Currently available murine Leydig cell lines can be applied to study early steps of steroidogenesis but not testosterone synthesis.

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.