Reactive oxygen disrupts mitochondria in MA-10 tumor leydig cells and inhibits steroidogenic acute regulatory (STAR) protein and steroidogenesis

Reactive oxygen species (ROS) are involved in a variety of pathophysiological conditions of the testis, and oxidative stress is known to inhibit ovarian and testicular steroidogenesis. The site of ROS-mediated inhibition of steroidogenesis in the corpus luteum and MA-10 tumor Leydig cells was shown to be the hormone-sensitive mitochondrial cholesterol transfer step. The purpose of this study was to examine the effects of ROS on steroidogenic acute regulatory (StAR) protein in MA-10 cells and determine the extent to which MA-10 cell mitochondria are sensitive to oxidative stress. cAMP-stimulated progesterone production was inhibited in a dose-dependent manner in MA-10 cells exposed to H₂O₂. StAR protein, but not mRNA levels, was decreased in parallel to changes in progesterone production. Even at the highest concentrations of H₂O₂ tested, there was no effect on P450 side-chain cleavage enzyme protein levels. Oxidative stress from exposure to exogenous xanthine oxidase and xanthine resulted in the inhibition of both progesterone production and StAR protein expression. The mature 30- and 32-kDa intramitochondrial forms of StAR were decreased relative to the 37-kDa extramitochondrial precursor form of STAR, indicating that the ROS-mediated inhibition of StAR protein was due, in part, to the inhibition of mitochondrial import and processing. Vital staining with the fluorescent dye tetramethylrhodamine ethyl ester was used to visualize changes in the mitochondrial electrochemical gradient-dependent membrane potential (Δψm). ROS caused a significant dissipation of Δψ_m and time-dependent loss of tetramethylrhodamine ethyl ester fluorescence. The inhibitory effects of H₂O₂ were transient. There was no evidence for ROS-induced cell death, and following H₂O₂ removal in the presence of continuous treatment with 8-bromo-cAMP, StAR protein levels and progesterone production were restored. In addition, there was no loss of cell viability following treatment with H₂O₂ or xanthine/xanthine oxidase as determined by trypan blue exclusion. H₂O₂ did not cause a significant decrease in total cellular ATP levels. These data indicate that oxidative stress-mediated perturbation of the mitochondria and dissipation of Δψ_m results in the inhibition of StAR protein expression and its import, processing, and cholesterol transfer activity. These findings confirm earlier studies demonstrating the requirement for maintenance of an intact Δψ_m for StAR protein function in cholesterol transport. The significant reduction in the 32- to 30-kDa mature forms of STAR, cessation of cholesterol transport, and loss of Δψ_m are consistent with mitochondrial perturbation because of oxidative stress. This mechanism likely contributes to a host of pathophysiological events evident in testicular disorders such as infection, reperfusion injury, aging, cryptorchidism, and varicocele.