Submission Number: JOH-4-13-7

Abstract Number: 338

REACTIVE OXYGEN DISRUPTS MITOCHONDRIA IN LEYDIG CELLS AND INHIBITS STEROIDOGENESIS.

J Allen ¹, Salil Ginde ¹, John Choi ¹, Thorsten Diemer* ^1,2, Karen Held-Hales ¹ and Dale Buchanan Hales* ¹

Department of Physiology & Biophysics, University of Illinois at Chicago, Chicago, IL ¹
Department of Urology, University Hospital, Justus-Liebig-University, Giessen, Germany ²

Abstract:
Previous investigations have established that reactive oxygen species (ROS) profoundly inhibit steroidogenesis in both mouse MA-10 tumor Leydig cells and rat luteal cells, but the mechanism of this inhibition has not been determined. Testicular macrophages are in close proximity to Leydig cells in the interstitium of the testis, and they are known to secrete ROS. The objective of this study is to determine the mechanism through which ROS inhibit Leydig cell steroidogenesis.

Both MA-10 cells and mouse Leydig cells in primary culture were stimulated with cAMP followed by treatment with varying concentrations of H2O2 for 3-6 hours. Synthesis of steroids was assessed by RIA. Northern and Western Blot analyses were performed to measure the expression of three key steroidogenic proteins: StAR, 3-HSD, and P450scc. Cells were stained with the potential-sensitive dye TMRE to assess disturbances in the mitochondrial electrochemical gradient (m).

RIA data indicated a significant (50%) decrease in progesterone synthesis in MA-10 cells after 250uM H2O2 treatment, and a significant (30%) decrease of testosterone production by Leydig cells. Northern Blot data showed StAR mRNA expression in MA-10 cells was not effected by H2O2 treatment for the concentration range used in the in vitro experiments (100uM-500uM). Western blot analysis of MA-10 cells showed 100uM H2O2 reduced StAR protein by 30% and 3-HSD protein by 30-40%. Treatment with 250uM H2O2 decreased StAR expression by 90% in MA-10 cells, and 50% in Leydig cells. In addition, StAR expression in MA-10 cells recovered 50% after H2O2 treatments were replaced with cAMP, demonstrating the transient nature of the effects of ROS. TMRE staining of MA-10 and Leydig cells indicated alterations of m after 100uM H2O2 treatment and profound disruption at 250uM H2O2.

These results indicate that ROS inhibit steroidogenesis in Leydig cells by decreasing expression of StAR and 3-HSD and disrupting m. We propose that disruption of mitochondrial function by reactive oxygen is a specific mechanism which inhibits testosterone biosynthesis in Leydig cells.

Source of Funding: NIH HD 35544 to D.B. Hales (USA);DFG Di 723/1-1 to T. Diemer (Ger) .

Keywords: reactive oxygen species, ROS, Leydig cells, steroidogenesis, mitochondria

This abstract is being presented at: 8:00 AM in session:
Gonadal Steriods