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.

Theca cells play a prominent role in ovarian steroidogenesis by producing aromatizable androgens under the primary control of LH, which are subsequently used by granulosa cells for estrogen biosynthesis. Thecal differentiation involves the regulated expression of a variety of specific genes coding for the LH receptor as well as the proteins and enzymes involved in the androgen biosynthesis pathway, such as steroidogenic acute regulatory protein (StAR) (encoded by STAR gene), cholesterol side chain cleavage enzyme (P450sc; encoded by the CYP11A1 gene), 3β-hydroxysteroid dehydrogenase (type I or II; encoded by Hsd3b1 gene in the rat and HSD3B2 gene in human, respectively), and cytochrome P450 17α-hydroxylase/C17-20 lyase (P450c17) (encoded by CYP17A1 gene) (1). Unlike other above-listed genes, CYP17A1 gene is expressed in theca but not granulosa cells and is the rate-limiting step required for androgen biosynthesis, catalyzing the conversion of progestins into androgens (2).

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder affecting approximately 5–10% of women of reproductive age (3, 4). Ovaries of women with PCOS are enlarged and characterized by prominent theca-interstitial hyperplasia and excessive production of androgens due to increased steroidogenic activity and excessive transcription of genes regulating androgen production, as reflected by increased levels of individual mRNA as well as enhanced promoter activities (5). Steroidogenic abnormalities associated with theca cells from women with PCOS were evaluated in long-term cultures, demonstrating increased 3β-hydroxysteroid dehydrogenase type II and P450c17 enzyme activities as well as increased CYP11A1, HSD3B2, and CYP17A1 mRNA expression (6–9). Clinical studies suggest that enhanced P450c17 enzyme activity and expression may account for hyperandrogenism in PCOS (10, 11). In addition, dysregulation of several signal-transduction pathways has been shown to play a pivotal role in androgen excess in PCOS women, including the MAPK, protein kinase C, and serine-threonine kinase (Akt)/protein kinase B (PKB) pathways (10, 12, 13).

Resveratrol (trans-3,5,4′-trihydroxystilbene) is a naturally occurring phenolic phytoalexin abundantly found in grapes, red wine, peanuts, and several medicinal plants with diverse biochemical and physiological properties, such as chemopreventive, antiinflammatory and antioxidant, as well as cardioprotective and neuroprotective effects (14–17). Additionally, rat in vitro studies have shown that resveratrol exerts an inhibitory effect on steroidogenesis in different cell types, including adrenocortical and Leydig cells (18, 19). Several signal transduction pathways are involved in resveratrol-mediated effects on cell functions. Recently, our group has demonstrated that resveratrol reduces proliferation of theca-interstitial cells, at least in part, by inhibition of the mevalonate pathway (20). We found that resveratrol inhibits both activity and expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting step of cholesterol synthesis, leading to a decreased availability of several downstream products, such as cholesterol and substrates of isoprenylation [farnesyl-pyrophosphate (FPP) and geranylgeranyl-pyrophosphate (GGPP)] (20). Other effects of resveratrol may involve different mechanisms. For example, resveratrol has been shown to extend lifespan in worms, flies, and mice through activation of sirtuins (21, 22), a family of nicotinamide adenine dinucleotide⁺-dependent deacetylases implicated in several important cellular processes, such as genomic stability, DNA repair, transcriptional silencing, and glucid and lipid metabolism (23, 24).

The aim of the present study was to evaluate the effects of resveratrol on rat theca-interstitial cell steroidogenesis and to determine the role of substrates of isoprenylation, inhibitors of sirtuins, and the Akt/PKB signaling pathway in this process.

Materials and Methods

Animals

Female Sprague Dawley rats were obtained at age 22 d from Charles River Laboratories (Wilmington, MA) and housed in an air-conditioned environment and a 12-h light, 12-h dark cycle. All animals received standard rat chow and water ad libitum. At the age of 27, 28, and 29 d, the rats were injected with 17β-estradiol (1 mg/0.3 ml of sesame oil sc) to stimulate ovarian development and growth of antral follicles. Twenty-four hours after the last injection, the animals were anesthetized using ketamine and xylazine (ip) and euthanized by intracardiac perfusion using 0.9% saline. All treatments and procedures were carried out in accordance with accepted standards of human animal care as outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals and a protocol approved by the Institutional Animal Care and Use Committee at the University of California, Davis.

