Impacto de la exposición a un exceso de testosterona durante el desarrollo fetal en el eje gonadal de machos ovinos

La función reproductiva de los animales y humanos puede ser modificada por numerosos factores ambientales, los cuales se harían presente desde la etapa fetal. Los factores ambientales podrían provenir de la madre, como también del medio ambiente externo y que se incorporan al microambiente uterino a través de diversos mecanismos. El período de desarrollo embrionario-fetal es particularmente sensible a estímulos de diverso orden. Durante este período, se desarrollan los diversos órganos y se programan sus funciones. La plasticidad del desarrollo es un concepto que permite explicar los cambios que se producen en el embrión o el feto y que alterarían la organogénesis y el funcionamiento de diversos órganos y cuya mayor expresión en las alteraciones funcionales, se observa en la vida postnatal, algunas de las cuales se pueden manifestar como síndromes. La hipótesis del origen fetal de las enfermedades del adulto descansa en este concepto. Según esta hipótesis, el patrón de desarrollo definido por el genoma, se altera producto de señales provenientes de la madre o del medio ambiente y desvían su trayectoria de desarrollo. El mecanismo por el cual se genera este fenómeno se denomina epigénesis. El aumento de las hormonas de origen materno puede constituir estímulos de programación fetal. Sin embargo, el término reprogramación fetal sería más adecuado ya que la reprogramación daría cuenta de los cambios generados por la epigénesis, a diferencia de la programación que estaría dada por el genoma. Una de esas hormonas es la testosterona. Estudios experimentales en monas y ovejas han establecido que si las madres son expuestas a niveles suprafisiológicos de testosterona, las crías hembras presentan retardo del crecimiento intrauterino, infertilidad, obesidad, insulino-resistencia y cambios de conducta en su vida postnatal. En las hembras ovinas con exposición prenatal a testosterona (EPT) en útero, se han observado alteraciones neuroendocrinas, ováricas, genitales y conductuales desde el nacimiento hasta la edad adulta. Las hembras ovinas prepuberales presentan menor peso corporal, inicio precoz de los ciclos estrales y mayor secreción de estradiol en respuesta al test de análogo de GnRH, en comparación a las hembras nacidas de madres controles. La mayor cantidad de estudios sobre los efectos de la reprogramación fetal inducida por testosterona en ovinos, se han enfocado en las crías hembras dado que los resultados obtenidos se pueden trasladar, como modelo experimental, a un síndrome endocrino-metabólico, que aqueja a un porcentaje elevado (10 al 15 %) de mujeres en edad fértil, conocido como Síndrome de Ovario Poliquístico. El efecto reprogramador de la testosterona sobre los machos ha sido menos explorado. Recientemente nuestro laboratorio demostró que los machos adultos nacidos de madres tratadas con testosterona, presentaban disminución en el porcentaje de espermios en el eyaculado, menor circunferencia escrotal y menor concentración de espermatocitos. Sorprendentemente, la cantidad de células de Sertoli en los túbulos seminíferos era mayor. Sin embargo, los mecanismos que conducen a estos fenómenos y la ontogenia de estos efectos se desconocen, lo que lleva a preguntarse si estos fenómenos de reprogramación o epigenéticos se establecen desde la etapa fetal, o se desarrollan en la vida postnatal. Para responder a esta interrogante, en la presente tesis se establecieron los siguientes objetivos específicos, a lo que se dará respuesta a través de los trabajos de investigación realizados. 1. Evaluar los efectos de la exposición fetal al exceso de andrógenos (testosterona o DHT) sobre el eje hipotálamo-pituitario mediante el estudio de la pulsatilidad de LH en diferentes fases del desarrollo sexual postnatal en machos. 2. Evaluar si la exposición fetal a un exceso de testosterona causa una disrupción temprana en la población celular testicular y en mediadores locales de la función testicular. 3. Evaluar la respuesta del eje pituitario-ganadal y el compromiso de la función testicular tras la administración de análogos a la GnRH en machos expuestos prenatalmente a testosterona. Los resultados obtenidos permitieron concluir que la exposición prenatal a un exceso de andrógenos altera el patrón normal de liberación de LH en machos, lo que sugiere que dichos andrógenos inducen cambios en la respuesta hipofisaria y/o en el patrón de liberación de GnRH hipotalámica. Los patrones de secreción de LH observados en machos expuestos prenatalmente a testosterona y a dihidrotestosterona fueron similares, y demuestra el origen androgénico (y no estrogénico) en la modificación de las características de liberación de LH. La exposición prenatal a un exceso de testosterona impide el desarrollo ontogénico de los testículos a través de su impacto sobre el número de cél. de Sertoli y la expresión de mediadores importantes para la diferenciación testicular. Finalmente, fue posible concluir que durante la época peripubertal, los machos expuestos a testosterona durante la gestación presentan una mayor sensibilidad hipofisaria a la GnRH, mientras que sus testículos redujeron su actividad/sensibilidad