Engeli Roger T, Fürstenberger Cornelia, Kratschmar Denise V, Odermatt Alex
Swiss Centre for Applied Human Toxicology and Division of Molecular and Systems Toxicology, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland.
Heliyon. 2018 Feb 1;4(2):e00527. doi: 10.1016/j.heliyon.2018.e00527. eCollection 2018 Feb.
Androgen biosynthesis in males occurs to a large extent in testicular Leydig cells. This study focused on the evaluation of three murine Leydig cell lines as potential screening tool to test xenobiotics interfering with gonadal androgen synthesis. The final step of testosterone (T) production in Leydig cells is catalyzed by the enzyme 17β-hydroxysteroid dehydrogenase 3 (17β-hsd3). The endogenous 17β-hsd3 mRNA expression and Δ4-androstene-3,17-dione (AD) to T conversion were determined in the murine cell lines MA-10, BLTK1 and TM3. Additionally, effects of 8-Br-cAMP and forskolin stimulation on steroidogenesis and T production were analyzed. Steroids were quantified in supernatants of cells using liquid chromatography-tandem mass spectrometry. Unstimulated cells incubated with AD produced only very low T but substantial amounts of the inactive androsterone. Stimulated cells produced low amounts of T, moderate amounts of AD, but high amounts of progesterone. Gene expression analyses revealed barely detectable 17β-hsd3 levels, absence of 17β-hsd5 (Akr1c6), but substantial 17β-hsd1 expression in all three cell lines. Thus, MA-10, BLTK1 and TM3 cells are not suitable to study the expression and activity of the gonadal T synthesizing enzyme 17β-hsd3. The low T production reported in stimulated MA-10 cells are likely a result of the expression of 17β-hsd1. This study substantiates that the investigated Leydig cell lines MA-10, BLTK1, and TM3 are not suitable to study gonadal androgen biosynthesis due to altered steroidogenic pathways. Furthermore, this study emphasizes the necessity of mass spectrometry-based steroid quantification in experiments using steroidogenic cells such as Leydig cells.
雄性体内的雄激素生物合成在很大程度上发生于睾丸间质细胞。本研究着重评估三种小鼠间质细胞系，将其作为检测干扰性腺雄激素合成的外源性物质的潜在筛选工具。间质细胞中睾酮（T）生成的最后一步由17β-羟基类固醇脱氢酶3（17β-hsd3）催化。在小鼠细胞系MA-10、BLTK1和TM3中测定了内源性17β-hsd3 mRNA表达以及Δ4-雄烯二酮（AD）向T的转化。此外，分析了8-溴环磷腺苷（8-Br-cAMP）和福司可林刺激对类固醇生成和T产生的影响。使用液相色谱-串联质谱法对细胞上清液中的类固醇进行定量。用AD孵育的未刺激细胞仅产生极低水平的T，但产生大量无活性的雄甾酮。刺激后的细胞产生少量的T、适量的AD，但产生大量的孕酮。基因表达分析显示，在所有三种细胞系中，17β-hsd3水平几乎检测不到，不存在17β-hsd5（Akr1c6），但17β-hsd1有大量表达。因此，MA-10、BLTK1和TM3细胞不适合用于研究性腺T合成酶17β-hsd3的表达和活性。刺激后的MA-10细胞中报道的低T产生可能是17β-hsd1表达的结果。本研究证实，由于类固醇生成途径改变，所研究的间质细胞系MA-10、BLTK1和TM3不适合用于研究性腺雄激素生物合成。此外，本研究强调了在使用诸如间质细胞等类固醇生成细胞的实验中，基于质谱的类固醇定量的必要性。
