MicroRNAs and GRK2 as modulators of Kiss1/GPR54 system: Physiopathological role in pubertal alterations and obesity induced hypogonadism

The reproductive function is governed by the so-called hypothalamic-pituitary-gonadal (HPG) axis, where an intricated network of central, peripheral and external factors determine hormonal balance and proper functioning of the reproductive system and the gonadal function, guaranteeing the perpetuation of the species 1–3. In recent years, it has been documented that a plethora of central (glutamate, GABA, NKB, NPY) 4–7, peripheral (insulin, leptin, ghrelin) 8,9 and external (nutritional availability, endocrine disruptors, circadian rhythms) 10–13 cues converge (either acting directly or indirectly) onto Kiss1 neurons in the hypothalamus, as major signaling hub of the HPG axis 14, whose products, kisspeptins, act on the GnRH neurons, via its canonical receptor, GPR5415, activating puberty onset and reproductive function. In addition, it is well recognized that reproductive function is altered under conditions of metabolic distress, ranging from subnutrition to obesity, type 2 diabetes and metabolic syndrome, which are bound to numerous perturbations, including disordered puberty, central hypogonadism (mainly in males) and cardiometabolic impairment16,17. MicroRNAs have been recently pointed out as essential players in the control of normal pubertal development18–21, although no study has addressed the specific regulation of Kiss1, at central levels, exerted by miRNAs 21,22. Further, the effects of miRNAs in the pathogenesis of central hypogonadism are completely unexplored. In parallel, the Kiss1/GPR54 system is a key element for the integration of the energetic status and reproductive capacity 23, where GPR54 inactivating mutations were described decades ago as underlying origin of hypogonadotropic hypogonadism 24,25. The G-protein coupled receptor kinase 2 (GRK2) 26, which is largely recognized as pleiotropic regulator of cellular signaling 27–29, has been suggested in vitro as a modulator of GPR5426. Nevertheless, no studies had addressed to date its potential roles in proper pubertal development and maintenance of reproductive capacity. In the above context, this Doctoral Thesis has addressed, as main aims, (i) the putative role of specific miRNAs in the physiological control of puberty via regulation of the Kiss1 system; (ii) the pathophysiological role of miRNAs in obesity-induced hypogonadism (OIH), their interplay with Kiss1 and their potential therapeutic implications; (iii) the role of GRK2 in the control of puberty and the HPG axis through regulation of GPR54 in normal conditions and under nutritional stress; and (iv) the implication of GRK2 in OIH through GPR54 regulation.La función reproductora está determinada por el correcto funcionamiento del eje hipotálamohipofiso-gonadal (HHG), donde una compleja red de factores centrales, periféricos y externos determinan el balance hormonal necesario para la adquisición de la capacidad reproductora y, en consecuencia, para el mantenimiento de las especies 1–3. De este modo, se ha documentado que una multitud de factores centrales (GABA, glutamato, NKB, NPY) 4–7, periféricos (leptina, insulina o ghrelina) 8,9 y externos (disruptores endocrinos, aporte energético, ritmos circadianos) 10–13 convergen (actuando directa o indirectamente) en las neuronas Kiss1 hipotalámicas como principal núcleo del eje HHG 14 y cuyo producto, las kisspeptinas, transmitirán información a la neurona GnRH, por medio de su receptor canónico, GPR5415 , activando la pubertad y la función reproductora. Al mismo tiempo, existen evidencias sobre la afectación de la función reproductora como consecuencia del desequilibrio homeostático presente en situaciones como la subnutrición o la obesidad, diabetes mellitus tipo 2 o síndrome metabólico, que se encuentran vinculadas a numerosos desórdenes, incluyendo alteraciones de la edad de pubertad, el hipogonadismo central (principalmente masculino) y la enfermedad cardiovascular 16,17. Datos recientes señalan que los microRNAs (miRNAs) son elementos implicados en la correcta transición puberal 18–21, aunque existen pocos estudios dirigidos a evaluar al papel de los miRNAs en la regulación específica de la expresión de Kiss1, a nivel central19,22. Además, la desregulación de determinados miRNAs en condiciones de hipogonadismo central, su impacto sobre el sistema Kiss1 y su implicación fisiopatológica en esta condición, permanecen inexplorados. En paralelo, el sistema Kiss1/GPR54 es un elemento clave en la integración del estado energético y la capacidad reproductora23, estando descrito que mutaciones inactivantes en GPR54 son una causa subyacente en determinados casos de hipogonadismo hipogonadotropo 24,25. La quinasa de receptor acoplado a proteína G (GRK2) 26 está reconocida como un regulador pleiotrópico de la señalización celular 27–29 y ha sido demostrada su capacidad para regular GPR54, in vitro 26. En cualquier caso, no existen estudios relacionados con su potencial implicación en la correcta maduración puberal y en el mantenimiento de la capacidad reproductora in vivo. En base a lo anterior, esta Tesis Doctoral ha abordado, como objetivos principales, (i) el estudio del papel específico de microRNAs en la regulación puberal a través de la regulación del sistema Kiss1; (ii) el papel fisiopatológico de los microRNAs en el hipogonadismo central inducido por obesidad (HIO) y sus potenciales implicaciones terapéuticas; (iii) el papel de GRK2 en el control de la puberal y el eje HHG mediante la regulación de GPR54 en condiciones control y de estrés nutricional; y (iv) la implicación de GRK2 en HIO a través de la regulación de GPR54

