Estudo das vias proteômicas do plasma seminal e dos espermatozoides associadas à infertilidade masculina
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Data
2018-05-08
Autores
Lopes, Paula Intasqui [UNIFESP]
Orientadores
Bertolla, Ricardo Pimenta [UNIFESP]
Tipo
Tese de doutorado
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Objetivo: A composição proteica do plasma seminal e dos espermatozoides é essencial para a sua função correta e, consequentemente, para a fertilidade. Assim, estudos proteômicos vêm sendo realizados a fim de entender os mecanismos pós-genômicos subjacentes e de identificar as proteínas alteradas na infertilidade masculina. Portanto, o objetivo deste estudo foi avaliar proteínas de plasma seminal e de espermatozoides e sua associação com alterações funcionais dos espermatozoides e com a infertilidade masculina primária ou secundária. Métodos: Este trabalho foi subdividido em dois estudos. No estudo 1, amostras seminais de 197 homens normozoospérmicos foram utilizadas. Após a coleta e liquefação do sêmen, uma alíquota foi utilizada para a análise seminal, outra para a avaliação da integridade funcional dos espermatozoides e o volume remanescente foi centrifugado para a separação do plasma seminal. Este foi então utilizado para a avaliação dos níveis seminais dos biomarcadores sugeridos: (i) de alterações na atividade mitocondrial dos espermatozoides – GSTM3, (ii) de defeitos no acrossoma dos espermatozoides – PLTP e (iii) de integridade do DNA dos espermatozoides – CRISPLD1, CRISPLD2 e RARRES1. No estudo 2, sete homens com fertilidade comprovada (n=7), nove homens com infertilidade primária (n=9) e sete homens com infertilidade secundária (n=7) foram incluídos. Após a coleta e liquefação do sêmen, uma alíquota foi utilizada para a análise seminal e o volume remanescente foi congelado a –80 °C. No momento da análise proteômica, a amostra foi descongelada e centrifugada para separação dos espermatozoides. Após a extração das proteínas dos espermatozoides, essas foram utilizadas para a análise proteômica shotgun por 1D-LC-MS/MS. As proteínas diferencialmente expressas foram utilizadas para análise de redes de interação proteína-proteína. As proteínas centrais nessas redes, envolvidas com funções biológicas alteradas em ambos os grupos de homens inférteis (BAG6, HSPA2 e SPA17), assim como as proteínas específicas de testículos (HIST1H2BA e SPA17) foram validadas nas amostras de espermatozoides. Nos dois estudos, os biomarcadores foram avaliados por Western blotting. No estudo 2, as proteínas foram também analisadas por imunocitoquímica utilizando microscopia confocal. Resultados: No estudo 1, o nível seminal da proteína GSTM3 estava aumentado em amostras com baixa atividade mitocondrial dos espermatozoides. Por outro lado, o nível das proteínas CRISPLD2 e RARRES1 estava diminuído no plasma seminal de homens com alta fragmentação de DNA dos espermatozoides. As proteínas PLTP e CRISPLD1 não variaram entre os grupos estudados. No estudo 2, um total de 1.305 proteínas de espermatozoides foram identificadas, sendo 102 diminuídas e 15 aumentadas em ambos os grupos de infertilidade. As proteínas diminuídas estavam relacionadas, principalmente, a modificações pós-traducionais e dobramento de proteínas. A proteína BAG6 demonstrou-se significativamente diminuída em homens inférteis, enquanto a proteína HIST1H2BA estava aumentada nesses pacientes. Os níveis das proteínas HSPA2 e SPA17 não diferiram entre os grupos. Na análise por imunocitoquímica, nenhuma diferença quanto à localização dessas proteínas nos espermatozoides foi observada. Conclusão: As proteínas do plasma seminal e dos espermatozoides refletem alterações funcionais dos espermatozoides e a infertilidade masculina.
