Analysis of the roles of neuropeptidergic system and micrornas in the control of puberty

La pubertad es la culminación de una cascada de eventos del desarrollo que conduce a la activación del eje hipotálamo-hipófiso-gonadal (HHG) y la adquisición de la capacidad reproductora . Dicha activación es desencadenada por un aumento en la secreción pulsátil de GnRH (hormona liberadora de gonadotropinas). Desde el punto de vista neuroendocrino, la adquisición y posterior mantenimiento de la función reproductora está controlada por un sofisticado sistema de regulación integrado por múltiples señales de origen central y periférico que actúan de forma coordinada sobre las neuronas GnRH, consideradas elemento clave en la integración final de dichas señales. En este contexto, cabe mencionar que la activación del eje reproductor en la pubertad, y el posterior mantenimiento de la función reproductora, depende críticamente del grado de reservas energéticas del organismo . En los últimos años, se ha profundizado en la caracterización de los mecanismos por los que el balance energético y el peso corporal influyen en el desarrollo puberal. Entre ellos, destaca el papel esencial de la leptina en la señalización de la magnitud de las reservas energéticas del organismo sobre los centros responsables de la activación del eje reproductor en pubertad . Sin embargo, a pesar del papel clave de las neuronas GnRH en el control metabólico del eje HHG, diferentes estudios sugieren que no existe una interacción directa entre las neuronas GnRH y la señalización por leptina . Los principales centros neuronales sugeridos como mediadores de los efectos de la leptina en las neuronas GnRH están localizados en el núcleo arcuato del hipotálamo (ARC) y expresan, entre otros péptidos POMC, NPY y kisspeptinas (neuropéptido esencial en la regulación de la función reproductora). Estudios recientes han señalado a las neuronas POMC, concretamente a al neuro-péptido derivado del gen POMC, -MSH, como mediador de los efectos anorexigénicos de la leptina, vía interacción con los receptores 3 y 4 de melanocortinas (MC3-R y MC4-R) . Además, también recientemente, estudios en oveja sugieren una posible interacción entre -MSH y kisspeptinas en la edad adulta . Sin embargo, el papel de -MSH en la regulación de la pubertad, y su posible interacción con kisspeptinas, son permanecen en gran medida desconocidos. Por otro lado, durante los últimos años, se han llevado a cabo numerosos trabajos con el fin de identificar los mecanismos responsables, en última instancia, del control de inicio de la pubertad. En este contexto, se ha planteado que la llegada a la pubertad está controlada por un grupo de genes jerárquicamente dispuestos dentro de redes conectadas funcionalmente, donde GnRH es el efector final. Algunos estudios publicados recientemente sugieren que diversos mecanismos epigenéticos, que incluyen tentativamente las acciones de RNAs pequeños, no codificantes (llamados globalmente microRNAs o miRNAs) podría modular la expresión de dichos genes, actuando como un sistema regulador capaz de integrar las diferentes redes implicadas en el control de la pubertad. Es sentido, en 2009, se publicaron una serie de estudios independientes de asociación del genoma completo (GWAS) que relacionaban la edad de menarquía con variaciones en el locus LIN28B, o alrededor de este . Uno de las principales funciones de Lin28b es la de inhibidor de la maduración de los miRNAs de la familia let-7 . Además, posteriormente, se demostró que la sobreexpresión de Lin28a en ratones conlleva un retraso en la edad de apertura vaginal, signo externo de la llegada a pubertad, y un retraso en el desarrollo sexual de estos animales, determinado por un menor peso del útero y ovarios respecto, a pesar de un mayor crecimiento y peso corporal . Sin embargo, a pesar de las evidencias indirectas que sugieren la implicación potencial del sistema Lin28/let-7 en el control de la edad de menarquia, se conoce relativamente poco sobre acerca de su función y sitios de acción en el control de la maduración puberal. En este contexto, el objetivo general de esta Tesis fue caracterizar el papel de ciertos sistemas neuropeptidérgios, concretamente -MSH y kisspeptinas, y del sistema Lin28/let-7 en el control fisiológico de la pubertad

Perturbation of hypothalamic MicroRNA expression patterns in male rats after metabolic distress: impact of obesity and conditions of negative energy balance

