Marcadores moleculares no sêmen suíno: identificação de novas proteínas no fluido epididimário e variação sazonal das defesas antioxidantes seminais
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Data
2017-08
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Orientador
Bustamante Filho, Ivan Cunha
Banca
Bustamante Filho, Ivan Cunha
Goettert, Marcia Ines
Oliveira, Ramatis Birnfeld De
Bortolozzo, Fernando Pandolfo
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Resumo
Durante o trânsito epididimário, os espermatozoides são expostos às secreções do epitélio, formando um ambiente natural essencial para a aquisição de motilidade e capacidade fertilizante pelas células espermáticas. O proteoma do fluido epididimário já vem sendo estudado por grupos de pesquisa há alguns anos e proteínas importantes vêm sendo identificadas no fluido da cauda do epidídimo; no entanto, uma investigação mais profunda da proteômica deste fluido nunca foi feita. No primeiro trabalho apresentado nesta dissertação, a técnica de identificação multidimensional de proteínas (MudPIT) foi utilizada para identificação de proteínas no fluido da cauda do epidídimo. Um total de 663 proteínas foi identificado, sendo que as proteínas mais abundantes observadas em uma análise semi-quantitativa foram as seguintes (contagens espectrais): epididymal-specific lipocalin-5 (1465), beta-hexosaminidase subunit beta precursor (1346), phosphatidylethanolamine-binding protein 4 precursor (367), lactotransferrin precursor (226), brain acid soluble protein 1 isoform 2 (134), di-N-acetylchitobiase (115), epididymis-specific alpha-mannosidase (114), epididymal secretory glutathione peroxidase precursor (112), reticulocalbin-1 isoform 2 (103) e alkaline phosphatase, tissue-nonspecific isozyme (101). A identificação de 663 proteínas no fluido da cauda do epidídimo de suínos possibilita uma maior compreensão dos processos de armazenamento à que estas células são submetidas neste ambiente e, a partir disso, possibilita o desenvolvimento técnicas de reprodução tanto para a solução de problemas de infertilidade humana quanto de produção animal. Em um segundo experimento, avaliou-se a atividade de proteínas antioxidantes presentes no plasma seminal durante as quatro estações do ano e sua variação estacional, em correlação com parâmetros de qualidade seminal. As enzimas previamente selecionadas para avaliação foram a superóxido-dismutase (SOD) e a glutationa peroxidase (GPx). Os parâmetros seminais avaliados apresentaram-se constantes durante todo o ano, sendo observada diferença entre estações apenas na concentração espermática e motilidade rápida após 5 dias de refrigeração a 17°C. As defesas antioxidantes do plasma seminal, medidas através das atividades de GPx e SOD se mantiveram constantes durante o ano. A avaliação destas enzimas nas células espermáticas evidenciou um aumento de atividade de SOD no verão e primavera (P < 0,05). As atividades das enzimas GPx e SOD apresentaram apenas duas correlações com motilidade total após 5 dias de preservação a 17°C. A atividade de SOD nos espermatozoides apresentou correlação negativa fraca (R2 = 0,4517; P = 0.0022). No plasma seminal, a GPx também apresentou correlação negativa (R2 = 0,2447; P = 0,0369). Conclui-se que, apesar de variações individuais, não ocorreram variações significativas na qualidade espermática ao longo do ano.