frente a LH. En resumen, la exposición prenatal de machos ovinos a testosterona altera el funcionamiento del eje gonadal ovino. Nuevos estudios serán necesarios para reconocer los mecanismos epigenéticos involucrados en las alteraciones de la funcionalidad del eje gonadal.The reproductive function of animals and humans can be modified by many environmental factors, which could affect the growing fetus. These environmental factors could come from the mother, as well as from the external environment and may reach the uterine microenvironment through various mechanisms. The period of embryo-fetal development is particularly sensitive to stimuli of different order. During this period, various organs are developing and their functions are programmed. The plasticity of development is a concept that allows to explain the changes that occur in the embryo or fetus and that they would alter the organogenesis and the functioning of various organs and whose greatest expression in functional alterations, is observed in postnatal life, some of which may be manifested as syndromes. The hypothesis of the fetal origin of adult diseases rests on this concept. According to this hypothesis, the pattern of development defined by the genome, is altered by signals derived from the mother or from the environment and could divert its pathway of development. The mechanism by which this phenomenon occurs is now called Epigenesis. The increase of hormones of maternal origen could constitute stimuli of fetal programming. However, the term fetal reprogramming would be more appropriate since the reprogramming would realize the changes generated by the epigenesis, in contrast to programming that would be given by the genome. One of these hormones is testosterone. Experimental studies in monkeys and sheep have established that if mothers are exposed to high levels of testosterone, their young female offspring exhibit intrauterine growth retardation, and in the other hand, infertility, obesity, insulin resistance and changes in the behavior during their postnatal life. In female sheep, with prenatal exposure to testosterone (PET) in uterus, it has been observed neuroendocrine, ovarian, genital and behavioural alterations from birth up to adulthood. The prepubertal female sheep have lower body weight, early onset of estrous cycles and greater secretion of estradiol in response to GnRH analogue test, in comparison to females born to control mothers. Most of studies about the fetal reprogramming by testosterone in ovines have been focused in female offspring, because their results coould be traslated, as an experimental model, to a endocrine-metabolic syndrome that affect to an elevated percentage of women in fertil age, known as Polycystic Ovary Sindrome (PCOS). The reprogramming effect of testosterone on males has been less explored. Our laboratory recently demonstrated that males born to mothers treated with testosterone presented a decrease in percentage of sperm in the ejaculate, less scrotal circumference, and less spermatocytes concentration. Surprensingly, the amount of Sertoli cells in the seminiferous tubules was higher. However, mechanisms that drive to this and the ontogenia of these findings are unknown. This leads to the questions if these reprogramming fenomena are established since the fetal stage or they are developed during the postnatal life. To anwer these interrogants, in the present tesis, the following specific objectives were defined to which, answers will be reached by the research work. 1. To evaluate the effects of the fetal exposure to an excess of androgens (testosterone and DHT) on the hypothalamus-pituitary axis by means of the LH pulsatility in different stages of the postnatal sexual development in males. 2. To evaluate if the fetal exposure to an excess of testosterone produces an early disruption in the testicular celular population and in the local mediators of the testicular function. . 3. To evaluate the pituitary-testicular axis and the compromise of the testicular function in response to a GnRH analogue in males prenatally exposed to testosterone. Results obtained allowed to conclude that the prenatal exposure to an excess of androgens in males alters the normal pattern of LH secretion.This suggests that prenatal androgens drives to changes in the pituitary responsiveness and/or in the GnRH secretion pattern. The pattern of LH secretion was similar in either males, prenatally exposed to testosterone or DHT, demonstrating an androgenic effect (and non-estrogenic) in the modification of the pulsatile LH secretion characteristics. The prenatal exposure to an excess of testosterone hinder the ontogenic development of testis by its impact on the Sertoli cells number and in the expression of local mediators important for the normal testicular differentiation. Finally, it was possible to conclude that males exposed to an excess of testosterone during gestation exhibited a higher pituitary responsiveness to the GnRH while testis showed a less sensitivity to the LH stimulus. In summary, the prenatal exposure to testosterone in male ovines alters the gonadal axis function. New studies are required to recognize the epigenetic mechanisms involved in the gonadal axis funtionality

