Resveratrol reduces steroidogenesis in rat ovarian theca-interstitial cells: the role of inhibition of Akt/PKB signaling pathway

Abstract

Polycystic ovary syndrome is characterized by theca-interstitial hyperplasia and increased expression of steroidogenic genes, leading to excessive androgen production. Resveratrol, a natural polyphenol, promotes apoptosis and reduces rat theca-interstitial cell growth, in part by inhibiting the mevalonate pathway and decreasing the availability of substrates of isoprenylation [farnesyl-pyrophosphate (FPP) and geranylgeranyl-pyrophosphate (GGPP)]. This study evaluated the effect of resveratrol on rat theca-interstitial cell steroidogenesis. Because resveratrol may activate sirtuins, this study also investigated whether steroidogenesis was affected by sirtuin inhibitors (nicotinamide, sirtinol). Theca-interstitial cells were cultured with or without resveratrol (1-10 μm), GGPP (30 μm), FPP (30 μm), nicotinamide (1 mm), and/or sirtinol (10 μm). Resveratrol did not affect progesterone levels but reduced androgen production in a concentration-dependent fashion (androstenedione by up to 78% and androsterone by up to 76%). This inhibitory effect correlated with a decrease in mRNA expression of genes regulating androgen production, especially Cyp17a1 (by up to 73%). GGPP and FPP had no effect on androgen levels and Cyp17a1 mRNA levels and did not alter the effects induced by resveratrol. Similarly, sirtuin inhibitors did not reverse resveratrol-induced inhibition of steroidogenesis. However, resveratrol decreased activity of serine-threonine kinase/protein kinase B pathway, a cell-signaling pathway involved in ovarian steroidogenesis. The present findings indicate that resveratrol reduces androgen production primarily by inhibiting Cyp17a1 mRNA expression, and this inhibition may be mediated, in part, by blocking the activity of the serine-threonine kinase/protein kinase B pathway. These findings may be of clinical relevance to conditions associated with excessive production of androgens by theca cells, such as polycystic ovary syndrome.

Figures

Fig. 1.

Effect of resveratrol (1–10 μm) on mRNA expression of Star (A), Cyp11a1 (B), Hsd3b1 (C), and Cyp17a1 (D). Theca-interstitial cells were cultured in chemically defined media supplemented with LH (5 ng/ml) for 48 h in the absence (control) or in the presence of resveratrol. Total cellular RNA was isolated, and mRNA expression was determined using quantitative real-time PCR reactions and normalized to Hprt mRNA levels. Results are presented as a percentage of control. Each bar represents mean ± sem from three independent experiments (each with n = 4). Means with no superscripts in common are significantly different (P < 0.05).

Fig. 2.

Effect of resveratrol (1–10 μm) on steroid production by theca-interstitial cell cultures: progesterone (A), androstenedione (B), and androsterone (C). The cells were cultured in chemically defined media supplemented with LH (5 ng/ml) for 48 h in the absence (control) or in the presence of resveratrol. Steroid levels were determined using liquid chromatography-mass spectrometry. The mean ± sem concentrations of steroids in control cultures were: progesterone, 769 ± 76 pg/well; androstenedione, 81 ± 12 pg/well; and androsterone, 677 ± 95 pg/well. Results are presented as a percentage of control. Each bar represents mean ± sem from three independent experiments (each with n = 4). Means with no superscripts in common are significantly different (P < 0.05).

Fig. 3.

Effect of resveratrol (10 μm), GGPP (30 μm), and FPP (30 μm) on mRNA expression of Star (A), Cyp11a1 (B), Hsd3b1 (C), and Cyp17a1 (D). Theca-interstitial cells were cultured in chemically defined media supplemented with LH (5 ng/ml) for 48 h in the absence (control) or in the presence of resveratrol and/or GGPP or FPP. The mRNA expression was determined as described in Fig. 1. Results are presented as a percentage of control. Each bar represents the mean ± sem from three independent experiments (each with n = 4). Means with no superscripts in common are significantly different (P < 0.05).

Fig. 4.

Effect of resveratrol (10 μm), GGPP (30 μm), and FPP (30 μm) on steroid production by theca-interstitial cells: progesterone (A), androstenedione (B), and androsterone (C). The cells were cultured in chemically defined media supplemented with LH (5 ng/ml) for 48 h in the absence (control) or in the presence of resveratrol and/or GGPP or FPP. Androgen levels were determined as described in Fig. 2. The mean ± sem concentrations of steroids in control cultures were: progesterone, 590 ± 64 pg/well; androstenedione, 55 ± 10 pg/well; and androsterone, 493 ± 40 pg/well. Results are presented as a percentage of control. Each bar represents mean ± sem from three independent experiments (each with n = 4). Means with no superscripts in common are significantly different (P < 0.05).

Fig. 5.

A, Effect of Akt inhibitor (1 μm) on steroid production by theca-interstitial cells. The cells were cultured in chemically defined media supplemented with LH (5 ng/ml) for 48 h in the absence (control) or in the presence of Akt inhibitor (124018 Akt Inhibitor VIII, Isozyme-Selective, Akti-1/2). Progesterone, androstenedione, and androsterone levels were determined as described in Fig. 2. The mean ± sem concentrations of steroids in control cultures were: progesterone, 517 ± 29 pg/well; androstenedione, 24 ± 2 pg/well; and androsterone, 51 ± 39 pg/well. Results are presented as a percentage of control. Each bar represents mean ± sem from three independent experiments (each with n = 4); *, Means significantly different from control (P < 0.01). B, Effect of Akt inhibitor (1 μm) on Cyp17a1 mRNA expression by theca-interstitial cells. The cells were cultured in chemically defined media supplemented with LH (5 ng/ml) for 48 h in the absence (control) or in the presence of Akt inhibitor. The mRNA expression was determined as described in Fig. 1. Results are presented as a percentage of control. Each bar represents mean ± sem from three independent experiments (each with n = 4); *, Means significantly different from control (P < 0.01). Akt inh, Akt inhibitor.

Fig. 6.

Effects of resveratrol (10 μm) on Akt/PKB phosphorylation. Theca-interstitial cells were cultured for 30 min in chemically defined media supplemented with LH (5 ng/ml) in the absence (control) or in the presence of resveratrol. The cells were lysed, and Western blot analysis was performed with antibodies specific for phospho- and total-Akt/PKB. Results are presented as a percentage of control. Each bar represents mean ± sem from three independent experiments. The bands of one representative experiment are shown.