Objective: The seminal plasma and sperm protein profile is essential for adequate sperm function, and, consequently, for male fertility. Thus, proteomic studies have been undertaken in order to understand underlying post-genomic mechanisms and to identify altered proteins in male infertility. Therefore, the aim of this study was to evaluate seminal plasma and sperm proteins and their association with sperm functional alterations, and with primary or secondary male infertility. Methods: This study was subdivided in two. In study 1, semen samples from 197 normozoospermic men were used. After semen collection and liquefaction, an aliquot was utilized for semen analysis, another for evaluation of sperm functional integrity, and the remaining volume was centrifuged for seminal plasma separation. Seminal plasma was then utilized for the evaluation of the proposed biomarkers: (i) of sperm mitochondrial activity alterations –GSTM3, (ii) of sperm acrosome defects – PLTP, and (iii) of sperm DNA integrity – CRISPLD1, CRIPSLD2 and RARRES1. In study 2, seven proven fertile men (n=7), nine men with primary infertility (n=9), and seven men with secondary infertility (n=7) were included. After semen collection and liquefaction, an aliquot was utilized for semen analysis and the remaining volume was frozen at -80 °C. Prior to their use, samples were thawed and centrifuged for sperm separation. After sperm proteins extraction, these were utilized for shotgun proteomics analysis by 1D-LC-MS/MS. Differentially expressed proteins were then used for protein-protein interaction network analysis. Central proteins in these networks, which were also involved with the altered biological functions in both infertility groups (BAG6, HSPA2 and SPA17), as well as the testis-specific proteins (HIST1H2BA and SPA17) were validated in sperm samples. In both studies, biomarkers were analyzed by Western blotting. In study 2, proteins were also evaluated by immunocytochemistry using confocal microscopy. Results: In study 1, GSTM3 seminal levels were increased in samples with low mitochondrial activity. Also, CRISPLD2 and RARRES1 levels were decreased in the seminal plasma of men with high sperm DNA fragmentation. PLTP and CRISPLD1 proteins did not vary between the studied groups. In study 2, a total of 1,305 sperm proteins were identified, of which 102 were decreased and 15 were increased in both groups of infertility. Decreased proteins were mostly related to protein post-translational modification and folding. BAG6 protein was significantly decreased in infertile men, whereas HIST1H2BA protein was increased in these patients. HSPA2 and SPA17 levels were not different between the groups. In immunocytochemistry analysis, no differences were observed regarding protein localization in sperm. Conclusion: Seminal plasma and sperm proteins reflect sperm functional alterations and male infertility.
Objective: The seminal plasma and sperm protein profile is essential for adequate sperm function, and, consequently, for male fertility. Thus, proteomic studies have been undertaken in order to understand underlying post-genomic mechanisms and to identify altered proteins in male infertility. Therefore, the aim of this study was to evaluate seminal plasma and sperm proteins and their association with sperm functional alterations, and with primary or secondary male infertility. Methods: This study was subdivided in two. In study 1, semen samples from 197 normozoospermic men were used. After semen collection and liquefaction, an aliquot was utilized for semen analysis, another for evaluation of sperm functional integrity, and the remaining volume was centrifuged for seminal plasma separation. Seminal plasma was then utilized for the evaluation of the proposed biomarkers: (i) of sperm mitochondrial activity alterations –GSTM3, (ii) of sperm acrosome defects – PLTP, and (iii) of sperm DNA integrity – CRISPLD1, CRIPSLD2 and RARRES1. In study 2, seven proven fertile men (n=7), nine men with primary infertility (n=9), and seven men with secondary infertility (n=7) were included. After semen collection and liquefaction, an aliquot was utilized for semen analysis and the remaining volume was frozen at -80 °C. Prior to their use, samples were thawed and centrifuged for sperm separation. After sperm proteins extraction, these were utilized for shotgun proteomics analysis by 1D-LC-MS/MS. Differentially expressed proteins were then used for protein-protein interaction network analysis. Central proteins in these networks, which were also involved with the altered biological functions in both infertility groups (BAG6, HSPA2 and SPA17), as well as the testis-specific proteins (HIST1H2BA and SPA17) were validated in sperm samples. In both studies, biomarkers were analyzed by Western blotting. In study 2, proteins were also evaluated by immunocytochemistry using confocal microscopy. Results: In study 1, GSTM3 seminal levels were increased in samples with low mitochondrial activity. Also, CRISPLD2 and RARRES1 levels were decreased in the seminal plasma of men with high sperm DNA fragmentation. PLTP and CRISPLD1 proteins did not vary between the studied groups. In study 2, a total of 1,305 sperm proteins were identified, of which 102 were decreased and 15 were increased in both groups of infertility. Decreased proteins were mostly related to protein post-translational modification and folding. BAG6 protein was significantly decreased in infertile men, whereas HIST1H2BA protein was increased in these patients. HSPA2 and SPA17 levels were not different between the groups. In immunocytochemistry analysis, no differences were observed regarding protein localization in sperm. Conclusion: Seminal plasma and sperm proteins reflect sperm functional alterations and male infertility.
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Citação
LOPES, Paula Intasqui. Estudo das vias proteômicas do plasma seminal e dos espermatozoides associadas à infertilidade masculina. 2018. Tese (Doutorado em Medicina: urologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2018.