Validação de um painel de biomarcadores proteicos no plasma seminal de adolescentes com e sem varicocele

Objective: To verify the seminal levels of potential biomarkers for testicular changes in adolescent varicocele. The specific objectives were to verify if the presence of varicocele and seminal alterations can alter the levels of the following proteins: Cab45 (45 kDa calcium-binding protein); LEFTY1 (Left-right determination factor 1); DNase I (deoxyribonuclease-1); PAP2-Alpha (Lipid phosphate phosphohydrolase 1); IFGBP7 (Insulin-like growth factor binding protein-7); IGHG3 (Ig gamma-3 chain C region); CRISP-3 (Cysteine-rich secretory protein 3). Method: This observational study included 61 adolescents, aged 15 to 17 years old with full sexual maturity (Tanner V), which were divided into 3 groups: control (without varicocele, n=20); VSN (with varicocele, normal semen analysis, n=22); and VSA (with varicocele and altered semen analysis, n=19). The adolescents collected one seminal sample after 2 to 5 days of ejaculatory abstinence. After semen liquefaction semen was centrifuged, in order to separate the seminal plasma from cellular constituents. Total protein in each sample was then quantified, and a volume correspondent to 50 ?g of protein for each patient was used for Western blotting for the follow proteins: Cab 45, LEFTY1, DNase I, PAP2-Alpha, IGFBP7, IGHG3 and CRISP-3 (all were normalized to the protein Epididymis Secretory Sperm Binding protein 67p Li [DJ-1]). Results: We observed lower sperm concentration (million/mL) and morphology (% normal sperm) in the VSA group when compared to the other groups. There was a decrease in the levels of Cab45 in both groups of adolescents with varicocele (VSN and VSA), with a lowest value in the VSA group. DNase I levels were lower in the group of adolescents with varicocele and altered semen analysis (VSA). There was an overexpression of IGFBP7 in the groups with varicocele when compared with control groups, and an overexpression of the two isoforms of CRISP-3 protein in the VSA group. Conclusion: Proteomics of seminal plasma reflects changes in testicular function in the adolescent varicocele. The levels of proteins LEFTY1, PAP2-Alpha, IGHG3 are not altered in the presence of a varicocele. Cab45 levels are decreased in the presence of varicocele; DNase I levels are decreased in the presence of seminal alterations and varicocele. Conversely, IGFBP7 is a positive marker of varicocele, and CRISP-3 is a positive marker of varicocele and altered semen quality. These results may help in the future to determine the medical management for early surgical intervention in varicocele.Objetivo: Verificar o nível de expressão de proteínas com potencial biomarcador para alteração testicular na varicocele em adolescentes. Os objetivos específicos foram verificar se a presença de varicocele e de alteração seminal trazem alterações nos níveis das seguintes proteínas: Cab45 (45 kDa calcium-binding protein); LEFTY1 (Left-right determination factor 1); DNase I (Deoxyribonuclease-1); PAP2-Alpha (Lipid phosphate phosphohydrolase 1); IFGBP7 (Insulin-like growth factor-binding protein 7); IGHG3 (Ig gamma-3 chain C region); CRISP-3 (Cysteine-rich secretory protein 3). Método: Este estudo observacional foi constituído por 61 adolescentes, com idade entre 15 e 17 anos e, maturidade sexual completa (Tanner V), que foram divididos em 3 grupos: Controle (sem varicocele, n=20); VSN (com varicocele e sêmen normal, n=22); e VSA (com varicocele e sêmen alterado, n=19). Os adolescentes coletaram 1 amostra seminal com 2 a 5 dias de abstinência ejaculatória. Após a liquefação, as amostras foram submetidas à centrifugação para que o plasma seminal fosse separado dos demais constituintes celulares e, então, após a quantificação total de proteínas, um volume correspondente a 50 µg de proteína por adolescente foi utilizado para a quantificação das seguintes proteínas Cab 45, LEFTY1, DNase I, PAP2-Alpha, IGFBP7, IGHG3 e CRISP-3 (todas foram normalizadas para a proteína Epididymis Secretory Sperm Binding Protein Li 67p [DJ-1]), pelo método de Western blotting. Resultados: Os indivíduos pertencentes ao grupo VSA apresentaram menor média de concentração de espermatozoides (milhões/mL) e células morfologicamente normais (% normais) com relação aos demais grupos experimentais. Observou-se diminuição nos níveis da proteína Cab45 em ambos os grupos de adolescentes com varicocele (VSN e VSA), sendo que o grupo VSA apresentou menor média. Os níveis da proteína DNase I estavam menores no grupo de adolescentes com varicocele e sêmen alterado (VSA). Observou-se uma hiperexpressão da proteína IGFBP7 nos grupos com varicocele quando comparados ao grupo controle e, uma hiperexpressão das duas isoformas da proteína CRISP-3 no grupo VSA. Conclusão: A proteômica do plasma seminal reflete alterações nas funções testiculares na varicocele em adolescentes. Os níveis das proteínas LEFTY1, PAP2-Alpha, IGHG3 não estão alterados com a presença de varicocele. Os níveis de Cab45 estão diminuídos na presença de varicocele; os níveis de DNase I estão diminuídos na presença de alterações seminais e varicocele. Por um lado, a proteína IFGBP7 é um marcador positivo para varicocele, e a CRISP-3 é um marcador positivo para variococele e qualidade seminal alterada. Esses resultados podem futuramente ajudar na determinação da conduta médica para uma intervenção cirúrgica precoce na varicocele.Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Dose-Dependent Effects and Reversibility of the Injuries Caused by Nandrolone Decanoate in Uterine Tissue and Fertility of Rats

This study is the first to investigate the effects of different doses of nandrolone decanoate (ND) upon uterine tissue and fertility, and if the reproductive alterations can be restored after cessation of the treatment. Wistar female rats were treated with ND at doses of 1.87, 3.75, 7.5, and 15mg/kg body weight, diluted in vehicle (n = 30/group), or received only mineral oil (control group, n = 45). The animals were divided into three periods of study: ND-treated receiving a daily subcutaneous injection for 15 consecutive days (1), and treatment with ND followed by 30-day recovery (2), and 60-day recovery (3). At the end of each period, five females per group were induced to death to histopathological analysis and the others were allowed to fertility evaluation (at 19th gestational day). Animals that received ND followed by 30-day recovery exhibited persistent diestrous and marked suppression of reproductive capacity. Conversely, after 60-day recovery, only lowest doses females (1.87 and 3.75mg/kg) exhibited restoration of normal estrous cyclicity. Uterine weights were increased after ND treatment similarly to that of the controls after 60-day recovery. The ND-treated groups showed histopathological changes in the endometrium, myometrium, and perimetrium, and an increase in the thickness of both muscular and serous layers. Notably, the recovery of uterine tissue after ND treatment was dose- and period-dependent. We reported that administration of ND promoted damage in uterine tissue and fertility of rats, and the recovery periods were insufficient to restore all of the side effects caused by ND under a dose-dependent responseFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP