Expressão de mRNA e identificação de proteínas das vias da arginina no endométrio, conceptos e placentomas em ovelhas
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2017-03-31
Autores
Nonato, Amanda [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A arginina é um aminoácido nutricionalmente essencial, importante na gestação de mamíferos, pois aumenta a sobrevivência, crescimento e desenvolvimento de embriões, fetos e neonatos. A arginina pode ser utilizada na síntese de poliaminas por uma via metabólica clássica, porém estudos recentes revelaram uma importante via alternativa. Todavia, ambas foram pouco investigadas. Nesse contexto, estudos são necessários a fim de identificar a ocorrência das vias metabólicas da arginina nos tecidos reprodutivos durante a gestação de ovelhas. Assim o presente estudo teve como objetivos: a) avaliar a expressão de genes das vias metabólicas da arginina em conceptos durante o período de peri-implantação em ovelhas; b) identificar a localização das proteínas das vias metabólicas da arginina em conceptos e no endométrio durante o período de peri-implantação em ovelhas; e c) identificar a localização das proteínas das vias metabólicas da arginina em placentomas em ovelhas. Para tanto, ovelhas da raça Rambouillet (n=72) foram sincronizadas e após detecção do estro por machos vasectomizados, foram cobertas por machos da raça Suffolk com fertilidade comprovada. No experimento 1, as ovelhas (n=20) foram histerectomizadas aos 13, 14, 15 ou 16 dias de gestação, e os fragmentos de conceptos coletados foram congelados em nitrogênio líquido a fim de se estudar a expressão gênica. No experimento 2, ovelhas (n=28) também foram histerectomizadas aos 13, 14, 15 ou 16 dias de gestação e, secções de conceptos e de endométrio, foram fixados a fim de avaliar a localização de proteínas por imunohistoquímica. No experimento 3, ovelhas (n=24) foram histerectomizadas aos 40, 60, 80, 100, 120 ou 140 dias de gestação e placentomas foram coletados e fixados para posterior análise imunohistoquímica. Os dados de expressão gênica foram analisados utilizando o procedimento GLM do SAS e as diferenças na intensidade da imunomarcação foram avaliadas com o software R, foi realizada a análise de variância (ANOVA). O nível de significância considerado foi <0,05. A expressão dos genes ADC e AGMAT (via alternativa), não foi alterada ao longos dos dias de gestação, e o mesmo ocorreu para o gene ARG1 (via clássica). O gene ODC1 (via clássica) apresentou maior expressão aos 13 dias de gestação (P=0,06). A análise imunohistoquímica identificou as proteínas ARG1, ODC1, ADC e AGMAT nos diferentes tecidos e dias de gestação, evidenciando a importância da ambas vias durante a gestação. A presença das proteínas no epitélio luminal do endométrio aumentou (p=0,038) após os 13 dias de gestação e foi significativamente maior aos 16 dias, sugerindo que essas proteínas são requeridas em maiores concentrações para o processo de alongamento do concepto e implantação. No epitélio glandular, a presença das proteínas não foi alterada (p=0,64) ao longo dos dias estudados. Não foi observada diferença (p=0,472) entre a identificação das proteínas no trofectoderma dos conceptos ao longo dos dias. Nos placentomas, a presença das proteínas foi menor (p<0,01) aos 40 dias de gestação, quando comparadas com os dias 60, 120 e 140. Os resultados do presente estudo evidenciam a importância de ambas vias metabólicas da arginina, para produção de poliaminas, durante o período gestacional de ovelhas.
Arginine is a nutritionally essential amino acid, important for pregnancy in mammalian, to increase the survival, growth and development of embryos, fetuses and neonates. Arginine can be used in the synthesis of polyamines by a classical metabolic pathway, though recent studies revealed an important alternative pathway, however, both have been poorly investigated. In this context, studies are necessary in order to identify an occurrence of arginine metabolic pathways in the reproductive tissues during a pregnancy in ewes. The present study had as objectives: a) evaluate the gene expression in the metabolic pathways of arginine, in concepts during the pre-implantation period of pregnancy in ewes; b) to identify the localization of proteins, from the metabolic pathways of arginine, in concepts and uterine endometrium during the pre-implantation period of pregnancy in ewes; and c) to identify the localization of proteins, from the metabolic pathway of arginine, in placentomes during the pre-implantation period of pregnancy in ewes. Rambouillet ewes (n=72) were synchronized and after estrus detection by vasectomized males, ewes were mated by Suffolk males with proven fertility. In experiment 1, ewes (n=20) were hysterectomized at days 13, 14, 15 or 16 of pregnancy, and the sections of concepts collected were frozen in liquid nitrogen in order to study gene expression. In the experiment 2, ewes (n=28) were also hysterectomized at days 13, 14, 15 or 16 of pregnancy, sections of conceptuses and uterine endometrium were fixed in order to identify the protein localization by immunohistochemical analysis. In experiment 3, ewes (n=24) were hysterectomized on days 40, 60, 80, 100, 120 or 140 days of pregnancy, placentomes were collected and fixed for immunohistochemical analysis. Data analysis of gene expression was analyzed using GLM procedure of SAS and differences in the intensity of the immunostaining were evaluated using software R, analysis of variance (ANOVA) was performed. Significance level was declared < 0.05. The expression of ADC and AGMAT genes (alternative pathway) did not alter across days of pregnancy, and the same happened with ARG1 gene (classic pathway). The ODC1 gene (classic pathway) showed greater expression at 13 days of pregnancy (P=0.06). The immunohistochemical analysis identified ARG1, ODC1, ADC and AGMAT proteins in all tissues and days of pregnancy studied, evidencing the relevance of both pathways during pregnancy. The identification of proteins on lumen epithelia from endometrium improved (p=0.038) after Day 13 of pregnancy and it was significantly greater on Day 16, suggesting that these proteins are required in higher concentrations for conceptuses elongation and implantation. On glandular epithelia, the proteins identification did not change (p=0.64) across days of pregnancy. No difference was observed (p=0.472) between the proteins identification on trofectoderm across days. On placentomes, the proteins identification was lower (p<0.01) on Day 40 of pregnancy compared with Days 60, 120 and 140. The results of the experiment indicate that relevance of both metabolic pathways of arginine to polyamines production during the pregnancy period in ewes.
Arginine is a nutritionally essential amino acid, important for pregnancy in mammalian, to increase the survival, growth and development of embryos, fetuses and neonates. Arginine can be used in the synthesis of polyamines by a classical metabolic pathway, though recent studies revealed an important alternative pathway, however, both have been poorly investigated. In this context, studies are necessary in order to identify an occurrence of arginine metabolic pathways in the reproductive tissues during a pregnancy in ewes. The present study had as objectives: a) evaluate the gene expression in the metabolic pathways of arginine, in concepts during the pre-implantation period of pregnancy in ewes; b) to identify the localization of proteins, from the metabolic pathways of arginine, in concepts and uterine endometrium during the pre-implantation period of pregnancy in ewes; and c) to identify the localization of proteins, from the metabolic pathway of arginine, in placentomes during the pre-implantation period of pregnancy in ewes. Rambouillet ewes (n=72) were synchronized and after estrus detection by vasectomized males, ewes were mated by Suffolk males with proven fertility. In experiment 1, ewes (n=20) were hysterectomized at days 13, 14, 15 or 16 of pregnancy, and the sections of concepts collected were frozen in liquid nitrogen in order to study gene expression. In the experiment 2, ewes (n=28) were also hysterectomized at days 13, 14, 15 or 16 of pregnancy, sections of conceptuses and uterine endometrium were fixed in order to identify the protein localization by immunohistochemical analysis. In experiment 3, ewes (n=24) were hysterectomized on days 40, 60, 80, 100, 120 or 140 days of pregnancy, placentomes were collected and fixed for immunohistochemical analysis. Data analysis of gene expression was analyzed using GLM procedure of SAS and differences in the intensity of the immunostaining were evaluated using software R, analysis of variance (ANOVA) was performed. Significance level was declared < 0.05. The expression of ADC and AGMAT genes (alternative pathway) did not alter across days of pregnancy, and the same happened with ARG1 gene (classic pathway). The ODC1 gene (classic pathway) showed greater expression at 13 days of pregnancy (P=0.06). The immunohistochemical analysis identified ARG1, ODC1, ADC and AGMAT proteins in all tissues and days of pregnancy studied, evidencing the relevance of both pathways during pregnancy. The identification of proteins on lumen epithelia from endometrium improved (p=0.038) after Day 13 of pregnancy and it was significantly greater on Day 16, suggesting that these proteins are required in higher concentrations for conceptuses elongation and implantation. On glandular epithelia, the proteins identification did not change (p=0.64) across days of pregnancy. No difference was observed (p=0.472) between the proteins identification on trofectoderm across days. On placentomes, the proteins identification was lower (p<0.01) on Day 40 of pregnancy compared with Days 60, 120 and 140. The results of the experiment indicate that relevance of both metabolic pathways of arginine to polyamines production during the pregnancy period in ewes.
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Palavras-chave
Poliaminas, Peri-implantação, Ovinos, qPCR, Polyamines, Perimplantation, Ovine