[Abstract] The hypothalamus plays a crucial role in body weight homeostasis through an intricate network of neuronal circuits that are under the precise regulation of peripheral hormones and central transmitters. Although deregulated function of such circuits might be a major contributing factor in obesity, the molecular mechanisms responsible for the hypothalamic control of energy balance remain partially unknown. MicroRNAs (miRNAs) have been recognized as key regulators of different biological processes, including insulin sensitivity and glucose metabolism. However, the roles of miRNA pathways in the control of metabolism have been mostly addressed in peripheral tissues, whereas the potential deregulation of miRNA expression in the hypothalamus in conditions of metabolic distress remains as yet unexplored. In this work, we used high-throughput screening to define to what extent the hypothalamic profiles of miRNA expression are perturbed in two extreme conditions of nutritional stress in male rats, namely chronic caloric restriction and high-fat diet–induced obesity. Our analyses allowed the identification of sets of miRNAs, including let-7a, mir-9*, mir-30e, mir-132, mir-145, mir-200a, and mir-218, whose expression patterns in the hypothalamus were jointly altered by caloric restriction and/or a high-fat diet. The predicted targets of these miRNAs include several elements of key inflammatory and metabolic pathways, including insulin and leptin. Our study is the first to disclose the impact of nutritional challenges on the hypothalamic miRNA expression profiles. These data will help to characterize the molecular miRNA signature of the hypothalamus in extreme metabolic conditions and pave the way for targeted mechanistic analyses of the involvement of deregulated central miRNAs pathways in the pathogenesis of obesity and related disorders.Instituto de Salud Carlos III; PI10/00088Ministerio de Economia y Competitividad; IN845B-2010/187Instituto de Salud Carlos III; PI13/00322FISXunta de Galicia; 10CSA916014PRXunta de Galicia; EM2013/011Ministerio de Ciencia e Innovación; BFU 2011-2502

Testicular expression of the Lin28/let-7 system: hormonal regulation and changes during postnatal maturation and after manipulations of puberty

[Abstract] The Lin28/let-7 system, which includes the RNA-binding proteins, Lin28a/Lin28b, and let-7 miRNAs, has emerged as putative regulator of puberty and male gametogenesis; yet, its expression pattern and regulation in postnatal testis remain ill defined. We report herein expression profiles of Lin28 and let-7 members, and related mir-145 and mir-132, in rat testis during postnatal maturation and in models of altered puberty and hormonal deregulation. Neonatal expression of Lin28a and Lin28b was low and rose markedly during the infantile period; yet, expression patterns diverged thereafter, with persistently elevated levels only for Lin28b, which peaked at puberty. Let-7a, let-7b, mir-132 and mir-145 showed profiles opposite to Lin28b. In fact, let-7b and mir-145 were abundant in pachytene spermatocytes, but absent in elongating spermatids, where high expression of Lin28b was previously reported. Perturbation of puberty by neonatal estrogenization reverted the Lin28/let-7 expression ratio; expression changes were also detected in other models of delayed puberty, due to early photoperiod or nutritional manipulations. In addition, hypophysectomy or growth hormone (GH) deficiency revealed regulation of this system by gonadotropins and GH. Our data document the expression profiles of the Lin28/let-7 system in rat testis along postnatal/pubertal maturation, and their perturbation in models of pubertal and hormonal manipulation.Ministerio de Economía y Competitividad; BFU2011-025021Ministerio de Economía y Competitividad; BFU2014-57581Junta de Andalucía; P08-CVI-03788Junta de Andalucía; P12-FQM-01943Xunta de Galicia; EM2013/01

Distinct expression patterns predict differential roles of the miRNA-binding proteins, Lin28 and Lin28b, in the mouse testis: studies during postnatal development and in a model of hypogonadotropic hypogonadism

[Abstract] Lin28 (also termed Lin28a) and Lin28b are related RNA-binding proteins, involved in the control of microRNA synthesis, especially of the let-7 family, with putative functions in early (embryo) development. However, their roles during postnatal maturation remain ill defined. Despite the general assumption that Lin28 and Lin28b share similar targets and functions, conclusive demonstration of such redundancy is still missing. In addition, recent observations suggest a role of Lin28 proteins in mammalian reproduction, which is yet to be defined. We document herein the patterns of RNA expression and protein distribution of Lin28 and Lin28b in mouse testis during postnatal development and in a model of hypogonadotropic hypogonadism as a result of inactivation of the kisspeptin receptor, Gpr54. Lin28 and Lin28b mRNAs were expressed in mouse testis across postnatal maturation, but their levels disparately varied between neonatal and pubertal periods, with peak Lin28 levels in infantile testes and sustained elevation of Lin28b mRNA in young adult male gonads, where relative levels of let-7a and let-7b miRNAs were significantly suppressed. In addition, Lin28 peptides displayed totally different patterns of cellular distribution in mouse testis: Lin28 was located in undifferentiated and type-A1 spermatogonia, whereas Lin28b was confined to spermatids and interstitial Leydig cells. These profiles were perturbed in Gpr54 null mouse testis, which showed preserved but irregular Lin28 signal and absence of Lin28b peptide, which was rescued by administration of gonadotropins, mainly hCG (as super-agonist of LH). In addition, increased relative levels of Lin28, but not Lin28b, mRNA and of let-7a/let-7b miRNAs were observed in Gpr54 KO mouse testes. Altogether, our data are the first to document the divergent patterns of cellular distribution and mRNA expression of Lin28 and Lin28b in the mouse testis along postnatal maturation and their alteration in a model of congenital hypogonadotropic hypogonadism. Our findings suggest distinct functional roles of these two related, but not overlapping, miRNA-binding proteins in the male gonad.Ministerio de Ciencia e Innovación; BFU 2008-00984Ministerio de Ciencia e Innovación; BFU2011-25021Junta de Andalucía; Project P08-CVI-03788European Commission; DEER FP7-ENV-2007-

