RESUMO
Our studies have focused on understanding how the initial segment is regulated, in particular, by testicular luminal fluid factor(s). Our working hypothesis is that testicular luminal fluid growth factors, e.g. FGFs, regulate initial segment function via activation of signal transduction pathways and PEA3 family transcription factors. These, in turn, regulate downstream genes that are important for providing an appropriate fluid milieu for the protection and survival of sperm and the initial segment. To test this hypothesis and to look for potential contraceptive targets, we used an in vivo electroporation technique to introduce dominant-negative plasmids of FGF Receptor 1 and ERM into the initial segment of the rat epididymis. The levels of several putative downstream genes were estimated using quantitative-PCR (q-PCR). Data suggests that initial segment genes are regulated by 5alpha-reductase-dependent and -independent pathways and that multiple growth factor pathways are activated to coordinate the expression of these genes.
Assuntos
Anticoncepcionais Masculinos , Proteínas de Ligação a DNA/genética , Epididimo/metabolismo , Fertilização/genética , Regulação da Expressão Gênica , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Fatores de Transcrição/genética , Animais , Colestenona 5 alfa-Redutase/genética , Colestenona 5 alfa-Redutase/metabolismo , Anticoncepcionais Masculinos/farmacologia , Eletroporação , Epididimo/efeitos dos fármacos , Epididimo/enzimologia , Fertilização/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Transferência de Genes , Masculino , Mutação , Plasmídeos/genética , Ratos , Transdução de SinaisRESUMO
Several genes expressed in the initial segment of the epididymis depend on factors from the testis that reach the epididymis via the luminal system. These include gamma-glutamyl transpeptidase mRNA IV (Ggt_pr4), steroid 5 alpha reductase (Srd5a1), glutathione peroxidase 5 (Gpx5), and cystatin-related epididymal spermatogenic (Cst8) genes. Promoter analyses indicated that these genes contain several ETS DNA-binding sites. Members of the polyomavirus enhancer activator 3 (ETV4) family bind to ETS sites on the promoter of target genes to regulate transcription. In this study, the role of ETV4 family members (ETV4, ETV5, ETV1) in the transcription of initial segment specific genes was evaluated. All three ETV4 family mRNAs are expressed in the principal cells of the initial segment and depend upon the presence of testicular luminal fluid factors. ETV4 protein was localized to principal cell nuclei and displayed the highest expression in the most proximal region of the initial segment. In addition, ETV4 protein levels were diminished after loss of testicular luminal fluid factors. A dominant-negative construct of ETV5 was in vivo electroporated into the initial segment to determine if ETV4 family members can regulate the transcription of testicular luminal fluid factor-regulated genes. Quantitative PCR indicated that 1 day postelectroporation, all three ETV4 family member mRNAs were significantly decreased. In addition, Ggt_pr4, Srd5a1, and Gpx5 mRNA levels were also significantly decreased. The data suggest that ETV4 family members regulate their own expression, and that they regulate transcription of a subset of genes that are dependent upon testicular luminal fluid factors.