During epididymal transit, sperm are exposed to epididymal secretions, which form an essential natural environment for the motility acquisition and fertilizing ability by sperm cells. The epididymal fluid proteome has already been studied by research groups for some years and important proteins have been identified in the cauda epididymal fluid (CEF); however, further investigation of this fluid proteomics was never made. In the first work presented multidimensional protein identification technique (mudPIT) was used to identify CEF proteins. A total of 663 proteins were identified, of which the most abundant proteins observed in a semi-quantitative analysis were as follows (spectral counts): epididymal-specific lipocalin-5 (1465), beta-hexosaminidase subunit precursor beta (1346), phosphatidylethanolamine -binding protein 4 precursor (367), lactotransferrin precursor (226), Brain acid soluble protein 1 isoform 2 (134), di-n-acetylchitobiase, partial (115), alpha-mannosidase epididymis-specific (114), epididymal secretory glutathione peroxidase precursor (112), reticulocalbin 2 isoform-1 (103) and alkaline phosphatase, tissue-nonspecific isozyme (101). The emergence of new techniques of proteomic analysis provides a better understanding of metabolic pathways and metabolic processes guided by proteins. Identify 663 proteins in the CEF of boars means understand the storage process to which sperm cells undergo and, from that, be able to develop breeding techniques both to solve problems of human infertility as of agricultural production. In a second experiment, the activity of antioxidant proteins present in the seminal plasma during the four seasons of the year and its seasonal variation in correlation with sperm quality parameters were evaluated. The enzymes previously selected for evaluation were superoxide dismutase (SOD) and glutathione peroxidase (GPx). The evaluated seminal parameters were constant throughout the whole year, with no difference between stations, only in sperm concentration and motility after 5 days after storage at 17 ° C. The antioxidant defenses of the seminal plasma, through measures of GPx and SOD activities remained constant during the year. The evaluation of these enzymes in the sperm showed an increase of SOD activity in spring and summer (P <0.05). The activities of GPx and SOD had only two correlations, total motility after 5 days of preservation at 17 ° C. The SOD activity in the sperm had a weak negative correlation (R2 = - 0.4517, P = 0.0022). In seminal plasma, GPx also showed a negative correlation (R 2 = 0.2447, P = 0.0369).
During epididymal transit, sperm are exposed to epididymal secretions, which form an essential natural environment for the motility acquisition and fertilizing ability by sperm cells. The epididymal fluid proteome has already been studied by research groups for some years and important proteins have been identified in the cauda epididymal fluid (CEF); however, further investigation of this fluid proteomics was never made. In the first work presented multidimensional protein identification technique (mudPIT) was used to identify CEF proteins. A total of 663 proteins were identified, of which the most abundant proteins observed in a semi-quantitative analysis were as follows (spectral counts): epididymal-specific lipocalin-5 (1465), beta-hexosaminidase subunit precursor beta (1346), phosphatidylethanolamine -binding protein 4 precursor (367), lactotransferrin precursor (226), Brain acid soluble protein 1 isoform 2 (134), di-n-acetylchitobiase, partial (115), alpha-mannosidase epididymis-specific (114), epididymal secretory glutathione peroxidase precursor (112), reticulocalbin 2 isoform-1 (103) and alkaline phosphatase, tissue-nonspecific isozyme (101). The emergence of new techniques of proteomic analysis provides a better understanding of metabolic pathways and metabolic processes guided by proteins. Identify 663 proteins in the CEF of boars means understand the storage process to which sperm cells undergo and, from that, be able to develop breeding techniques both to solve problems of human infertility as of agricultural production. In a second experiment, the activity of antioxidant proteins present in the seminal plasma during the four seasons of the year and its seasonal variation in correlation with sperm quality parameters were evaluated. The enzymes previously selected for evaluation were superoxide dismutase (SOD) and glutathione peroxidase (GPx). The evaluated seminal parameters were constant throughout the whole year, with no difference between stations, only in sperm concentration and motility after 5 days after storage at 17 ° C. The antioxidant defenses of the seminal plasma, through measures of GPx and SOD activities remained constant during the year. The evaluation of these enzymes in the sperm showed an increase of SOD activity in spring and summer (P <0.05). The activities of GPx and SOD had only two correlations, total motility after 5 days of preservation at 17 ° C. The SOD activity in the sperm had a weak negative correlation (R2 = - 0.4517, P = 0.0022). In seminal plasma, GPx also showed a negative correlation (R 2 = 0.2447, P = 0.0369).
Descrição
Palavras-chave
Preservação espermática; Espectrometria de massa; Sazonalidade
Citação
ARGENTI, Laura Espíndola. Marcadores moleculares no sêmen suíno: identificação de novas proteínas no fluido epididimário e variação sazonal das defesas antioxidantes seminais. 2016. Dissertação (Mestrado) – Curso de Biotecnologia, Universidade do Vale do Taquari - Univates, Lajeado, 15 dez. 2016. Disponível em: http://hdl.handle.net/10737/1627.