Efeitos da exposição intrauterina à betametasona sobre a reprodução de ratos Wistar
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Data
2017-02-07
Autores
Borges, Cibele dos Santos [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A betametasona é o corticosteroide de escolha para o amadurecimento pulmonar fetal diminuindo a incidência de síndrome de angústia respiratória e mortalidade neonatal. Estudos realizados em parceria com o nosso laboratório demonstraram que a exposição pré-natal a este fármaco promoveu alterações nas concentrações de testosterona e parâmetros espermáticos da prole masculina de ratos. Recentemente, efeitos sobre o eixo hipotalâmico-hipofisário-adrenal foram observados por gerações. Assim, o objetivo do presente trabalho foi avaliar os efeitos nos parâmetros reprodutivos da prole feminina e masculina de ratos, provocados pela exposição in utero à betametasona. Para tanto, ratas Wistar prenhes foram alocadas em grupo controle (n=11) e tratado (n=13) com 0,1mg/kg de betametasona, via intramuscular, nos dias gestacionais 12, 13, 18 e 19. No experimento 1, foram avaliados: no dia pós-natal (DPN) 1 o peso inicial e a distância ano-genital da prole feminina, a partir do DPN 30, a instalação da puberdade, e a partir do DPN 70, a ciclicidade estral. No DPN 85, procedeu-se a eutanásia de uma fêmea em estro, e foram coletados uma alíquota de sangue para a determinação das concentrações hormonais e os órgãos reprodutores para pesagem. Ovário e útero foram fixados para análise histológica e morfométrica. No DPN 90 foi realizado comportamento sexual seguido de teste de fertilidade e no DPN 95 um segmento uterino de uma fêmea em estro por ninhada foi utilizado para a reatividade farmacológica. No experimento 2, foram avaliados: o consumo de ração e ganho de peso materno durante o tratamento, o peso inicial e a distância ano-genital da prole masculina no DPN 1, a instalação da puberdade a partir do DPN 30. No DPN 45 e no DPN110 procedeu-se a eutanásia de um animal de cada ninhada e foram coletados uma alíquota de sangue para a determinação das concentrações hormonais e os órgãos reprodutores para pesagem. O testículo foi fixado em Bouin e analisado para histologia, morfometria e imunohistoquímica para proteínas Cx43 e PCNA. No DPN 90, um animal de cada ninhada foi utilizado para a realização do teste de fertilidade natural. No experimento 3, dois machos da F1 foram separados e utilizados para a obtenção dos seguintes parâmetros: um animal de cada ninhada no DPN 90 foi utilizado para a realização do teste de comportamento sexual, e após 30 dias de recuperação procedeu-se a eutanásia dos animais e uma alíquota de sangue foi obtida para a determinação das concentrações hormonais e os órgãos reprodutores para pesagem. O testículo esquerdo teve a túnica albugínea removida e foi utilizado para a determinação da produção diária espermática. O epidídimo esquerdo foi utilizado para a determinação dos parâmetros espermáticos (contagem, motilidade e morfologia) assim como para o teste de fertilidade por meio da inseminação artificial in utero. O outro animal foi eutanasiado no DPN 120 e direcionado para os testes de reatividade farmacológica das glândulas sexuais acessórias. No experimento 4, um filhote macho de cada ninhada da F1 foi utilizado para acasalar com fêmeas não tratadas para a obtenção da segunda geração (F2). A prole obtida da F2 foi avaliada quanto os mesmos parâmetros descritos nos experimentos 2 e 3. No experimento 5, os epidídimos direito dos animais tanto da F1 quanto da F2, no DPN 45 e 110, foram coletados e processados para histopatologia, imunomarcação para proteína Cx43 e p63 e estereologia, e o epidídimo esquerdo para reatividade farmacológica. A exposição in utero à betametasona promoveu uma redução no consumo de ração e no ganho de peso materno durante o tratamento. Na prole feminina foram observados: redução do peso da ninhada ao nascimento, atraso na instalação da puberdade, redução no número de estros, aumento do peso uterino, nas concentrações de LH, comprimento do ciclo estral, na contratilidade uterina e na área do miométrio. Em contrapartida, houve uma redução nas concentrações de FSH, no quociente de lordoses, na área endometrial e na fertilidade das ratas adultas. Na prole masculina da F1, foram observados redução do peso inicial ao nascimento, atraso na instalação da puberdade, redução das concentrações de testosterona no DPN 45, aumento do peso gonadal e alteração no padrão de organização das células de Sertoli e germinativas (DPN 45 e DPN 110). A produção diária espermática foi significativamente reduzida, assim como o diâmetro dos túbulos seminíferos, além da alteração na dinâmica da espermatogênese. No epidídimo foi observado um atraso na diferenciação das células do epitélio (DPN 45), redução da densidade volumétrica do epitélio, aumento no número de espermatozoides imóveis e malformados e alteração na expressão das proteínas Cx43 e p63. O teste de contratilidade das glândulas sexuais acessórias monstrou um aumento na atividade contrátil da vesícula seminal, cujo peso absoluto foi reduzido. Os testes de fertilidade revelaram redução no potencial fértil dos animais expostos à betametasona, tanto pelo acasalamento natural quanto pela inseminação artificial in utero. Na F2, ocorreu um padrão similar de alterações sobre o desenvolvimento sexual da prole masculina: redução do peso ao nascimento, atraso na instalação da puberdade e aumento no peso dos testículos. Além disso, foi observada redução no peso da vesícula seminal, assim como na sua atividade contrátil. As concentrações hormonais foram reduzidas para FSH e aumentadas para LH. Foi observada redução no volume das células de Leydig, redução na produção espermática, atraso na diferenciação celular do epitélio epididimário, alteração na expressão de Cx43, e redução no número de espermatozoides móveis e normais. Houve redução da frequência ejaculatória e de fertilidade. Diante do exposto, concluiu-se que a exposição de ratos à betametasona, em períodos críticos do desenvolvimento intrauterino promoveu uma reprogramação reprodutiva multigeracional. Do ponto de vista translacional estes resultados alertam para possíveis riscos reprodutivos da exposição pré-natal aos glicocorticoides na terapia antenatal.
