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

Abstract

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.

A Testis-Specific Long Noncoding RNA, Start, Is a Regulator of Steroidogenesis in Mouse Leydig Cells

“…In TM3 cells, Star gene expression was significantly increased by Start overexpression (Figure 10B), and in MA-10 cells, not only Star but also Cyp17a1, Hsd3b1, and Hsd17b3 genes were significantly upregulated by Start (Figure 10D). We failed to detect testosterone in the culture media of MA-10 cells overexpressing Start, probably due to low capability of testosterone production of this cell line as previously reported (67,68). These results supported the notion that Start acts as an activator of steroidogenic genes in Leydig cells, as shown by results for 8-day-old mice (Figure 9A).…”

Androgenic effect of aqueous leaf extract of Moringa oleifera on Leydig TM3 cells in vitro

“…Several studies had previously established that TM3 cells are suitable for use in the determination of testosterone production (Ilfergane & Henkel, 2018; Jambor et al., 2019; Lin et al., 2019; Liu et al., 2016; Xia et al., 2020). However, another study demonstrated that unstimulated and stimulated TM3 cells produced a low amount of testosterone and a high amount of progesterone, suggesting that TM3 cells are unsuitable for studying the gonadal androgen biosynthesis due to altered steroidogenic pathways (Engeli, Fürstenberger, Kratschmar, & Odermatt, 2018). However, the effect of MO on progesterone levels in the TM3 cells was not included in this present study.…”

A 35-bp Conserved Region Is Crucial for Insl3 Promoter Activity in Mouse MA-10 Leydig Cells

“…Contrary to primary Leydig cells, MA-10 cells proliferate and contain an aberrant chromosome number [ 32 ]. Despite these issues, MA-10 cells nonetheless respond to hormonal stimulation, like primary Leydig cells, with an increase in steroid hormone production [ 30 , 33 , 34 , 35 , 36 ], indicating that the signalling cascades, kinases, and transcription factors required for this response are present in MA-10 Leydig cells. More relevant to our current work, MA-10 Leydig cells constitutively express Insl3 [ 13 , 19 , 22 , 23 , 27 , 37 , 38 , 39 ] and have been validated as a suitable model to study Insl3 gene transcription [ 27 ].…”