Changes in hypothalamic expression of the Lin28/let-7 system and related MicroRNAs during postnatal maturation and after experimental manipulations of puberty

[Abstract] Lin28 and Lin28b are related RNA-binding proteins that inhibit the maturation of miRNAs of the let-7 family and participate in the control of cellular stemness and early embryonic development. Considerable interest has arisen recently concerning other physiological roles of the Lin28/let-7 axis, including its potential involvement in the control of puberty, as suggested by genome-wide association studies and functional genomics. We report herein the expression profiles of Lin28 and let-7 members in the rat hypothalamus during postnatal maturation and in selected models of altered puberty. The expression patterns of c-Myc (upstream positive regulator of Lin28), mir-145 (negative regulator of c-Myc), and mir-132 and mir-9 (putative miRNA repressors of Lin28, predicted by bioinformatic algorithms) were also explored. In male and female rats, Lin28, Lin28b, and c-Myc mRNAs displayed very high hypothalamic expression during the neonatal period, markedly decreased during the infantile-to-juvenile transition and reached minimal levels before/around puberty. A similar puberty-related decline was observed for Lin28b in monkey hypothalamus but not in the rat cortex, suggesting species conservation and tissue specificity. Conversely, let-7a, let-7b, mir-132, and mir-145, but not mir-9, showed opposite expression profiles. Perturbation of brain sex differentiation and puberty, by neonatal treatment with estrogen or androgen, altered the expression ratios of Lin28/let-7 at the time of puberty. Changes in the c-Myc/Lin28b/let-7 pathway were also detected in models of delayed puberty linked to early photoperiod manipulation and, to a lesser extent, postnatal underfeeding or chronic subnutrition. Altogether, our data are the first to document dramatic changes in the expression of the Lin28/let-7 axis in the rat hypothalamus during the postnatal maturation and after different manipulations that disturb puberty, thus suggesting the potential involvement of developmental changes in hypothalamic Lin28/let-7 expression in the mechanisms permitting/leading to puberty onset.Ministerio de Economia y Competitividad; BFU 2008-00984Ministerio de Economia y Competitividad; BFU 2011-25021Junta de Andalucía; P08-CVI-03788United States. National Institutes of Health HD025123-ARRAUnited States. National Science Foundation; IOS1121691Instituto de Salud Carlos III; PI10/00088Xunta de Galicia; IN845B-2010/187Xunta de Galicia; 10CSA916014P

Defining a novel leptin–melanocortin–kisspeptin pathway involved in the metabolic control of puberty

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 onsetThis work was supported by grants BFU2011-025021 & BFU2014-57581-P (Ministerio de Economía y Competitividad, Spain; co-funded with EU funds from FEDER Program); project PIE-00005 (Flexi-Met, Instituto de Salud Carlos III, Ministerio de Sanidad, Spain); Projects P08-CVI-03788 and P12-FQM-01943 (Junta de Andalucía, Spain); EU research contract DEER FP7-ENV-2007-1 and the New Zealand Health Research Council. CIBER Fisiopatología de la Obesidad y Nutrición is an initiative of Instituto de Salud Carlos III. Senior authors are indebted with Dr. R.A. Steiner (University of Washington, Seattle, USA) and Dr. U. Boehm (University of Saarland School of Medicine, Homburg, Germany) for provision of relevant mouse lines, essential for conduction of some of the experiments included in this studyS

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

[Abstract] 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.Ministerio de Ciencia e Innovación; BFU 2008-00984Ministerio de Ciencia e Innovación; BFU 2011-25021Andalucía. Junta, P08-CVI-0060

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