Estudo proteômico das células espermáticas de touros
Carregando...
Arquivos
Data
2017-08-25
Autores
Scott, Caroline [UNESP]
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
O estudo proteômico é utilizado como ferramenta na reprodução animal como auxílio para compreesão da fisiologia das células. Neste sentido esta técnica é empregada em espermatozoides na tentativa de elucidar os processos biológicos e assim determinar os mecanismos moleculares envolvidos na separação do sexo. Assim, o objetivo deste estudo foi descrever as proteínas das células espermáticas de bovinos em diferentes aspectos. No estudo 1, investigou-se a influência do tampão de extração, associado ou não ao método mecânico (flash-frozen), e da concentração celular sobre a quantidade de proteínas extraídas de espermatozoides de bovinos. Foram utilizados como tampões TRIS contendo Nonidet P-40 (NP), RIPA modificado (RP) e uréia/tiuréia/CHAPS (UT), e as concentrações de 2, 4, 6, 8 e 10 x 106 espermatozoides/mL em grupos submetidos ou não ao flash-frozen. As concentrações de proteína total foram quantificadas e gel de eletroforese SDS-1D foi confeccionado. O tratamento UT recuperou maior concentração de proteínas, porém no RP as proteínas apresentaram melhor resolução no gel de eletroforese. A concentração protéica aumentou de acordo com a concentração de células no NP e UT. A influência do flash-frozen variou de acordo com o tratamento. No estudo 2, o objetivo foi traçar o perfil proteico de células espermáticas sexadas (X e Y) de bovinos. Foram utilizadas amostras comerciais de espermatozoides sexados X (n = 6) e Y (n = 6). As proteínas foram solubilizadas, submetidas a espectromia de massas, análise SWATH. Foram identificadas 459 proteínas comuns aos grupos, 10 variaram a abundância relativa (p < 0,05) De acordo com os resultados obtidos nos 2 estudos conclui-se que a concentração de proteína recuperada em uma amostra varia de acordo com o tratamento e concentração celular. Não foram identificadas proteinas exclusivas presentes nos espermatozoides sexados para X ou Y, entretanto deve-se ressaltar que há uma contaminação de 10% com espermatozoides portadores dos cromossomos opostos, ainda assim, este estudo poderá ser utilizado como embasamento científico para novas pesquisas em busca de marcadores sexo-específicos.
The proteomic study is used as a tool in animal reproduction as an aid to understanding of the cells physiology. In this regard, this technique is used in spermatozoa in an attempt to elucidate the biological processes and thus determine the molecular mechanisms involved in the sex separation. Therefore the aim of this study was to describe the bovine sperm cell proteins in different aspects. In study 1, the influence of the extraction buffer, associated or not to mechanical method (flash-frozen), and cellular concentration on the amount of proteins extracted from bovine spermatozoa were investigated. TRIS buffers containing Nonidet P-40 (NP), modified RIPA (RP) and urea/thiourea/CHAPS (UT) were used as well as concentrations of 2, 4, 6, 8 and 10 x 106 spermatozoa/mL in groups submitted or not to flash-frozen. Total protein concentrations were quantified and SDS-1D gel electrophoresis was prepared. The UT treatment recovered a higher concentration of proteins, but in RP the proteins showed better resolution in electrophoresis gel. The protein concentration increased according to the concentration of cells in the NP and UT. The influence of flash-frozen varied according to the treatment. In study 2, the objective was to map the protein profile of sexed sperm cells (X and Y) of cattle. Commercial samples of sexed spermatozoa X (n = 6) and Y (n = 6) were used. The proteins were solubilized, submitted to mass spectrometry SWATH analisys. 459 proteins common to the groups were identified, 10 varied in relative abundance (p <0.05). According to the results obtained in the 2 studies it is concluded that the concentration of recovered protein in a sample varies according to the treatment and cellular concentration. No exclusive proteins were identified in spermatozoa sexed for X or Y, but it should be noted that there is a 10% contamination with spermatozoa bearing the opposite chromosomes, however, this study could be used as a scientific basis for further research in search of sex-specific markers.
The proteomic study is used as a tool in animal reproduction as an aid to understanding of the cells physiology. In this regard, this technique is used in spermatozoa in an attempt to elucidate the biological processes and thus determine the molecular mechanisms involved in the sex separation. Therefore the aim of this study was to describe the bovine sperm cell proteins in different aspects. In study 1, the influence of the extraction buffer, associated or not to mechanical method (flash-frozen), and cellular concentration on the amount of proteins extracted from bovine spermatozoa were investigated. TRIS buffers containing Nonidet P-40 (NP), modified RIPA (RP) and urea/thiourea/CHAPS (UT) were used as well as concentrations of 2, 4, 6, 8 and 10 x 106 spermatozoa/mL in groups submitted or not to flash-frozen. Total protein concentrations were quantified and SDS-1D gel electrophoresis was prepared. The UT treatment recovered a higher concentration of proteins, but in RP the proteins showed better resolution in electrophoresis gel. The protein concentration increased according to the concentration of cells in the NP and UT. The influence of flash-frozen varied according to the treatment. In study 2, the objective was to map the protein profile of sexed sperm cells (X and Y) of cattle. Commercial samples of sexed spermatozoa X (n = 6) and Y (n = 6) were used. The proteins were solubilized, submitted to mass spectrometry SWATH analisys. 459 proteins common to the groups were identified, 10 varied in relative abundance (p <0.05). According to the results obtained in the 2 studies it is concluded that the concentration of recovered protein in a sample varies according to the treatment and cellular concentration. No exclusive proteins were identified in spermatozoa sexed for X or Y, but it should be noted that there is a 10% contamination with spermatozoa bearing the opposite chromosomes, however, this study could be used as a scientific basis for further research in search of sex-specific markers.
Descrição
Palavras-chave
Bovino, Extração-proteína, Proteína-sexo-específica, Bovine, Protein-extraction, Sex-specific-proteina