Betamethasone is the glucocorticoid of choice for fetal lung maturation, reducing the incidence of respiratory distress syndrome and neonatal mortality. Studies conducted in association with our laboratory have shown that prenatal exposure to this drug promoted changes in testosterone levels and sperm parameters of male rats offspring. Recently, effects on the hypothalamic-pituitary-adrenal axis were observed for generations. The aim of this study was to evaluatethe effects on reproductive parameters of male and female rats, caused by in utero exposure to betamethasone. Therefore, pregnant Wistar rats were divided into control group (n=11) and treated (n=13) with 0.1mg / kg betamethasone, intramuscularly, on gestational days 12, 13, 18 and 19. In the experiment 1, it was evaluated: the initial weight and the anogenital distance at PND 1, the puberty installation from PND 30, and from PND 70 as the estrus cyclicity. On PND 85, one female was euthanized in estrus and an aliquot of blood was collected for hormone levels and the reproductive organs were weighted. Ovary and uterus were fixed for histological and morphometric analysis. In DPN 90, sexual behavior was performed followed by fertility test, and in PND 95 an uterine segment from an animal per litter was used for pharmacological reactivity. In experiment 2, it was evaluated: the maternal food intake and weight gain during treatment, the body weight and anogenital distance of male offspring on PDN 1 and the puberty installation from PND 30. In DPN 45 and DPN110 an animal per litter was euthanized and an aliquot of blood was collected to hormone levels and the reproductive organs were weighted. The right testis was fixed in Bouin and processed: histology, morphometry and immunohistochemistry for PCNA and Cx43 protein. At PND 90 one animal from each litter was used to perform natural fertility test. In experiment 3, two males of F1 were used to obtain the following parameters: an animal from each litter on PND 90 was used to perform the sexual behavior test and after 30 days of recovery, it was euthanized and an aliquot of blood was obtained for hormone levels and the reproductive organs were weighted. The left testis had its tunica albuginea removed and used to determine daily sperm production. The left epididymis was used for the determination of sperm parameters (count, motility and morphology) as well as the fertility test by in utero artificial insemination. The other animal was euthanized in DPN 120 for pharmacological reactivity tests of male sex glands. In experiment 4, one male pup from F1 was used to mating with untreated females to obtain the second generation (F2). The offspring obtained from F2 was evaluated for the same parameters described in the experiments 2 and 3. In Experiment 5, the right epididymis from F1 and F2 animals, on PND 45 and PND 110 were processed for histopathology, immunostaining for Cx43 and p63 protein and stereology, and the left epididymis for pharmacology reactivity test. In utero exposure to betamethasone promoted a reduction in food intake and in the weight gain of mothers during the treatment. In the female pups were observed: a reduction on weight at birth, delayed on puberty onset, reduction in estrus numbers, increase in uterine weight, LH levels, estrous cycle length, uterine contractility, and area of the myometrium. In contrast, there was a reduction in FSH levels, lordosis quotient, endometrial area and fertility. In F1 male offspring, there was a reduction in the initial weight of male offspring, delayed puberty onset, low testosterone levels in PND 45, a gonadal weight increase and alteration in the pattern of Sertoli and germ cells organization on PND 45 and 110. Reduction on daily sperm production, in the diameter of the seminiferous tubules and also an alteration in the dynamics of spermatogenesis. In the epididymis, there was a delay in epithelial cells differentiation (PND 45), reduction on epithelium density volume, an increase in the number of immobile and abnormal spermatozoa and alteration in the expression of Cx43 and p63. The seminal vesicle showed a decrease in its weight and an increase in contractility activity. Fertility tests revealed reduction in the fertility potential of animals exposed to betamethasone, either by natural mating and by in utero artificial insemination. In F2, there was a similar pattern of changes on the sexual development of male offspring, as well as in sperm quality, characterized by reduced birth weight, delay in puberty onset and increase in weight of the testicles. Furthermore, there was a reduction in weight of the seminal vesicle, as well as their contractility activity. Hormone levels showed reduced FSH and increased LH. It was observed decrease in Leydig cells volume, reduction on sperm production, delay in the epithelial cells differentiation, alteration in the expression of Cx43 and reduction in the number of normal and mobile spermatozoa. There was a reduction in the ejaculatory frequency and fertility potential. From the foregoing, it is concluded that betamethasone exposure, in critical periods of intrauterine development in rats, promoted a reproductive multigenerational reprogramming. From the translational perspective these results warn of possible adverse reproductive outcomes of prenatal exposure to glucocorticoids in the human antenatal therapy.
