Efeitos da restrição proteica materna sobre o padrão vascular e expressão de proteínas no epidídimo de ratos Wistar machos em diferentes fases do desenvolvimento pós-natal
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Data
2019-08-02
Autores
Cavariani, Marilia Martins
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Universidade Estadual Paulista (Unesp)
Resumo
O estado nutricional materno desempenha papel crucial na saúde e no bem-estar do feto. Alterações testiculares, prostáticas e espermáticas foram observadas em animais adultos, cujas mães sofreram restrição de proteína. No entanto, não há trabalhos com este modelo experimental que enfoquem o desenvolvimento, padrão de vascularização e seus reflexos na expressão de proteínas no epidídimo. O objetivo deste estudo é investigar o padrão das respostas morfológicas, imuno-histoquímicas e expressão de proteínas do epidídimo da prole de ratos Wistar de mães submetidas à restrição proteica no período de gestação e de lactação. Fêmeas prenhes foram alocadas nos grupos experimentais normoproteico (NP) e hipoproteico (HP) que receberam, respectivamente, dietas contendo 17% e 6% de proteínas durante gestação e lactação. Após o desmame, os ratos receberam dieta padrão para roedores até as idades de 21, 44 e 120 dias pós-natais (DPN) quando foram eutanasiados. Os epidídimos foram coletados e processados segundo técnicas histológicas, imuno-histoquímicas e de western blotting. Nos filhotes HP, o tamanho reduzido e baixo peso observados ao nascimento se mantiveram até o DPN 120, acompanhados da redução dos órgãos do sistema genital masculino para todas as idades analisadas. A dieta hipoproteica materna diminuiu os níveis séricos de testosterona nos animais no DPN 44, aumentou os níveis de aldosterona nos animais no DPN 21 e não alterou os níveis de estradiol em nenhuma das idades. Nos animais HP, a expressão de AR foi reduzida em todo epidídimo no DPN 44, enquanto a expressão de ERα mostrou-se aumentada no corpo e cauda nos DPNs 21 e 44 e a expressão de ERβ foi reduzida no segmento inicial e cabeça no DPN 120. A imunolocalização destes receptores hormonais foi observada no núcleo e citoplasma das células epiteliais epididimárias e nas células do interstício do órgão em todas as idades. Apesar da diminuição na expressão de AQP9 no segmento inicial e cabeça assim como o aumento na expressão de AQP1 e AQP9 no corpo e cauda terem sido significativos somente para os animais HP no DPN 44, essas alterações também foram observadas nos animais HP nos DPNs 21 e 120. Para animais NP e HP de todas as idades, a imunolocalização de AQP1 apareceu nas células endoteliais de canais vasculares ao longo de todo órgão, enquanto a imunolocalização de AQP9 foi observada nos estereocílios das células principais do epidídimo. A Expressão do VEGFa não foi diferente entre os animais NP e HP em nenhuma das idades. Já a expressão do seu receptor (VEGFR-2) foi menor no segmento inicial e cabeça dos animais HP no DPN 21. A restrição proteica materna aumentou a expressão da Src 416 em todo epidídimo no DPN 21, enquanto diminuiu a expressão desta proteína no segmento inicial e cabeça no DPN 44 e no corpo e cauda no DPN 120. Já a expressão da Src 527 foi diminuída no segmento inicial e cabeça dos animais HP no DPN 21. Observou-se o aumento da expressão da Cldn-1 somente no corpo e cauda epididimários dos animais HP no DPN 44. O índice de densidade microvascular (DMV) e o índice de DMV/estroma dos animais HP nos DPNs 21 e 44 foram menores do que nos animais NP nestas idades. Mesmo que algumas das alterações observadas para os filhotes HP não tenham se mantido até a vida adulta, a oferta insuficiente de proteínas no início da vida alterou a estrutura e o funcionamento do epidídimo em períodos importantes de seu desenvolvimento pós-natal.