Cell culture and reagents

The collection and purification of ovarian theca-interstitial cells were performed as described previously (25, 26). Briefly, the ovaries were removed from the animals and dissected free of oviducts and fat under a dissecting microscope. After a 60-min collagenase digestion, theca-interstitial cells were purified using discontinuous Percoll gradient centrifugation. The cells were counted, and viability, as assessed by the trypan blue exclusion test, was routinely in the 90–95% range. Theca-interstitial cells were incubated in 24-well fibronectin-coated plates at a density of 400,000 cells/well. The cultures were carried out for 48 h at 37 C in an atmosphere of 5% CO₂ humidified air in serum-free McCoy's 5A culture medium supplemented with 1% antibiotic/antimycotic mix, 0.1% BSA, and 2 mm l-glutamine. The above-mentioned time point was chosen based on our previous work evaluating rat theca-interstitial cell steroidogenesis (27). The cells were incubated in the absence (control) or in the presence of resveratrol (1–10 μm), FPP (30 μm), GGPP (30 μm), nicotinamide (1 mm), sirtinol (10 μm), insulin (0.1 μg/ml), and Akt inhibitor (1 μm). The concentrations of these compounds were selected based on previous studies (28, 29). All cultures were carried out in the presence of ovine LH (5 ng/ml). All above chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) except for Akt inhibitor (Akt inhibitor VIII, Isozyme-Selective, Akti-1/2), which was purchased from Calbiochem Co. (Gibbstown, NJ), and LH, which was obtained from the National Hormone and Pituitary Program at the Harbor-University of California, Los Angeles Medical Center (Torrance, CA).

Androstenedione, androsterone, progesterone, and the deuterated derivative of androstenedione-d7 were obtained from Steraloids (Newport, RI), whereas testosterone-d3 was obtained from Cerillient (Round Rock, TX). Acetonitrile and methanol were HPLC grade and obtained from Burdick and Jackson (Muskesgon, MI). Acetone, isopropanol, and ammonium hydroxide were Optima grade and obtained from Fisher (St. Louis, MO). Formic acid was American Chemical Society grade and obtained from EMD (Gibbstown, NJ).

Total RNA isolation and quantitative real-time PCR

Total RNA was isolated using the MagMAX-96 Total RNA Isolation kit (Applied Biosystems, Foster City, CA) and the KingFisher robot (Thermo Scientific, Vantaa, Finland). RT of total RNA to cDNA was performed using High-Capacity cDNA Reverse Transcription kit for RT-PCR (Applied Biosystems). PCR were set up in 28-μl volumes, consisting of 5 μl of cDNA, 4.5-μl forward and 4.5-μl reverse 900 nm primers, and 14 μl of 2× SYBR Green PCR Master Mix (Applied Biosystems).

Quantitative RT-PCR reactions were performed in triplicate using the ABI 7300 Real-Time PCR System (Applied Biosystems). Separate cDNA dilutions were included in each PCR run to generate standard curves. Data were analyzed using SDS 1.4 software (Applied Biosystems). The relative amount of target mRNA was expressed as a ratio normalized to hypoxanthine phosphoribosyltransferase (Hprt). The primer sequences were as follows: rat Star forward (5′-GCC TGA GCA AAG CGG TGT C-3′) and reverse (5′-CTG GCG AAC TCT ATC TGG GTC TGT-3′); rat Cyp11a1 forward (5′-GCT GGA AGG TGT AGC TCA GG-3′) and reverse (5′-CAC TGG TGT GGA ACA TCT GG-3′); rat Hsd3b1 forward (5′-CCA GAA ACC AAG GAG GAA T-3′) and reverse (5′-CCA GAA ACC AAG GAG GAA T-3′); rat Cyp17a1 forward (5′-ACT GAG GGT ATC GTG GAT GC-3′) and reverse (5′-CCG TCA GGC TGG AGA TAG AC-3′); and rat Hprt forward (5′-TTG TTG GAT ATG CCC TTG ACT-3′) and reverse (5′-CCG CTG TCT TTT AGG CTT TG-3′).

