Effect of Glutathione Depletion on Leydig Cell Steroidogenesis in Young and Old Brown Norway Rats

Abstract

Changes in the oxidant/antioxidant environment of aging Leydig cells have been shown to be correlated with the reduced ability of these cells to produce testosterone. With this in mind, we hypothesized that the experimental depletion of glutathione (GSH), an abundant Leydig cell intracellular antioxidant, might 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-tertbutyl-␣-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 LHstimulated cAMP production, steroidogenic acute regulatory protein, cholesterol side-chain cleavage, 3␤-hydroxysteroid dehydrogenase, and 17␣-hydroxylase/17,20-lyase. The results of these studies, taken together, are consistent with the hypothesis that alteration in the oxidant/antioxidant environment may play a significant, causative role in the age-related reduced ability of Leydig cells to produce testosterone. (Endocrinology 149: 2612-2619, 2008) A GING IN RODENTS and men is associated with reduced serum testosterone concentration (1, 2). Testosterone is produced by the Leydig cells in the interstitial compartment of the mammalian testis. Studies of the Brown Norway rat have demonstrated that reduced steroidogenesis by individual Leydig cells, rather than loss of the cells, accounts for reduced serum levels of testosterone (3, 4). Multiple defects have been identified in the steroidogenic pathway of old Leydig cells, including reductions in each of LH-stimulated cAMP production, the cholesterol transport proteins steroidogenic acute regulatory protein (StAR) and peripheral benzodiazepine receptor (also referred to as translocator protein), and downstream steroidogenic enzymes of the mitochondria and smooth endoplasmic reticulum (5-8).Although the mechanism(s) by which age-related defects in the Leydig cell steroidogenic pathway occur remains uncertain, there is correlative evidence that changes in the oxidant/antioxidant status of the cells is involved. Thus, the antioxidant defense molecules superoxide dismutase-1 and -2, glutathione peroxidase, and glutathione (GSH) are significantly reduced as Leydig cells age (9, 10). Additionally, the superoxide content of the mitochondria of aging Leydig cells is significantly increased, compared with young Leydig cells (11), as is lipid pero...

Deleterious Cholesterol Hydroperoxide Trafficking in Steroidogenic Acute Regulatory (StAR) Protein-expressing MA-10 Leydig Cells

“…Leydig cells, which reside in the testicular interstitium, are reported to be particularly suscepti- ble to oxidative damage in vivo due to their close proximity to reactive oxygen species-producing testicular interstitial macrophages (44). Decreased antioxidant enzyme activities, gene expression, and protein levels, along with lower glutathione, have been reported to play a key role in the diminished ability of Leydig cells from aging rat populations to synthesize testosterone (43,(45)(46)(47). Other work showed that steroidogenic luteal cells in vitro became damaged and dysfunctional when exposed to cumene hydroperoxide or a fatty acid hydroperoxide (48).…”

Arsenic induced progesterone production in a caspase‐3‐dependent manner and changed redox status in preovulatory granulosa cells

“…In addition, GSH is involved in Leydig cell steroidogenesis (Diemer et al, 2003;Chen et al, 2008). Here, we wondered whether stimulation of GSHmediated progesterone production and the anti-apoptotic effect in preovulatory GCs exposed to arsenite.…”

“…Thus, we can conclude that the protective effect of arsenite on GCs can be partially due to GSH production augmented by progesterone. Both ROS and the depletion of GSH inhibit steroidogenesis in Leydig cells (Diemer et al, 2003;Chen et al, 2008). Chen et al (2010) reported that GSH depletion by BSO led to significant reductions of progesterone level in MA-10 Leydig cells exposured to the pro-oxidant tert-butyl hydroperoxide (t-BuOOH), although BSO alone did not affect progesterone production.…”

“…Reactive oxygen disrupts mitochondria and inhibits StAR protein and steroidogenesis in MA-10 tumor Leydig cells (Diemer et al, 2003). It has been shown that the depletion of the antioxidant glutathione (GSH) resulted in reduced Leydig cell steroidogenesis in rats (Chen et al, 2008). Dale et al suggested that mitochondria must be energized, polarized, and actively respiring to support steroidogenesis in Leydig cells (Hales et al, 2005).…”

Knockout of the transcription factor Nrf2: Effects on testosterone production by aging mouse Leydig cells

“…A number of deficits have been identified in the steroidogenic pathway of old Leydig cells that might account for the reduced LH-stimulated testosterone production, including reductions in cAMP production, steroidogenic acute regulatory protein (STAR), translocator protein (TSPO), cholesterol translocation from the cytosol into the mitochondria, cholesterol metabolism to pregnenolone in the mitochondria, and downstream steroidogenic enzymes of the mitochondria and smooth endoplasmic reticulum (Culty et al, 2002; Leers-Sucheta et al, 1999; Liao et al 1993; Luo et al, 2005, 2001, 1996; Zirkin and Chen, 2000). Although the mechanism by which these changes occur remains uncertain, imbalance between the production of reactive oxygen species (ROS) and intracellular antioxidant defenses has been proposed (Cao et al, 2004; Chen et al, 2001, 2008; Chen and Zirkin, 1999; Lacombe et al, 2006; Luo et al, 2006). …”