Long-term testosterone treatment during pregnancy does not alter insulin or glucose profile in a sheep model of polycystic ovary syndrome

© 2017, © 2017 Informa UK Limited, trading as Taylor & Francis Group.The administration of testosterone to pregnant sheep to resemble fetal programming of the polycystic ovary syndrome could alter other hormones/factors of maternal origin with known effects on fetal growth. Hence, we studied the weekly profile of insulin, progesterone and glucose during a treatment with testosterone propionate given biweekly from weeks 5 to 17 of pregnancy (term at 21 weeks) and checked the outcome of their fetuses at 17 weeks of gestation after C-section. Control dams were only exposed to the vehicle of the hormone. The testosterone administration did not cause any significant change in the maternal weekly profile of insulin, progesterone or glucose concentration, although the plasma levels of testosterone in the treated dams were inversely correlated to the levels of progesterone. Testosterone treatment also induced an inverse correlation between mean maternal insulin levels and fetal insulin levels

Prenatal testosterone exposure disrupts Insulin secretion and promotes insulin resistance

Hyperandrogenemia and metabolic disturbances during postnatal life are strongly linked both to polycystic ovary syndrome and other conditions that arise from prenatal exposure to androgen excess. In an animal model of this condition, we reported that insulin sensitivity (IS) was lower in young female sheep born to testosterone-treated mothers versus sheep born to non-exposed mothers (control). This lower insulin sensitivity remains throughout reproductive life. However, it is unknown whether abnormal postnatal levels of testosterone (T) further decrease IS derived from prenatal exposure to testosterone. Therefore, we assessed the effects of an acute testosterone administration (40 mg) on IS and insulin secretion during an intravenous glucose tolerance test performed at 40 weeks of age (adulthood) in previously ovariectomized sheep at 26 weeks of age (prepuberty), that were either prenatally exposed to testosterone (T-females, n = 6) or not (C-females, n = 6). The incremental area under the curve of insulin was greater in C-females both with or without the acute testosterone treatment (P < 0.05). The ISI-Composite was lower after an acute testosterone treatment, only in T-females. We conclude that prenatal exposure to testosterone disrupts pancreatic insulin secretion in response to glucose and that in this setting further hyperandrogenemia may predispose to lower insulin sensitivity.Comisión Nacional de Investigación Científica y Tecnológica (CONICYT) CONICYT FONDECYT 114043

Altered testicular development as a consequence of increase number of sertoli cell in male lambs exposed prenatally to excess testosterone

The reprograming effects of prenatal testosterone (T) treatment on postnatal reproductive parameters have been studied extensively in females of several species but similar studies in males are limited. We recently found that prenatal T treatment increases Sertoli cell number and reduced spermatogenesis in adult rams. If such disruptions are manifested early in life and involve changes in testicular paracrine environment remain to be explored. This study addresses the impact of prenatal T excess on testicular parameters in infant males, including Sertoli cell number and expression of critical genes [FSH receptor (FSHR), androgen receptor (AR), transforming growth factor beta 1 (TGFB1), 3 (TGFB3), transforming growth factor beta type 1 receptor, (TGFBR1), and anti-Mullerian hormone (AMH)] modulating testicular function. At 4 week of age, male lambs born to dams treated with 30 mg of T propionate twice weekly from day 30 to 90, followed by 40 mg of T propionate from day 90 to 120 of pregnancy (T-males), had a higher number of Sertoli cells/ testis compared to C-males. Expression level of AMH, TGFB1, and AR also tended to be lower in T-males. These findings provide evidence that impact of fetal exposure to T excess is evident early in postnatal life, mainly characterized by an increase in Sertoli cell number. This could explain the testicular dysfunction observed in adult rams.Fil: Rojas García, Pedro P.. Universidad de Concepción; ChileFil: Recabarren, Mónica P.. Universidad de Concepción; ChileFil: Sir Petermann, Teresa. Universidad de Santiago de Chile; ChileFil: Rey, Rodolfo Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas; ArgentinaFil: Palma, Sergio. Universidad de Concepción; ChileFil: Carrasco, Albert. Universidad de Concepción; ChileFil: Pérez Marín, Carlos C.. Universidad de Córdoba; ArgentinaFil: Padmanabhan, Vasantha. University Of Michigan; Estados UnidosFil: Recabarren, Sergio E.. Universidad de Concepción; Chil