Deregulation of miR-324/KISS1/kisspeptin in early ectopic pregnancy: mechanistic findings with clinical and diagnostic implications

[Abstract] BACKGROUND: Ectopic pregnancy is a life-threatening condition for which novel screening tools that would enable early accurate diagnosis would improve clinical outcomes. Kisspeptins, encoded by KISS1, play an essential role in human reproduction, at least partially by regulating placental function and possibly embryo implantation. Kisspeptin levels are elevated massively in normal pregnancy and reportedly altered in various gestational pathologic diseases. Yet, the pathophysiologic role of KISS1/kisspeptin in ectopic pregnancy has not been investigated previously. OBJECTIVE: The purpose of this study was to evaluate changes of KISS1/kisspeptin levels in ectopic pregnancy and their underlaying molecular mechanisms and to ascertain the diagnostic implications of these changes. STUDY DESIGN: A total of 122 women with normal pregnancy who underwent voluntary termination of pregnancy and 84 patients who experienced tubal ectopic pregnancy were recruited. Measurements of plasma kisspeptins and KISS1 expression analyses in human embryonic/placental tissue were conducted in ectopic pregnancy and voluntary termination of pregnancy control subjects during the early gestational window (<12 weeks). Putative microRNA regulators of KISS1 were predicted in silico, followed by expression analyses of selected microRNAs and validation of repressive interactions in vitro. Circulating levels of these microRNAs were also assayed in ectopic pregnancy vs voluntary termination of pregnancy. RESULTS: Circulating kisspeptins gradually increased during the first trimester of normal pregnancy but were reduced markedly in ectopic pregnancy. This profile correlated with the expression levels of KISS1 in human embryonic/placental tissue, which increased in voluntary termination of pregnancy but remained suppressed in ectopic pregnancy. Bioinformatic predictions and expression analyses identified miR-27b-3p and miR-324-3p as putative repressors of KISS1 in human embryonic/placental tissue at <12 weeks gestation, when expression of microRNAs was low in voluntary termination of pregnancy control subjects but significantly increased in ectopic pregnancy. Yet, a significant repressive interaction was documented only for miR-324-3p, occurring at the predicted 3'-UTR of KISS1. Interestingly, circulating levels of miR-324-3p, but not of miR-27b-3p, were suppressed distinctly in ectopic pregnancy, despite elevated tissue expression of the pre-microRNA. A decision-tree model that used kisspeptin and miR-324-3p levels was successful in discriminating ectopic pregnancy vs voluntary termination of pregnancy, with a receiver-operating characteristic area under the curve of 0.95±0.02 (95% confidence interval). CONCLUSION: Our results document a significant down-regulation of KISS1/kisspeptins in early stages of ectopic pregnancy via, at least partially, a repressive interaction with miR-324-3p. Our data identify circulating kisspeptins and miR-324-3p as putative biomarkers for accurate screening of ectopic pregnancy at early gestational ages.Ministerio de E$conomía y Competitividad (España); BFU2014-57581-PMinisterio de Economía y Competitividad ; BFU2017-83934-PInstituto de Salud Carlos III; PIE-00005Junta de Andalucía; P08-CVI-03788Junta de Andalucía; P12-FQM-0194

Kisspeptins and the neuroendocrine control of reproduction: Recent progress and new frontiers in kisspeptin research.

In late 2003, a major breakthrough in our understanding of the mechanisms that govern reproduction occurred with the identification of the reproductive roles of kisspeptins, encoded by the Kiss1 gene, and their receptor, Gpr54 (aka, Kiss1R). The discovery of this unsuspected reproductive facet attracted an extraordinary interest and boosted an intense research activity, in human and model species, that, in a relatively short period, established a series of basic concepts on the physiological roles of kisspeptins. Such fundamental knowledge, gathered in these early years of kisspeptin research, set the scene for the more recent in-depth dissection of the intimacies of the neuronal networks involving Kiss1 neurons, their precise mechanisms of regulation and the molecular underpinnings of the function of kisspeptins as pivotal regulators of all key aspects of reproductive function, from puberty onset to pulsatile gonadotropin secretion and the metabolic control of fertility. While no clear temporal boundaries between these two periods can be defined, in this review we will summarize the most prominent advances in kisspeptin research occurred in the last ten years, as a means to provide an up-dated view of the state of the art and potential paths of future progress in this dynamic, and ever growing domain of Neuroendocrinology

