Efeitos da restrição proteica materna sobre o desenvolvimento do ducto mesonéfrico e do epidídimo nas fases iniciais em ratos wistar
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Data
2019-07-19
Autores
Santos, Talita de Mello
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Universidade Estadual Paulista (Unesp)
Resumo
O ambiente desempenha papel crucial durante o desenvolvimento fetal, podendo influenciar diretamente a saúde da prole. Assim, o estado nutricional materno é essencial para a saúde e bem-estar do feto. Há relatos de alterações em parâmetros relacionados à função epididimária, em animais adultos, cujas mães sofreram restrição de proteína durante as fases de gestação e lactação. A origem das alterações funcionais no epidídimo pode estar relacionada à fatores hormonais, bem como na expressão de seus receptores, e por meio da modulação de moléculas de sinalização como FGFs (Fator de crescimento de Fibroblastos), vias da ERK e Wnt, e à funcionalidade da Src, ou seja, proteínas relacionadas aos processos do desenvolvimento epididimário. No entanto, não há informações que esclareçam as causas dessas alterações ou como a restrição proteica atua ao longo do desenvolvimento pré e pós-natal do epididímo. Desta forma, o objetivo deste trabalho foi avaliar o impacto da restrição proteica materna, no ducto mesonéfrico e no epidídimo nas fases iniciais do desenvolvimento. Assim, ratas Wistar prenhes foram divididas em dois grupos experimentais que receberam uma dieta de normoproteica (NP; 17% de proteína; n=19) ou hipoproteica (HP; 6% de proteína; n=19) durante a gestação e a lactação. Cinco ratas prenhes de cada grupo foram eutanasiadas para coleta dos ductos mesonéfricos dos embriões no dia gestacional (DG)17,5 e as demais permaneceram até os dias pós-natais (DPN) 7 ou 14. Os filhotes machos tiveram seus parâmetros biométricos aferidos e, em seguida, foram eutanasiados. Procedeu-se a coleta de sangue para análise dos hormônios sexuais e dos órgãos genitais, os quais foram pesados e armazenados. Durante toda a fase de gestação e lactação parâmetros maternos, como consumo alimentar e peso corpóreo, também foram aferidos. O epidídimo foi processado segundo técnicas histológicas, imuno-histoquímicas e de Western blotting para detecção de AR, ERα, ERβ, 5α-red, P450aro, Wnt9b, Src Y416 e Y527, Phospho-p44/42 MAPK (Erk1/2), AQP1 e 9, FGFR1 e 2, VEGF, VEGFR, PCNA. As mães restritas tiveram seus parâmetros alimentares e corpóreos alterados pela dieta, assim como os filhotes machos, os quais nasceram menores e com redução da distância anogenital (DAG) e do peso dos órgãos genitais. Os ductos mesonéfricos do grupo HP tiveram comprimento menor nos tempos de 0h e 24h quando comparado ao grupo NP. No DPN7 houve aumento de estradiol sérico e dos níveis proteicos da ERK1/2, Src-426, ERα, ERβ, P450 e VEGFa e diminuição de AR, 5α-red, FGFR1, FGFR2, Src-527, AQP9 e VGFR2, e redução no diâmetro luminal e tubular na região proximal no grupo HP. No DPN14, houve redução dos níveis proteicos de AR, AQP9 e Wnt9b e aumento de ERα, P450aro e ERK2 nos animais restritos. Neste mesmo grupo, a altura do epitélio foi menor nas regiões do segmento inicial (SI) e corpo, e do diâmetro tubular no corpo. Além disso, o presente estudo foi o primeiro a realizar a imunolocalização da AQP1 no epidídimo de animais de 14 dias, bem como observar diminuição da microdensidade vascular no grupo HP quando comparado ao NP. Concluímos que a restrição proteica materna altera a morfogênese epididimária durante o desenvolvimento pré e pós-natal. Essas alterações estão associadas ao suprimento sanguíneo prejudicado, a falhas nas vias hormonais e a alterações na expressão de proteínas necessárias para a angiogênese, formação e manutenção do microambiente epididimário, bem como em proteínas responsáveis pelo crescimento e diferenciação epididimário. Assim, nossos dados sugerem que essas alterações durante os estágios iniciais do desenvolvimento do epidídimo podem estar relacionadas a alterações na qualidade dos gametas, já observada em animais adultos.
