TGF-β1 signaling regulates steroidogenic gene expression, affecting testicular testosterone levels in mice.

Abstract

<p>(<b>A</b>) Decreased Tgfbr2<sup>fl</sup>°<sup>x</sup> allele in purified primary Leydig cells isolated from mice harboring the Cyp17iCre transgene. The genomic DNA isolated from primary Leydig cells of Tgfbr2<sup>flox/flox</sup> and Tgfbr2<sup>flox/flox</sup> Cyp17iCre mice was amplified for Tgfbr2 intron region containing the LoxP site. A pair of β-actin primers was used as the control for the amount of genomic DNA. (<b>B</b>) Decreased TGF-β1-mediated repression of steroidogenic gene expression with Tgfbr2 silencing. Purified primary Leydig cells from the testes of 12-week-old Tgfbr2<sup>flox/flox</sup> (n = 6) and Tgfbr2<sup>flox/flox</sup> Cyp17iCre (n = 6) mice were treated with 300 µM of 8-Br-cAMP and 2 ng/ml of TGF-β1 for 24 hours, and mRNA expression levels were measured using qRT-PCR. β-actin expression was used as a loading control. The data are presented as the mean ± SEM. **, P<0.01; ***, P<0.01. (<b>C</b>) Testicular testosterone levels were measured by RIA in the testes of 5 week-old Tgfbr2<sup>flox/flox</sup> and Tgfbr2<sup>flox/flox</sup> Cyp17iCre mice. (<b>D</b>) Total protein (100 µg) from the testes of 5 week-old Tgfbr2<sup>flox/flox</sup> and Tgfbr2<sup>flox/flox</sup> Cyp17iCre mice was subjected to western blot analysis for protein levels of steroidogenic genes. The relative level of each protein/GAPDH was quantified by densitometric analysis using Image J software. In panels C and D, the data are presented as the mean ± SD (n = 10). **, P<0.01.</p

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