Neonatal exposure to androgens dynamically alters gut microbiota architecture

Gonadal steroids strongly contribute to the metabolic programming that shapes the susceptibility to the manifestation of diseases later in life, and the effect is often sexually dimorphic. Microbiome signatures, together with metabolic traits and sex steroid levels, were analyzed at adulthood in neonatally androgenized female rats, and compared with those of control male and female rats. Exposure of female rats to high doses of androgens on early postnatal life resulted in persistent altera tions of the sex steroid profile later on life, namely lower progesterone and higher estr adiol and estrone levels, with no effect on endogenous androgens. Neonatally androgenized females were heavier (10% at early adulthood and 26% at adulthood) than controls and had impaired glucose homeostasis observed by higher AUC of glucose in GTT and ITT when subjected to obesogenic manipulations. Androgenized female displayed overt alterations in gut microbiota, indicated especially by higher Bacteroidetes and lower Firmicutes abundance at early adulthood, which disappeared when animals were concurrently overfed at adulthood. Notably, these changes in gut microbiota were related with the intestinal expression of several miRNAs, such as miR-27a-3p, miR-29a-5p, a nd miR-100-3p. Our results suggest that nutritional and hormonal disruption at early developmental periods not only alters the metabolic programming of the individual later in life but also perturbs the architecture of gut microbiota, which may interact with the host by a cross-talk mediated by intestinal miRNAs; phenomena that may contribute to amplify the metabolic derangement caused by obesity, as seen in neonatally androgenized female rats

Interplay between gonadal hormones and postnatal overfeeding in defining sex-dependent differences in gut microbiota architecture

Aging is associated with a decline in sex hormones, variable between sexes, that has an impact on many different body systems and might contribute to age-related disease progression. We aimed to characterize the sex differences in gut microbiota, and to explore the impact of depletion of gonadal hormones, alone or combined with postnatal overfeeding, in rats. Many of the differences in the gut microbiota between sexes persisted after gonadectomy, but removal of gonadal hormones shaped several gut microbiota features towards a more deleterious profile, the effect being greater in females than in males, mainly when animals were concurrently overfed. Moreover, we identified several intestinal miRNAs as potential mediators of the impact of changes in gut microbiota on host organism physiology. Our study points out that gonadal hormones contribute to defining sex-dependent differences of gut microbiota, and discloses a potential role of gonadal hormones in shaping gut microbiota, as consequence of the interaction between sex and nutrition. Our data suggest that the changes in gut microbiota, observed in conditions of sex hormone decline, as those caused by ageing in men and menopause in women, might exert different effects on the host organism, which are putatively mediated by gut microbiota-intestinal miRNA cross-talk

Interplay between gonadal hormones and postnatal overfeeding in defining sex-dependent differences in gut microbiota architecture

Aging is associated with a decline in sex hormones, variable between sexes, that has an impact on many different body systems and might contribute to age-related disease progression. We aimed to characterize the sex differences in gut microbiota, and to explore the impact of depletion of gonadal hormones, alone or combined with postnatal overfeeding, in rats. Many of the differences in the gut microbiota between sexes persisted after gonadectomy, but removal of gonadal hormones shaped several gut microbiota features towards a more deleterious profile, the effect being greater in females than in males, mainly when animals were concurrently overfed. Moreover, we identified several intestinal miRNAs as potential mediators of the impact of changes in gut microbiota on host organism physiology. Our study points out that gonadal hormones contribute to defining sex-dependent differences of gut microbiota, and discloses a potential role of gonadal hormones in shaping gut microbiota, as consequence of the interaction between sex and nutrition. Our data suggest that the changes in gut microbiota, observed in conditions of sex hormone decline, as those caused by ageing in men and menopause in women, might exert different effects on the host organism, which are putatively mediated by gut microbiota-intestinal miRNA cross-talk.This work was supported by research grants from Ministerio de Economia y Competitividad (CP14/00114 to A.C.; PIE14/00005 to JLM and MTS; AGL2015/- 67896-P to JL-M and AC); Ministerio de Ciencia e Innovacion (FIS PI19/00299 to AC; DTS19/00007 to AC; PI16/01777 to PPM); Fondo Europeo de Desarrollo Regional (FEDER). A.C. is supported by an ISCIII research contract, Instituto de Salud Carlos III (Programa Miguel-Servet CP14/00114 and CPII19/ 00007)