Epigenetics of polycystic ovary syndrome

Polycystic ovarian syndrome (PCOS) is an endocrine and metabolic dysfunction, highly prevalent in women in their reproductive years. Hyperandrogenism, oligo-ovulation, polycystic ovarian morphology are the main features of this syndrome. PCOS is a genetic disorder with a multifactorial etiology and has a strong link with environmental components. It is frequently associated with obesity and insulin resistance. Recently, epigenetic mechanisms have been involved in the pathogenesis of PCOS. Several studies showed that methylation in DNA and miRNAs is altered in women with PCOS in blood, serum, adipose tissue, granulose cells and theca. This evidence indicates that women with PCOS have a different epigenetic regulation, which might be triggered by an adverse intrauterine environment or by postnatal environmental elements such as diet and or obesity.Fondecyt 1151531 Fondecyt 1130240 Fondecyt 114043

Secretory patterns of leptin and luteinizing hormone in food-restricted young female sheep

Leptin, the product of the ob gene, has been proposed as a metabolic signal that regulates the secretion of GnRH/LH. This may be critical during prepubertal development to synchronize information about energy stores and the secretion of GnRH/LH. This study aimed to assess the effect of food restriction on the episodic secretion of leptin and LH in young female sheep. Five 20-week-old prepubertal females were fed a low-level diet for 10 weeks to maintain the body weight. Control females of the same age received food ad libitum. Blood samples were collected at 10-min intervals for six hours at 20, 26, and 30 weeks of age, and plasma leptin, LH, insulin and cortisol concentrations were measured. In the control group, no changes were found in pulsatile LH secretion characteristics. Mean LH concentrations and LH amplitude were lower in the food-restricted group than in the control group at 26 and 30 weeks of age. In the control group, pulsatile Ieptin secretion did not change. When compare

Prenatal testosterone excess alters Sertoli and germ cell number and testicular FSH receptor expression in rams

Exposure to excess testosterone (T) during fetal life has a profound impact on the metabolic and reproductive functions in the female's postnatal life. However, less is known about the effects of excess testosterone in males. The aim of the present study was to evaluate the impact (consequences) of an excess of T during fetal development on mature male testis. The testicular evaluation was by histological analysis and by determination of mRNA expression of the FSH receptor (FSH-R), transforming growth factor-β type I receptor (TβR-I), and two members of the TGF-β superfamily, transforming growth factor-β3 (TGFβ3) and anti-Müllerian hormone (AMH) in males born to mothers receiving an excess of T during pregnancy. At 42 wk of age, postpubertal males born to mothers treated with 30 mg of T propionate twice weekly from day 30 to 90, followed by 40 mg of T propionate from day 90 to 120 of pregnancy (T males), showed higher concentrations of FSH in response to a GnRH analog, a higher number of Sertoli cells/seminiferous tubule cross-section, and a lower number of germ cells/tubules (P < 0.05) than control males (C males) born to mothers treated with the vehicle. The mRNA expression of FSH-R and of TβR-I was higher in T males compared with C males (P < 0.05). Moreover, in T males, AMH expression level correlated negatively with the expression level of TGFβ3. In C males, this latter correlation was not observed. These results suggest that prenatal exposure to an excess of T can negatively modify some histological and molecular characteristics of the mature testis.Fil: Rojas García, Pedro P.. Universidad de Concepción; ChileFil: Recabarren, Mónica. Universidad de Concepción; ChileFil: Sarabia, Luis. Universidad de Chile; ChileFil: Schön, Jennifer. Freie Universität; AlemaniaFil: Gabler, Christoph. Freie Universität; AlemaniaFil: Einspanier, Ralf. Freie Universität; AlemaniaFil: Maliqueo, Manuel. Universidad de Chile; ChileFil: Sir Petermann, Teresa. Universidad de Chile; ChileFil: Rey, Rodolfo Alberto. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Biología Celular e Histología; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Recabarren, Sergio E.. Universidad de Concepción; Chil