Sirt1 and Nrf2: regulation of Leydig cell oxidant/antioxidant intracellular environment and steroid formation†

Abstract

Previous studies reported that, with aging, Leydig cell intracellular antioxidants are reduced in concentration and intracellular ROS levels increase, suggesting that oxidant/antioxidant imbalance may contribute to the reduced testosterone production that characterizes the aging cells. As yet, little is known about how the Leydig cell oxidant/antioxidant environment is regulated. Sirt1, an enzyme that deacetylates transcription factors, and the transcription factor Nrf2, have been shown to be associated with cellular response to oxidative stress. We hypothesized that Sirt1 and/or Nrf2 might be involved in regulating the oxidant/antioxidant environment of Leydig cells, and therefore, the testosterone production. We found that Sirt1 and Nrf2 are present in the Leydig cells of Brown Norway rats, though reduced in aged cells. In MA-10 cells in which Sirt1 or Nrf2 were suppressed by nicotinamide (NAM) or ML385, respectively, or in which siRNAs were used for knockdown of Sirt1 or Nrf2, increased ROS levels and decreased progesterone production occurred. In rat Leydig cells, inhibition of Sirt1 by culturing the cells with NAM resulted in increased ROS and reduced testosterone production, and subsequent removal of NAM from the culture medium resulted in increased testosterone production. Activation of rat Leydig cells Sirt1 with honokiol or of Nrf2 with sulforaphane resulted in the maintenance of testosterone production despite the exposure of the cells to oxidizing agent. These results, taken together, suggest that Sirt1 and Nrf2 are involved in maintaining the Leydig cell oxidant/antioxidant environment, and thus in maintaining steroid production.

Keywords: Leydig cell; Nrf2; Sirt1; oxidative stress; testosterone.

Figure 1

Sirt1 and Nrf2 protein in young and old Brown Norway rat Leydig cells. (A) Western blots of Sirt1 and Nrf2 proteins in Leydig cells isolated from the testes of young (3-month-old) and aged (21-month-old) rats. Actin was used as a loading control. Quantification of Sirt1 (B) and Nrf2 (C) in Leydig cells isolated from young (n = 3) and old (n = 3) rats.

Figure 2

Effect of the Sirt1 inhibitor nicotinamide (NAM) and the Nrf2 inhibitor ML385 on MA-10 cell viability and steroid (progesterone) production. Cell viability of MA-10 cells in response to increasing concentrations of NAM (A) and ML385 (B) was determined by the MTT assay. Progesterone production by MA-10 cells was determined after culturing cells with increasing concentrations of NAM (C) or ML385 (D) in the presence of LH for 2 h. In each case, at least three independent experiments were performed. Data shown are the mean ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared to LH-treated control cells.

Figure 3

Effect of Sirt1 and Nrf2 knockdowns by siRNAs on LH-induced steroid (progesterone) and ROS production by MA-10 cells. The cells (5x10⁵) were transfected with Sirt1 or Nrf2 siRNA, respectively, for 12 h before culturing with LH (2 h). Control (scrambled) siRNA was used as a negative control. TA: transfection agent only. Equal amounts of protein were separated by SDS-PAGE and immunoblotted using Sirt1 and Nrf2 antibodies. Actin was used as a loading control. LH-induced progesterone production was determined after Sirt1 (A) or Nrf2 (B) knockdowns. Progesterone concentrations were measured by RIA. Generation of ROS in response to LH (10 ng/ml) was measured by DCF-DA treatment after knockdown of Sirt1 or Nrf2 (C). H₂O₂ was used as a positive control. Data represent percent of control. In each case, at least three independent experiments were performed. Data shown are the mean ± SEM. ^***P < 0.001 compared with LH-treated cells.

Figure 4

Long-term effect of the Sirt1inhibitor nicotinamide (NAM) on testosterone and ROS production by stem cell-derived, seminiferous tubule-associated Leydig cells. (A) Leydig cell testosterone production at times after culturing isolated seminiferous tubules with LH. The inset shows Leydig cells stained for 3β-HSD after culture of the tubules with LH for 40 days. (B) Western blots of Sirt1 and antioxidant enzymes GPx and CAT after treatment of the tubule-associated, stem cell-derived Leydig cells with the oxidant H₂O₂ or with increasing concentrations of the Sirt1 inhibitor NAM (days 42–49). Actin was used as a loading control. (C) ROS production by the seminiferous tubule-associated Leydig cells incubated with LH plus NAM. Seminiferous tubules were cultured with LH for 6 weeks and then with NAM for 1 week. H₂O₂ was used as a positive control. ROS was detected by incubating the tubules with DCF-DA (30 min). DCF-DA values are expressed as fluorescence units. Each bar represents the mean ± SEM. ^*P < 0.05, ^***P < 0.001 compared with LH-treated cells. (D) Testosterone production by Leydig cells in response to culture with LH for 105 days; after culture with LH for 42 days and then with LH plus NAM (10 mM) from days 42–70; and after culture with LH for 42 days, then with LH plus NAM from days 42–70, and then after removal of NAM. Testosterone was assayed in the medium by RIA. Data are expressed as mean ± SEM of at least 3 independent experiments. To confirm the presence of Leydig cells on the surfaces of the tubules, the cells were stained for 3β-HSD after tubules were cultured with LH for 70 days or with LH for 42 days and then with LH plus NAM from days 42–70.

Figure 5

Effect of Sirt1 or Nrf2 activators honokiol and sulforaphane, respectively, on H₂O₂ or NaIO₃-induced ROS production. Seminiferous tubules were cultured with LH for 7 weeks, and then H₂O₂ or NaIO₃ was added in the presence of LH plus honokiol or sulforaphane for 1 week. The seminiferous tubules then were incubated with DCF-DA for 30 min. DCF-DA values are expressed as percent of control. Data are expressed as mean ± SEM of at least three independent experiments.

Figure 6

Long-term effects of the Sirt1 and Nrf2 pharmacological activators honokiol and sulforaphane on testosterone production by stem cell-derived, seminiferous tubule-associated Leydig cells cultured with the oxidants H₂O₂ or NaIO₃. (A) Western blots of Sirt1 and Nrf2 protein in Leydig cells after treatment of the cells with honokiol or sulforaphane. (B and C) Seminiferous tubules were cultured with LH for 7 weeks, after which H₂O₂ (B) or NaIO₃ (C) was added to the culture in the culture medium in the presence of LH plus honokiol or sulforaphane. Culture continued to week 13. During the culture from 7 to 13 weeks, medium was collected each week for testosterone measurement. Data are expressed as mean ± SEM of at least three independent experiments. Statistical analyses are shown in the tables associated with Figures 6B and6C. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.