Willi syndrome (PWS) is characterised by impairments of multiple systems including the growth hormone (GH) axis and skeletal growth. To address our lack of knowledge of the influence of PWS on skeletal integrity in mice, we have characterised the endocrine and skeletal phenotype of the PWS-ICdel mouse model for "full" PWS and determined the impact of thermoneutrality. Tibial length, epiphyseal plate width and marrow adiposity were reduced by 6%, 18% and 79% in male PWS-ICdel mice, with osteoclast density being unaffected. Similar reductions in femoral length accompanied a 32% reduction in mid-diaphyseal cortical diameter. Distal femoral Tb.N was reduced by 62%, with individual trabeculae being less plate-like and the lattice being more fragmented (Tb.Pf increased by 63%). Cortical strength (Ultimate moment) was reduced by 26% as a result of reductions in calcified tissue strength and the geometric contribution. GH and prolactin contents in PWS-ICdel pituitaries were reduced in proportion to their smaller pituitary size, with circulating IGF-1 concentration reduced by 37-47%. Conversely, while pituitary LH content was halved, circulating gonadotropin concentrations were unaffected. Although longitudinal growth, marrow adiposity and femoral geometry were unaffected by thermoneutrality, strengthened calcified tissue reversed weakened cortex of PWS-ICdel femora. While underactivity of the GH-axis may be due to loss of Snord116 expression and impaired limb bone geometry and strength due to loss of Magel2 expression, comprehensive analysis of skeletal integrity in the single gene deletion models is required. Our data imply that thermoneutrality may ameliorate the elevated fracture risk associated with PWS.

Gonadal hormone-dependent vs. -independent effects of kisspeptin signaling in the control of body weight and metabolic homeostasis.

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.

Intergenerational Influence of Paternal Obesity on Metabolic and Reproductive Health Parameters of the Offspring: Male-Preferential Impact and Involvement of Kiss1-Mediated Pathways.

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.

Expression of hypothalamic KiSS-1 system and rescue of defective gonadotropic responses by kisspeptin in streptozotocin-induced diabetic male rats.

Hypogonadotropism is a common feature of uncontrolled diabetes, for which the ultimate mechanism remains to be elucidated. Kisspeptins, ligands of G protein-coupled receptor 54 (GPR54) encoded by the KiSS-1 gene, have recently emerged as major gatekeepers of the gonadotropic axis. Alteration in the hypothalamic KiSS-1 system has been reported in adverse metabolic conditions linked to suppressed gonadotropins, such as undernutrition. However, its potential contribution to defective gonadotropin secretion in diabetes has not been evaluated. We report herein analyses of luteinizing hormone (LH) responses to kisspeptin and hypothalamic expression of the KiSS-1 gene in streptozotocin (STZ)-induced diabetic male rats. In addition, functional studies involving kisspeptin replacement or continuous administration of leptin and insulin to diabetic male rats are presented. Kisspeptin administration evoked robust LH and testosterone bursts and enhanced postgonadectomy LH concentrations, despite prevailing attenuation of gonadotropic axis in diabetic animals. In addition, hypothalamic KiSS-1 mRNA levels were unambiguously decreased in diabetic male rats, and the postorchidectomy rise in KiSS-1 mRNA was severely blunted. Repeated administration of kisspeptin to diabetic rats evoked persistent LH and testosterone responses and partially rescued prostate and testis weights. In addition, central infusion of leptin, but not insulin, was sufficient to normalize hypothalamic KiSS-1 mRNA levels, as well as LH and testosterone concentrations. In summary, we provide evidence for altered expression of the hypothalamic KiSS-1 system in a model of uncontrolled diabetes. This observation, together with the ability of exogenous kisspeptin to rescue defective LH responses in diabetic rats, unravel the physiopathological implication, and potential therapeutic intervention, of the KiSS-1 system in altered gonadotropin secretion of type 1 diabetes.

Development and validation of a method for precise dating of female puberty in laboratory rodents: The puberty ovarian maturation score (Pub-Score).

Puberty is a key developmental event whose primary regulatory mechanisms remain poorly understood. Precise dating of puberty is crucial for experimental (preclinical) studies on its complex neuroendocrine controlling networks. In female laboratory rodents, external signs of puberty, such as vaginal opening (VO) and epithelial cell cornification (i.e., first vaginal estrus, FE), are indirectly related to the maturational state of the ovary and first ovulation, which is the unequivocal marker of puberty. Whereas in rats, VO and FE are almost simultaneous with the first ovulation, these events are not so closely associated in mice. Moreover, external signs of puberty can be uncoupled with first ovulation in both species under certain experimental conditions. We propose herein the Pubertal Ovarian Maturation Score (Pub-score), as novel, reliable method to assess peripubertal ovarian maturation in rats and mice. This method is founded on histological evaluation of pre-pubertal ovarian maturation, based on antral follicle development, and the precise timing of first ovulation, by retrospective dating of maturational and regressive changes in corpora lutea. This approach allows exact timing of puberty within a time-window of at least two weeks after VO in both species, thus facilitating the identification and precise dating of advanced or delayed puberty under various experimental conditions.

Metabolic control of female puberty: potential therapeutic targets.

INTRODUCTION: The onset of puberty in females is highly sensitive to the nutritional status and the amount of energy reserves of the organism. This metabolic information is sensed and transmitted to hypothalamic GnRH neurons, considered to be ultimately responsible for triggering puberty through the coordinated action of different peripheral hormones, central neurotransmitters, and molecular mediators. AREAS COVERED: This article will review and discuss (i) the relevant actions of the adipose hormone leptin, as a stimulatory/permissive signal, and the gut hormone ghrelin, as an inhibitory factor, in the metabolic control of female puberty; (ii) the crucial role of the hypothalamic kisspeptin neurons, recently emerged as essential gatekeepers of puberty, in transmitting this metabolic information to GnRH neurons; and (iii) the potential involvement of key cellular energy sensors, such as mTOR, as molecular mediators in this setting. EXPERT OPINION: The thorough characterization of the physiological roles of the above elements in the metabolic control of female puberty, along with the discovery of novel factors, pathways, and mechanisms involved, will promote our understanding of the complex networks connecting metabolism and puberty and, ultimately, will aid in the design of target-specific treatments for female pubertal disorders linked to conditions of metabolic stress.

Animal Modeling of Early Programming and Disruption of Pubertal Maturation.

Puberty is a fascinating developmental transition that gates the attainment of reproductive capacity and culminates the somatic and sexual maturation of the organism. Rather than a circumscribed phenomenon, puberty is the endpoint of a long-lasting developmental continuum, which initiates in utero. Besides important genetic determinants, the tempo of puberty is influenced by numerous endogenous and exogenous factors that, acting at different levels of the developing hypothalamic-pituitary-gonadal (HPG) axis along the maturational continuum indicated above, can influence puberty onset. Among the different modifiers of puberty, in this chapter we will focus our attention on two major groups of signals, sex steroids and nutritional cues, and how these interplay mostly with the central elements of the HPG axis, and especially with gonadotropin-releasing hormone neurons and their key upstream afferents, Kiss1 neurons, to influence the timing of puberty. Special emphasis will be given to summarize information emerging from relevant preclinical (mostly rodent) animal models, and how this information might be relevant in terms of translational medicine, as it may help for a better understanding and eventually management of pubertal disorders of escalating prevalence worldwide.