Sample preparation and processing for quantification of steroids

Each sample was directly assayed; the following extraction procedure was applied to each specimen. Each sample aliquot (300 μl) was placed in a 2.0-ml autosampler vial and spiked with 150 μl of internal standard solution, i.e. androstenedione-d7 and testosterone-d3. Detection and quantitation of all analytes was accomplished using selective reaction monitoring.

Mass spectrometry

We have developed a novel turbulent flow chromatography HPLC-mass spectrometry/mass spectrometry method that allows the simultaneous detection of androstenedione, androsterone, and progesterone. It consists of a high-pressure liquid chromatography instrument configuration multiplexing Thermo Aria TLX-2 turbulent flow chromatography (two loading pumps and two eluting pumps, Shimadzu LC-10AD) system and an autosampler outfitted with a 300-position Peltier tray (Franklin, MA), coupled to a Thermo Scientific TSQ Vantage triple quadrupole mass spectrometer (Thermo Scientific, San Jose, CA), equipped with a heated electrospray ionization source. The instrument was controlled using Aria software (version 1.6.1). A Thermo Cyclone P extraction column (0.5 × 50 mm, 60-μm particle size; Franklin, MA) was used for online sample extraction of diluted serum, and HPLC separation was carried out by a 2.1 × 100-mm, 3-μm particle size ACE C18 column protected by a reverse phase guard cartridge (Mac-Mod, Chadds Ford, PA) contained within a Hot Pocket column heater (Thermo Scientific).

Precursor and product ions for each target analyte were chosen for selective reaction monitoring transitions, and the related parameters for the different analytes were isolated by HPLC separation based on the following mobile-phase gradient: solvent A, water containing 0.1% formic acid; B, methanol; C, acetonitrile/isopropyl alcohol/acetone 60/30/10; and D, water/acetonitrile (98/2 vol/vol) with 0.1% ammonium hydroxide.

Detection and quantification employed select reaction monitoring liquid chromatography-mass spectrometry/mass spectrometry transitions of initial precursor ions for androstenedione, androsterone, and progesterone mass to charge ratio (m/z) 287.2, 291.4, and 315.2, respectively. The response for the major product ions for each of the analytes was plotted and peaks at the proper retention time integrated using LCQuan. This software was used to generate calibration curves and quantitate the analytes in all samples. The concentrations of androstenedione, androsterone, and progesterone in each sample (e.g. calibrators, quality control, and unknowns) were determined by an internal standard method using the peak area ratio and linear regression analysis. The responses for androstenedione, androsterone, and progesterone were linear and gave correlation coefficients of 0.99 or better.

Western blot analysis

Cells were incubated in microcentrifuge tubes without (control) or with resveratrol (10 μm) for 30 min. At the end of the treatment, cells lysates were prepared as previously described (30). Protein concentrations were determined using Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). For immunoblot analysis, samples containing equal amounts of protein (65 μg) were resolved in 12% SDS-PAGE gels and transferred to a nitrocellulose membrane by electroblotting. Blots were blocked at room temperature for 1 h using blocking buffer (LI-COR Biosciences, Lincoln, NE) and incubated with rabbit monoclonal phospho-PKB antibody [phospho-Akt (Ser473) (D9E) XP rabbit monoclonal antibody no. 4060S; Cell Signaling Technology, Beverly, MA] overnight at 4 C (1:500 in blocking buffer). After incubation, the membranes were washed three times with PBS and incubated with goat antirabbit IgG antibody for 1 h at room temperature (1:10,000 in blocking buffer). To determine the total amount of PKB in each sample, membranes were incubated with mouse monoclonal total-PKB antibody [Akt (pan) 40D4 mouse monoclonal antibody no. 2920; Cell Signaling Technology] overnight at 4 C (1:500 in blocking buffer) and primary antibody to β-actin (Monoclonal Anti-Actin Clone AC-40 Purified Mouse Immunoglobulin A3853; 1:40,000; Sigma-Aldrich, St. Louis, MO) for 1 h. After washing three times with PBS, the blots were incubated with goat antimouse secondary antibody for 1 h at room temperature (1:40,000 in blocking buffer). Blots were developed with the Odyssey Infrared Imaging System (LI-COR Biosciences).

