Most aging men and many young men have reduced circulating testosterone levels, referred to as hypogonadism. Hypogonadism is estimated to affect about 5 million American men, including both aged and young. Low testosterone can have negative effects on mood, cognition, cardiovascular health, energy, libido, muscle mass, and bone density.Leydig cells are the testicular cells responsible for testosterone biosynthesis.A number of cellular changes have been identified in the steroidogenic pathway of aged Leydig cells that are associated with reduced testosterone formation, including reductions in luteinizing hormone (LH)-stimulated cAMP production, the cholesterol transport proteins steroidogenic acute regulatory (STAR) protein and translocator protein (TSPO), and downstream steroidogenic enzymes of the mitochondria and smooth endoplasmic reticulum. Many of the changes in steroid formation that characterize aged Leydig cells can be elicited by the experimental alteration of the redox environment of young cells, suggesting that changes in the intracellular redox balance may cause reduced testosterone production. As yet, cause-effect relationships have not been established, however.Reduced glutathione (GSH), the most abundant intracellular small molecule thiol present in mammalian cells, serves as a potent intracellular antioxidant and is particularly abundant in Leydig cells. GSH decreases significantly as Leydig cells age. Given the abundance of GSH in Leydig cells, we hypothesized that the experimental depletion of GSH would result in an increasingly pro-oxidant intracellular environment, and that this would cause reduced steroidogenic function. Buthionine sulfoximine (BSO), a specific γ-glutamylcysteine synthetase inhibitor, can block the rate-limiting step of GSH biosynthesis, and by doing so deplete the intracellular GSH pool in both cultured cells and whole animals. To test the effect of decreased GSH on steroid formation, we first cultured MA-10 Leydig cells with BSO short-term (24 hours). This treatment reduced intracellular levels of GSH somewhat but had no effect on progesterone production by these cells at this early time. Nor did treatment of the cells with tert-butyl hydroperoxide (t-BuOOH, 2 hours), an oxidant. However, when the GSH-depleted cells subsequently were exposed acutely to t-BuOOH, intracellular reactive oxygen species concentration was significantly increased, and this was accompanied by reductions in steroid production. These results suggested that alteration of the intracellular redox environment can result in the increased sensitivity of MA-10 cells to oxidative stress under circumstances in which the cells have reduced antioxidant capacity. In a second series of studies, cells were cultured for days to weeks with BSO alone. Over time, at times after GSH was reduced, steroid synthesis decreased, reminiscent of natural aging of primary Leydig cells. These results suggest that, as in aging, exposure to an increasingly pro-oxidant environment, over time, can have a negative impact on Leydig cell steroidogenic function, and that, indeed, increases in oxidative stress contribute to or cause the reduced testosterone production that characterizes Leydig cells aging.As of yet, the molecular mechanisms by which aging results in an altered redox environment remains unclear.Also uncertain is the molecular mechanism by which reductions in GSH and other antioxidant molecules in Leydig cells, and thus increased oxidative stress, elicit reduced sensitivity to LH and thus reduced steroid formation.
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Effects of Glutathione Depletion on Leydig Cell Aging and Steroidogenesis
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