Betamethasone is the glucocorticoid of choice for fetal lung maturation, reducing the incidence of respiratory distress syndrome and neonatal mortality. Studies conducted in association with our laboratory have shown that prenatal exposure to this drug promoted changes in testosterone levels and sperm parameters of male rats offspring. Recently, effects on the hypothalamic-pituitary-adrenal axis were observed for generations. The aim of this study was to evaluatethe effects on reproductive parameters of male and female rats, caused by in utero exposure to betamethasone. Therefore, pregnant Wistar rats were divided into control group (n=11) and treated (n=13) with 0.1mg / kg betamethasone, intramuscularly, on gestational days 12, 13, 18 and 19. In the experiment 1, it was evaluated: the initial weight and the anogenital distance at PND 1, the puberty installation from PND 30, and from PND 70 as the estrus cyclicity. On PND 85, one female was euthanized in estrus and an aliquot of blood was collected for hormone levels and the reproductive organs were weighted. Ovary and uterus were fixed for histological and morphometric analysis. In DPN 90, sexual behavior was performed followed by fertility test, and in PND 95 an uterine segment from an animal per litter was used for pharmacological reactivity. In experiment 2, it was evaluated: the maternal food intake and weight gain during treatment, the body weight and anogenital distance of male offspring on PDN 1 and the puberty installation from PND 30. In DPN 45 and DPN110 an animal per litter was euthanized and an aliquot of blood was collected to hormone levels and the reproductive organs were weighted. The right testis was fixed in Bouin and processed: histology, morphometry and immunohistochemistry for PCNA and Cx43 protein. At PND 90 one animal from each litter was used to perform natural fertility test. In experiment 3, two males of F1 were used to obtain the following parameters: an animal from each litter on PND 90 was used to perform the sexual behavior test and after 30 days of recovery, it was euthanized and an aliquot of blood was obtained for hormone levels and the reproductive organs were weighted. The left testis had its tunica albuginea removed and used to determine daily sperm production. The left epididymis was used for the determination of sperm parameters (count, motility and morphology) as well as the fertility test by in utero artificial insemination. The other animal was euthanized in DPN 120 for pharmacological reactivity tests of male sex glands. In experiment 4, one male pup from F1 was used to mating with untreated females to obtain the second generation (F2). The offspring obtained from F2 was evaluated for the same parameters described in the experiments 2 and 3. In Experiment 5, the right epididymis from F1 and F2 animals, on PND 45 and PND 110 were processed for histopathology, immunostaining for Cx43 and p63 protein and stereology, and the left epididymis for pharmacology reactivity test. In utero exposure to betamethasone promoted a reduction in food intake and in the weight gain of mothers during the treatment. In the female pups were observed: a reduction on weight at birth, delayed on puberty onset, reduction in estrus numbers, increase in uterine weight, LH levels, estrous cycle length, uterine contractility, and area of the myometrium. In contrast, there was a reduction in FSH levels, lordosis quotient, endometrial area and fertility. In F1 male offspring, there was a reduction in the initial weight of male offspring, delayed puberty onset, low testosterone levels in PND 45, a gonadal weight increase and alteration in the pattern of Sertoli and germ cells organization on PND 45 and 110. Reduction on daily sperm production, in the diameter of the seminiferous tubules and also an alteration in the dynamics of spermatogenesis. In the epididymis, there was a delay in epithelial cells differentiation (PND 45), reduction on epithelium density volume, an increase in the number of immobile and abnormal spermatozoa and alteration in the expression of Cx43 and p63. The seminal vesicle showed a decrease in its weight and an increase in contractility activity. Fertility tests revealed reduction in the fertility potential of animals exposed to betamethasone, either by natural mating and by in utero artificial insemination. In F2, there was a similar pattern of changes on the sexual development of male offspring, as well as in sperm quality, characterized by reduced birth weight, delay in puberty onset and increase in weight of the testicles. Furthermore, there was a reduction in weight of the seminal vesicle, as well as their contractility activity. Hormone levels showed reduced FSH and increased LH. It was observed decrease in Leydig cells volume, reduction on sperm production, delay in the epithelial cells differentiation, alteration in the expression of Cx43 and reduction in the number of normal and mobile spermatozoa. There was a reduction in the ejaculatory frequency and fertility potential. From the foregoing, it is concluded that betamethasone exposure, in critical periods of intrauterine development in rats, promoted a reproductive multigenerational reprogramming. From the translational perspective these results warn of possible adverse reproductive outcomes of prenatal exposure to glucocorticoids in the human antenatal therapy.
Descrição
Palavras-chave
Betametasona, Exposição intrauterina, Fertilidade, Gerações, Reprodução