Maternal nutrition status plays a crucial role in the health and well-being of the fetus. Changes of testes and prostate as well as spermatic disorders were observed in adult animals whose mothers were subjected to protein restriction. However, there are no studies with this experimental model that focus the development and the vascular pattern of epididymis, as well as their reflexes in the expression of proteins of the epididymis. The aim of this study is to investigate the pattern of morphometric, immunohistochemical and protein expression of the epididymis of the Wistar rat offspring whose mothers were subjected to a low-protein diet during gestation and lactation. Pregnant females were divided into normoprotein (NP) group and low-protein (LP) group that received, respectively, diets containing 17% and 6% of protein during gestation and lactation. After weaning, the LP and NP male pups received the standard diet for rodents until the ages of 21, 44 and 120 days (PND), when they were euthanized. The epididymides were collected and processed according to histological, immunohistochemical and western blotting techniques. In the LP offspring, the smaller body size and low weight observed at birth remained until the PND 120, as well as the reduction of the male genital system organs weight for all analyzed ages. Maternal low-protein diet decreased testosterone serum levels in animals at PND 44, increased aldosterone serum levels in animals at PND 21 and did not altered estradiol serum levels in any age. In LP animals, the AR expression was decreased throughout epididymis at PND 44, whereas ERα expression was increased in epididymis corpus plus cauda at PND 21 and 44, and ERβ expression was reduced in epididymis initial segment plus caput at PND 120. Immunolocalization of these hormone receptors were observed in the nucleus and cytoplasm of epididymis epithelial cells and in the interstitial cells of this organ at all ages. The decrease of AQP9 expression on the initial segment plus caput as well as the increase of AQP1 and AQP9 expression on the corpus plus cauda were significant only in LP animals at PND 44. However, these alterations were also observed in LP animals at PND 21 and 120. The immunolocalization pattern of AQP1 and AQP9 was similar between LP and NP animals at all ages, in with AQP1 staining appearing on endothelial cells of vascular channels throughout the organ, while the immunolocalization of AQP9 was observed in the stereocilia of epididymis principal cells. The VEGFa expression in the epididymis was not different between NP and LP animals in none of the analyzed ages. However, the expression of its receptor (VEGFr-2) was lower on epididymis initial segment plus caput of LP animals at PND 21. Maternal protein restriction increased the Src 416 expression throughout epididymis at PND 21, whereas decreased the expression of this protein on epididymis initial segment plus caput at PND 44 and on epididymis corpus plus cauda at PND 120. Conversely, Src 527 expression was decreased in epididymis initial segment plus caput in LP animals at PND 21. Cldn-1 expression was increased only in the epididymis corpus plus cauda of LP animals at PND 44. The microvascular density (MVD) and the MDV/stroma index of LP animals at PND 21 and 44 were lower than those of NP animals at these ages. Although some of the changes observed for LP pups were not maintained until adulthood, we observed that the insufficient supply of proteins in early life altered the structure and the functioning of the epididymis in important periods of postnatal epididymal development.
Maternal nutrition status plays a crucial role in the health and well-being of the fetus. Changes of testes and prostate as well as spermatic disorders were observed in adult animals whose mothers were subjected to protein restriction. However, there are no studies with this experimental model that focus the development and the vascular pattern of epididymis, as well as their reflexes in the expression of proteins of the epididymis. The aim of this study is to investigate the pattern of morphometric, immunohistochemical and protein expression of the epididymis of the Wistar rat offspring whose mothers were subjected to a low-protein diet during gestation and lactation. Pregnant females were divided into normoprotein (NP) group and low-protein (LP) group that received, respectively, diets containing 17% and 6% of protein during gestation and lactation. After weaning, the LP and NP male pups received the standard diet for rodents until the ages of 21, 44 and 120 days (PND), when they were euthanized. The epididymides were collected and processed according to histological, immunohistochemical and western blotting techniques. In the LP offspring, the smaller body size and low weight observed at birth remained until the PND 120, as well as the reduction of the male genital system organs weight for all analyzed ages. Maternal low-protein diet decreased testosterone serum levels in animals at PND 44, increased aldosterone serum levels in animals at PND 21 and did not altered estradiol serum levels in any age. In LP animals, the AR expression was decreased throughout epididymis at PND 44, whereas ERα expression was increased in epididymis corpus plus cauda at PND 21 and 44, and ERβ expression was reduced in epididymis initial segment plus caput at PND 120. Immunolocalization of these hormone receptors were observed in the nucleus and cytoplasm of epididymis epithelial cells and in the interstitial cells of this organ at all ages. The decrease of AQP9 expression on the initial segment plus caput as well as the increase of AQP1 and AQP9 expression on the corpus plus cauda were significant only in LP animals at PND 44. However, these alterations were also observed in LP animals at PND 21 and 120. The immunolocalization pattern of AQP1 and AQP9 was similar between LP and NP animals at all ages, in with AQP1 staining appearing on endothelial cells of vascular channels throughout the organ, while the immunolocalization of AQP9 was observed in the stereocilia of epididymis principal cells. The VEGFa expression in the epididymis was not different between NP and LP animals in none of the analyzed ages. However, the expression of its receptor (VEGFr-2) was lower on epididymis initial segment plus caput of LP animals at PND 21. Maternal protein restriction increased the Src 416 expression throughout epididymis at PND 21, whereas decreased the expression of this protein on epididymis initial segment plus caput at PND 44 and on epididymis corpus plus cauda at PND 120. Conversely, Src 527 expression was decreased in epididymis initial segment plus caput in LP animals at PND 21. Cldn-1 expression was increased only in the epididymis corpus plus cauda of LP animals at PND 44. The microvascular density (MVD) and the MDV/stroma index of LP animals at PND 21 and 44 were lower than those of NP animals at these ages. Although some of the changes observed for LP pups were not maintained until adulthood, we observed that the insufficient supply of proteins in early life altered the structure and the functioning of the epididymis in important periods of postnatal epididymal development.
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Palavras-chave
epidídimo, restrição proteica, desenvolvimento pós-natal, programação fetal, epididymis, protein restriction, postnatal development, fetal programming