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Transfection of Sertoli cells with androgen receptor alters gene expression without androgen stimulation.

Fietz D, Markmann M, Lang D, Konrad L, Geyer J, Kliesch S, Chakraborty T, Hossain H, Bergmann M - BMC Mol. Biol. (2015)

Bottom Line: Microarray results were confirmed by quantitative RT-PCR analysis for 22 candidate genes.We 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Veterinary Anatomy, Histology and Embryology, Justus Liebig University, Frankfurter Straße 98, 35392, Giessen, Germany. Daniela.Fietz@vetmed.uni-giessen.de.

ABSTRACT

Background: Androgens 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.

Results: Transfection 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.

Conclusion: We 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.

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Related in: MedlinePlus

Illustration of eight genes and their association to known pathways in IPA. Green color denotes down-regulation, whereas red color denotes up-regulation
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Fig5: Illustration of eight genes and their association to known pathways in IPA. Green color denotes down-regulation, whereas red color denotes up-regulation

Mentions: Not only metabolism and immune response gene expression seem to be altered in transfected Sertoli cells, but also cell cycle and development genes (desert hedge hog (Dhh) FC = −2.032; fibroblast growth factor receptor 2 (Fgfr2) FC = −8.239; follistatin (Fst) FC = 2.162; inhibin beta b (Inhbb) FC = −3.126). Dhh is involved in various areas of embryonic development, including testicular cord formation. It is expressed in mouse Sertoli cell precursors during mid- to late gestation [33] and also is important for germ cell development after puberty in mouse [34] and rat testis [35]. In the mouse, a lack of Dhh results in a severe impairment of spermatogenesis due to a lack of spermatogonial development beyond primary spermatocytes [34]. Fgfr2 is a known differentiation factor in prenatal Sertoli cells as it is concomitantly expressed with Sry and is essential for subsequent expression of anti-muellerian hormone (Amh) and Sox9 [36]. IPA analysis of upstream regulation predicted an inhibition of transcription factor Sox9 with a z-score of −2.2 (Table 6). Moreover, lack of Fgfr2 might cause a partial XY sex reversal, as loss of Fgfr2 leads to an up-regulation of Follistatin (Fst), a female somatic cell marker [37], which was confirmed by microarray and RT-qPCR. A down-regulation of the Sertoli cell marker Inhbb (for review see [38]) also points to a decreased Sertoli cell function and a severe disturbance of spermatogenesis in the rat [39]. Figure 5 shows the association of Inhbb, Fst, Dhh, Pmepa1, Fgfr2, Ptgs2, Tf and Myc as especially interesting genes on known pathways as predicted by IPA. Fig. 5


Transfection of Sertoli cells with androgen receptor alters gene expression without androgen stimulation.

Fietz D, Markmann M, Lang D, Konrad L, Geyer J, Kliesch S, Chakraborty T, Hossain H, Bergmann M - BMC Mol. Biol. (2015)

Illustration of eight genes and their association to known pathways in IPA. Green color denotes down-regulation, whereas red color denotes up-regulation
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4696255&req=5

Fig5: Illustration of eight genes and their association to known pathways in IPA. Green color denotes down-regulation, whereas red color denotes up-regulation
Mentions: Not only metabolism and immune response gene expression seem to be altered in transfected Sertoli cells, but also cell cycle and development genes (desert hedge hog (Dhh) FC = −2.032; fibroblast growth factor receptor 2 (Fgfr2) FC = −8.239; follistatin (Fst) FC = 2.162; inhibin beta b (Inhbb) FC = −3.126). Dhh is involved in various areas of embryonic development, including testicular cord formation. It is expressed in mouse Sertoli cell precursors during mid- to late gestation [33] and also is important for germ cell development after puberty in mouse [34] and rat testis [35]. In the mouse, a lack of Dhh results in a severe impairment of spermatogenesis due to a lack of spermatogonial development beyond primary spermatocytes [34]. Fgfr2 is a known differentiation factor in prenatal Sertoli cells as it is concomitantly expressed with Sry and is essential for subsequent expression of anti-muellerian hormone (Amh) and Sox9 [36]. IPA analysis of upstream regulation predicted an inhibition of transcription factor Sox9 with a z-score of −2.2 (Table 6). Moreover, lack of Fgfr2 might cause a partial XY sex reversal, as loss of Fgfr2 leads to an up-regulation of Follistatin (Fst), a female somatic cell marker [37], which was confirmed by microarray and RT-qPCR. A down-regulation of the Sertoli cell marker Inhbb (for review see [38]) also points to a decreased Sertoli cell function and a severe disturbance of spermatogenesis in the rat [39]. Figure 5 shows the association of Inhbb, Fst, Dhh, Pmepa1, Fgfr2, Ptgs2, Tf and Myc as especially interesting genes on known pathways as predicted by IPA. Fig. 5

Bottom Line: Microarray results were confirmed by quantitative RT-PCR analysis for 22 candidate genes.We 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Veterinary Anatomy, Histology and Embryology, Justus Liebig University, Frankfurter Straße 98, 35392, Giessen, Germany. Daniela.Fietz@vetmed.uni-giessen.de.

ABSTRACT

Background: Androgens 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.

Results: Transfection 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.

Conclusion: We 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.

Show MeSH
Related in: MedlinePlus