| Leydig cells, the testicular cells responsible for testosterone production, do not turn over once formed during the peripubertal period. Previous studies have shown that intracellular reactive oxygen species (ROS) increase as Leydig cells age, and that this increase is correlated with reduced Leydig cell testosterone production. Although this has suggested that increased ROS might cause decreased testosterone, a cause-effect relationship has not been proven. Moreover, the mechanisms by which ROS increase with age are poorly understand, as are the effect of ROS increases on Leydig cell macromolecules, and on how aging Leydig cells respond to acute changes in their environment. These issues are addressed in the thesis.;The redox environment of a cell results from a combination of ROS production and the ability of the antioxidant system to counter the effects of oxidizing molecules and thus protect cellular macromolecules. We hypothesized that the experimental depletion of glutathione (GSH), an abundant Leydig cell intracellular antioxidant, would result in reduced testosterone production. Incubation of Leydig cells isolated from the testes of adult Brown Norway rats with buthionine sulfoximine (BSO) reduced GSH content by more than 70% and testosterone production by about 40%. The antioxidants vitamin E, N-tert-butyl-α-phenylnitrone and Trolox countered BSO's effect on steroidogenesis but not on GSH depletion. Together, BSO and glutathione ethyl ester maintained intracellular GSH and also testosterone production, whereas 1,2-dithiole-3-thione, which increases intracellular GSH, increased testosterone production. In vivo studies also were conducted. Young (4 month old) and old (24 month old) rats were injected with BSO twice a day for 7 d, after which Leydig cells were isolated and analyzed in vitro. BSO treatment reduced Leydig cell GSH content by 70% and the ability of the Leydig cells to produce testosterone by more than 50%. As with aging, decreases were seen in LH-stimulated cAMP production, steroidogenic acute regulatory protein, cholesterol side-chain cleavage, 3β-hydroxysteroid dehydrogenase, and 17α-hydroxylase/17,20-lyase. The results of these studies, taken together, are consistent with the conclusion that alteration in the oxidant/antioxidant environment plays a causative role in the age-related reduced ability of Leydig cells to produce testosterone.;Next, we hypothesized that aging cells, with an increasingly prooxidant intracellular environment, might be particularly susceptible to acute oxidative stress. To test this, MA-10 Leydig tumor cells were incubated with BSO or diethyl maleate (DEM) so as to deplete glutathione (GSH), and then exposed to the prooxidant tert-butylhydroperoxide (t-BuOOH). This increased intracellular ROS concentration and resulted in reduced progesterone production. In contrast, treatment of control cells with t-BuOOH had no effect. Depletion of GSH and subsequent treatment of the cells with t-BuOOH induced the phosphorylation of each of ERK1/2, JNK and p38, members of the MAPK family. Inhibition of p38 phosphorylation largely prevented the t-BuOOH-induced down-regulation of progesterone production in GSH-depleted cells. These results indicate that alteration of the intracellular GSH redox environment results in the increased sensitivity of MA-10 cells to oxidative stress, and that this is mediated by activation of one or more redox-sensitive MAPK members.;In the final study, we hypothesized that luteinizing hormone (LH), the pituitary gonadotropin that stimulates testosterone production, affects the oxidant-to-antioxidant balance in Leydig cells, resulting in greater damage in aged than young cells because of the reduced antioxidant capacity that occurs with aging. To address this, we tested the effects of LH stimulation on stress response genes, intracellular ROS formation, and ROS-induced damage to ROS-susceptible macromolecules (DNA) in young and aged cells. Microarray analysis indicated that LH stimulation of Leydig cells resulted in significant increases in genes associated with stress response and anti-apoptotic/cell protective pathways. Short-term LH treatment of primary Leydig cells isolated from young rats resulted in increased levels of ROS compared to controls. Shortly thereafter, ROS production by the young cells decreased to control levels. Aged Leydig cells also showed increased ROS levels soon after their stimulation by LH. However, in contrast to the young cells, ROS production peaked later, and the time to recovery was increased as well. In both young and aged cells, treatment with LH resulted in increased levels of DNA damage, as assessed by Comet assay, but significantly more so in the aged cells. DNA damage levels reflected the levels of intracellular ROS. These results indicate that LH stimulation causes increased ROS production by young and aged Leydig cells, and that while DNA damage occurred in cells of both ages, greater damage was seen in the aged cells. Thus the results are consistent with those obtained with MA-10 cells.;These studies, taken together, provide strong evidence that the intracellular oxidant/anti-oxidant redox environment of aging Leydig cells affects intraceullar macromolecules and plays a very important role in the functional decline of these cells over time. |
