The Effects of Reactive Oxygen Species on Steroidogenesis in MA-10 Cells

Salil Ginde, and Dale Buchanan, University of Illinois-Chicago, Chicago, IL, 60607

Injection (i.p.) of lipopolysaccharide (LPS) causes a rapid decrease in testosterone production in mice, corresponding to a decrease in Steroidogenic Acute Regulatory (StAR) protein expression and a perturbation of the mitochondrial electrochemical gradient in Leydig cells. LPS is thought to induce the production of reactive oxygen species (ROS) by interstitial testicular macrophages that may disrupt the mitochondrial gradient in adjacent Leydig cells. The activity of StAR protein is dependent upon an intact gradient. Thus we hypothesize that the rapid decrease in testosterone levels in LPS-injected mice results from the acute inhibition in the function of StAR due to a transiently disturbed mitochondrial electrochemical gradient.

To test this hypothesis, MA-10 cells (mouse tumor Leydig cells) were incubated for three hours with treatments of H2O2, a known ROS donor. Radioimmunoassay (RIA) of the media indicated more than 50% inhibition of progesterone synthesis evident at levels of 50uM H2O2, and complete inhibition observed at levels greater than 100uM. Western blot analysis of protein lysates showed that StAR protein is largely inhibited at 50uM H2O2 and completely inhibited at levels of more than 50uM. In parallel TMRE (tetramethylrhodamine ethyl ester) staining of treated cells indicated a complete disruption of mitochondrial electrochemical gradient after treatments with 100uM H2O2, 100uM tert-butyl H2O2, and 50uM cumene H2O2.

Reactive oxygen species inhibit steroidogenesis in MA-10 cells by disrupting the mitochondrial electrochemical gradient, thus causing a decrease in StAR protein function. These results support the hypothesis that LPS injection in vivo inhibits testosterone production in Leydig cells via induction of production of ROS.