The environment plays a crucial role during fetal development and can directly influence the health of the offspring. Thus, maternal nutritional status is essential for fetus' health and well-being. There have been reports of alterations in parameters related to epididymal function in adult animals whose mothers suffered protein restriction during the gestation and lactation. The origin of the functional alterations in the epididymis can be related to the hormonal factors, as well as in the expression of its receptors, and on signaling molecules modulation like FGFs (Fibroblast Growth Factor), ERK and Wnt pathways, and functionality of Src, or proteins related to epididymal development. However, there is no information to clarify the causes of these changes or how protein restriction acts throughout pre and postnatal development. The aim of this study was to evaluate the impact of maternal protein restriction on the mesonephric duct and in the early stages of epididymal development. Pregnant female Wistar rats were divided into two groups that received either normoprotein (17%; NP) or low-protein (6%; LP) diet ad libitum during gestation and lactation. Five pregnant rats from each group were euthanized to collect the mesonephric ducts on gestational day (DG) 17.5 and the others rats remained until the postnatal days (PND) 7 or PND14. The male offspring had their biometric parameters verified and were euthanized. Blood was collected for sex hormones analysis and genital organs were weighted and stored. Throughout the gestation and lactation maternal parameters, such as food consumption and body weight, were also measured. The epididymis was processed according to histological, immunohistochemical and Western blotting techniques for AR, ERα, ERβ, 5α-RD, P450aro, Wnt9b, Src Y416 and Y527, Phospho-p44/42 MAPK (ERK1/2), AQP1 and 9, FGFR1 and 2, VEGF, VEGFR and PCNA detection. Restricted mothers had their food consumption and body parameters altered by low-protein diet, as did the male offspring, which were born smaller and with anogenital distance (DAG) reduced. The HP mesonephric ducts lengths were shorter at 0h and 24h when compared to the NP group. At PND7 there was an increase in serum estradiol and in the ERK1 / 2, Src-426, ERα, ERβ, P450 and VEGFα protein levels and a reduction in the AR, 5α-red, FGFR1, FGFR2, Src-527, AQP9 and VGFR2 expression. Besides a reduction in the luminal and tubular diameter in the HP epididymal proximal region. At PND14, there was a reduction of AR, AQP9 and Wnt9b protein levels and an increase of ERα, P450aro and ERK2 expression in the restricted animals. In this same group, the epithelium height was smaller in initial segment (SI) and caput, and in the corpus' tubular diameter. In addition, the present study was the first to perform the AQP1 immunolocalization at 14-day-old animals epididymis, as well as, to observe a decrease in vascular microdensity in the HP group when compared to NP. We conclude that maternal protein restriction alters epididymal morphogenesis during pre and postnatal development. These changes are associated with blood supply impairment and changes in hormonal pathways and proteins expression of those that are required for angiogenesis, formation and maintenance of the epididymal microenvironment, as well as proteins responsible for epididymal growth and differentiation. Thus, our data suggest that these changes during the early stages of epididymal development may be related to changes in spermatozoa quality already observed in adult animals.
The environment plays a crucial role during fetal development and can directly influence the health of the offspring. Thus, maternal nutritional status is essential for fetus' health and well-being. There have been reports of alterations in parameters related to epididymal function in adult animals whose mothers suffered protein restriction during the gestation and lactation. The origin of the functional alterations in the epididymis can be related to the hormonal factors, as well as in the expression of its receptors, and on signaling molecules modulation like FGFs (Fibroblast Growth Factor), ERK and Wnt pathways, and functionality of Src, or proteins related to epididymal development. However, there is no information to clarify the causes of these changes or how protein restriction acts throughout pre and postnatal development. The aim of this study was to evaluate the impact of maternal protein restriction on the mesonephric duct and in the early stages of epididymal development. Pregnant female Wistar rats were divided into two groups that received either normoprotein (17%; NP) or low-protein (6%; LP) diet ad libitum during gestation and lactation. Five pregnant rats from each group were euthanized to collect the mesonephric ducts on gestational day (DG) 17.5 and the others rats remained until the postnatal days (PND) 7 or PND14. The male offspring had their biometric parameters verified and were euthanized. Blood was collected for sex hormones analysis and genital organs were weighted and stored. Throughout the gestation and lactation maternal parameters, such as food consumption and body weight, were also measured. The epididymis was processed according to histological, immunohistochemical and Western blotting techniques for AR, ERα, ERβ, 5α-RD, P450aro, Wnt9b, Src Y416 and Y527, Phospho-p44/42 MAPK (ERK1/2), AQP1 and 9, FGFR1 and 2, VEGF, VEGFR and PCNA detection. Restricted mothers had their food consumption and body parameters altered by low-protein diet, as did the male offspring, which were born smaller and with anogenital distance (DAG) reduced. The HP mesonephric ducts lengths were shorter at 0h and 24h when compared to the NP group. At PND7 there was an increase in serum estradiol and in the ERK1 / 2, Src-426, ERα, ERβ, P450 and VEGFα protein levels and a reduction in the AR, 5α-red, FGFR1, FGFR2, Src-527, AQP9 and VGFR2 expression. Besides a reduction in the luminal and tubular diameter in the HP epididymal proximal region. At PND14, there was a reduction of AR, AQP9 and Wnt9b protein levels and an increase of ERα, P450aro and ERK2 expression in the restricted animals. In this same group, the epithelium height was smaller in initial segment (SI) and caput, and in the corpus' tubular diameter. In addition, the present study was the first to perform the AQP1 immunolocalization at 14-day-old animals epididymis, as well as, to observe a decrease in vascular microdensity in the HP group when compared to NP. We conclude that maternal protein restriction alters epididymal morphogenesis during pre and postnatal development. These changes are associated with blood supply impairment and changes in hormonal pathways and proteins expression of those that are required for angiogenesis, formation and maintenance of the epididymal microenvironment, as well as proteins responsible for epididymal growth and differentiation. Thus, our data suggest that these changes during the early stages of epididymal development may be related to changes in spermatozoa quality already observed in adult animals.
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Palavras-chave
Desenvolvimento epididimário, Fatores de crescimento, Programação fetal, Receptores hormonais, Restrição proteica