Endocrine Abstracts

Androgen signalling plays a central role in the regulation of male reproduction. Androgens act predominantly by activating the classical intracellular androgen receptor (AR). In addition, alternative pathways may account for rapid effects of androgens via cytoplasmic or membrane-localized androgen receptor − ZIP9. In the testis, the main source of androgens, in particular testosterone, are Leydig cells. The proper synthesis of testosterone depends on the structural condition of Leydig cells, and any disturbance in the biosynthesis and availability of steroids may affect the secretory activity of these cells. The initial steps of steroidogenesis occur in the Leydig cells mitochondria, which need to be fully functional. It should be noted that the dynamic changes in the mitochondria (their fission and fusion) are associated with the steroidogenic activity of Leydig cells. Therefore, the aim of this study was to test whether the disruption of androgen signalling may have an effect on the expression of mitochondrial dynamics-involved proteins Drp1, Mfn2 and Tom20 in rodent Leydig cells. Experiments were performed using rat primary Leydig cells and mouse TM3 Leydig cell line. To determine the role of AR signalling in the mitochondria dynamics, pharmacological antagonists − hydroxyflutamide (HF) and bicalutamide (BIC) were used to inhibit activation of AR, and ZIP9 (i) or/and siRNA was used to knockdown the expression of these receptors (ii). Immunofluorescence, western blot, qRT-PCR were used for detection Drp1, Mfn2, and Tom20 expression. We found that testosterone increased the expression of Mfn2 and Drp1, whereas the expression of Tom20 decreased in both primary Leydig cells and TM3 cells. HF, BIC, and both androgen receptors silencing partly blocked testosterone effect on Mfn2 expression, which indicates that Mfn2 is controlled by both receptors. The increase of Drp1 expression was inhibited only by BIC and ZIP9 silencing, which suggests that in Leydig cells Drp1 expression is regulated by ZIP9. In contrast, only HF and AR silencing blocked the effect of testosterone on Tom20 expression demonstrating that this protein is dependent on AR signalling. The above findings were confirmed by the results of immunofluorescence analysis. Collectively, our results indicate a crosstalk between androgen and mitochondrial protein expression in Leydig cells and point to cooperation of classical and non-classical androgen signalling pathways in controlling Leydig cell function. This study was supported by a research Grant OPUS 12 2016/23/B/NZ4/01788 from the National Science Centre. The conference attendance was supported by “Jagiellonian Interdisciplinary PhD Programme” (POWR.03.05.00-00-Z309/17-00) funded by the European Union.