RESUMO
<p><b>AIM</b>To analyze the functional interactions of Cyclin with p53 and Atm in spermatogenesis and DNA double-strand break repair.</p><p><b>METHODS</b>Two lines of double knockout mice were generated. Spermatogenesis and double strand break repair mechanisms were analyzed in Cyclin A1 (Ccna1); p53- and Ccna1; Atm-double knockout mice.</p><p><b>RESULTS</b>The block in spermatogenesis observed in Cyclin A1-/- (Ccna1-/-) testes at the mid-diplotene stage is associated with polynucleated giant cells. We found that Ccna1-deficient testes and especially the giant cells accumulate unrepaired DNA double-strand breaks, as detected by immunohistochemistry for phosphorylated H2AX. In addition, the giant cells escape from apoptosis. The development of giant cells occurred in meiotic prophase I, because testes lacking ATM, which are known to develop spermatogenic arrest earlier than prophase I, do not develop giant cells in the absence of cyclin A1. Cyclin A1 interacted with p53 and phosphorylated p53 in complex with CDK2. Interestingly, p53-deficiency significantly increased the number of giant cells in Ccna1-deficient testes. Gene expression analyses of a panel of DNA repair genes in the mutant testes revealed that none of the genes examined were consistently misregulated in the absence of cyclin A1.</p><p><b>CONCLUSION</b>Ccna1-deficiency in spermatogenesis is associated with defects in DNA double-strand break repair, which is enhanced by loss of p53.</p>
Assuntos
Animais , Masculino , Camundongos , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular , Genética , Fisiologia , Proteínas de Ciclo Celular , Genética , Metabolismo , Ciclina A , Genética , Metabolismo , Ciclina A1 , Ciclina B , Ciclina B2 , DNA , Genética , Reparo do DNA , Genética , Fisiologia , Proteínas de Ligação a DNA , Genética , Metabolismo , Regulação da Expressão Gênica , Genética , Fisiologia , Camundongos Knockout , Proteínas Serina-Treonina Quinases , Genética , Metabolismo , Espermatogênese , Genética , Fisiologia , Testículo , Biologia Celular , Metabolismo , Proteína Supressora de Tumor p53 , Genética , Metabolismo , Proteínas Supressoras de Tumor , Genética , MetabolismoRESUMO
<p><b>AIM</b>Although epidermal growth factor receptors are expressed in the testes, whether they signal through epidermal growth factor receptor pathway substrate 8 (Eps8) is unknown. Here we evaluated the expression pattern of Eps8 in the maturing testis.</p><p><b>METHODS</b>The expression of Eps8 was analysed by Northern blotting, immunocytochemistry and Western blotting in primary Sertoli cell cultures and in testicular tissue of rodents.</p><p><b>RESULTS</b>Eps8 is specifically expressed in gonocytes, Leydig and Sertoli cells of the neonatal rats and in Leydig and Sertoli cells of the adult rats and mice. Although gonocytes express Eps8, no signal was found in prepubertal or mature spermatogonia and the expression level of Eps8 in Sertoli cells increases with age. No regulation of Eps8 expression in primary immature rat Sertoli cells by Follicle stimulating hormone (FSH) was detected by Western blotting.</p><p><b>CONCLUSION</b>Eps8 seems to be involved in the growth factor-controlled regulation of cell proliferation and differentiation in the seminiferous epithelium. Eps8 is a possible marker for gonocytes and in Sertoli cells it could be involved in crosstalk with other growth factor pathways.</p>
Assuntos
Animais , Masculino , Ratos , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Bases , Northern Blotting , Linhagem Celular , Primers do DNA , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Células Intersticiais do Testículo , Biologia Celular , Fisiologia , Proteínas , Genética , RNA , Genética , Ratos Sprague-Dawley , Células de Sertoli , Fisiologia , Maturidade Sexual , Espermatócitos , Biologia Celular , Fisiologia , TestículoRESUMO
<p><b>AIM</b>To specifically express the Asp567Gly human follicle-stimulating hormone receptor (FSHR) under the control of its promoter to evaluate the phenotypic consequences in the presence of normal pituitary function.</p><p><b>METHODS</b>We produced transgenic mice overexpressing the Asp567Gly human FSHR under the control of a 1.5kb 5'-flanking region fragment of its promoter.</p><p><b>RESULTS</b>Mice were phenotypically normal and fertile. In males, mRNA could be detected in the testis and the brain, indicating that the 1.5kb promoter fragment drives expression not only in the gonads. The testis weight/body weight ratio and the testosterone levels in transgenic and non-transgenic littermates were similar. By in situ hybridisation we found that the transgenic FSHR was highly expressed in Sertoli cells, spermatocytes and round spermatids. However, a radioligand receptor assay failed to show a significant difference in total FSHR binding sites in testis homogenates of transgenic and wild type animals, suggesting that the transgenic FSHR is probably not translated into functional receptor protein.</p><p><b>CONCLUSION</b>A 1.5kb 5'-region of the human FSHR drives mRNA expression of the transgene in the testis but leads to ectopic expression in germ cells and in the brain. No phenotypic consequences could be documented due to the lack of protein expression.</p>