Abstract
BackgroundAndrogens play an important role for the development of male fertility and gained interest as growth and survival factors for certain types of cancer. Androgens act via the androgen receptor (AR/Ar), which is involved in various cell biological processes such as sex differentiation. To study the functional mechanisms of androgen action, cell culture systems and AR-transfected cell lines are needed. Transfection of AR into cell lines and subsequent gene expression analysis after androgen treatment is well established to investigate the molecular biology of target cells. However, it remains unclear how the transfection with AR itself can modulate the gene expression even without androgen stimulation. Therefore, we transfected Ar-deficient rat Sertoli cells 93RS2 by electroporation using a full length human AR.ResultsTransfection success was confirmed by Western Blotting, immunofluorescence and RT-PCR. AR transfection-related gene expression alterations were detected with microarray-based genome-wide expression profiling of transfected and non-transfected 93RS2 cells without androgen stimulation. Microarray analysis revealed 672 differentially regulated genes with 200 up- and 472 down-regulated genes. These genes could be assigned to four major biological categories (development, hormone response, immune response and metabolism). Microarray results were confirmed by quantitative RT-PCR analysis for 22 candidate genes.ConclusionWe conclude from our data, that the transfection of Ar-deficient Sertoli cells with AR has a measurable effect on gene expression even without androgen stimulation and cause Sertoli cell damage. Studies using AR-transfected cells, subsequently stimulated, should consider alterations in AR-dependent gene expression as off-target effects of the AR transfection itself.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-015-0051-7) contains supplementary material, which is available to authorized users.
Highlights
Androgens play an important role for the development of male fertility and gained interest as growth and survival factors for certain types of cancer
Transfection of 93RS2 cells with the human AR Performing reverse transcription polymerase chain reaction (RT-PCR) with primers specific for mouse and rat Ar, respectively, rat Sertoli cells (93RS2, [18]) proved to lack endogenous Ar (Fig. 1) and were chosen for further experiments
Success of transfection with full length human AR coding DNA sequence (CDS) was validated by immunofluorescence (IF, Fig. 2a), Western Blot (Fig. 2b) and RT-PCR (Fig. 2c)
Summary
Androgens play an important role for the development of male fertility and gained interest as growth and survival factors for certain types of cancer. Transfection of AR into cell lines and subsequent gene expression analysis after androgen treatment is well established to investigate the molecular biology of target cells. It remains unclear how the transfection with AR itself can modulate the gene expression even without androgen stimulation. The action of the most important androgens testosterone (T) and dihydrotestosterone (DHT) is mediated by the androgen receptor (AR/Ar). Since germ cells do not express AR/Ar, the androgen action has to be mediated towards the germ cells by Sertoli cells. No meiotic germ cells were observed in SCARKO mice, showing the importance of a functional AR/Ar on Sertoli cell biology and for the development of germ cells
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