Statistical analysis

Statistical analysis was performed using JMP 9.0 software (SAS, Cary, NC). Data are presented as the mean ± sem. Means were compared by ANOVA followed by post hoc testing using Tukey's honestly significant difference test. When appropriate, data were logarithmically transformed. A value of P < 0.05 was considered statistically significant. Each experiment was repeated at least three times.

Results

Effect of resveratrol on steroidogenesis

To determine whether resveratrol affects the expression of the key genes involved in the regulation of steroidogenesis, theca-interstitial cells were cultured for 48 h in the absence (control) or presence of resveratrol (1–10 μm). As shown in Fig. 1A, resveratrol had no significant effect on Star mRNA expression, except for a slight decrease by 15% (P < 0.05) at a concentration of 10 μm.

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).

In the same experiments, the exposure of cells to resveratrol induced a concentration-dependent decrease in both Cyp11a1 and Hsd3b1 mRNA levels, respectively, by up to 38% (P < 0.001) and 42% (P < 0.001) at the highest concentration (Fig. 1, B and C).

However, the most profound resveratrol-induced inhibitory effect on mRNA expression was found in Cyp17a1. As presented in Fig. 1D, resveratrol at a concentration of 10 μm decreased the level of Cyp17a1 transcripts by 73% (P < 0.001) compared with control.

To evaluate effects of resveratrol on individual steroids, levels of progesterone, androstenedione, and androsterone were evaluated in spent media using liquid chromatography-mass spectrometry. Forty-eight-hour exposure of cells to resveratrol did not affect progesterone production at any of the tested concentrations. In contrast, resveratrol decreased androgen levels in a concentration-dependent fashion. As shown in Fig. 2, B and C, resveratrol at the highest concentration (10 μm) induced a significant inhibitory effect on both androstenedione and androsterone production, respectively, by 78% (P < 0.001) and 74% (P < 0.001). Because high concentrations of resveratrol (≥30 μm) have been shown to reduce proliferation of theca-interstitial cells (30), present results were also analyzed accounting for possible effect on the growth of cells. However, resveratrol had no significant effect on the growth of theca-interstitial cells at concentrations evaluated in this study (≤10 μm). Inhibitory effects of resveratrol on the level of androgens persisted when expressing steroid levels per unit of protein determined at the end of the culture period.

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).

Effect of substrates of isoprenylation

To determine whether the effect of resveratrol on steroidogenesis is related to decreased isoprenylation, theca-interstitial cells were cultured for 48 h without (control) or with resveratrol (10 μm), GGPP (30 μm), and/or FPP (30 μm) (Fig. 3). The concentration of resveratrol was selected based on our previous experiments, where resveratrol at 10 μm induced an inhibitory effect on mRNA expression of the relevant genes regulating steroidogenesis.

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).

As presented in Fig. 3A, neither resveratrol nor a substrate of isoprenylation, FPP, had any significant effect on Star mRNA levels. A decrease by 16% (P < 0.05) induced by GGPP was observed. Moreover, the addition of either GGPP or FPP to resveratrol-treated cultures did not alter Star mRNA expression compared with the effects of resveratrol alone.

In agreement with our previous experiments (Fig. 1), the exposure of cells to resveratrol significantly inhibited Cyp11a1, Hsd3b1, and Cyp17a1 mRNA levels, respectively, by 36, 37, and 69% (P < 0.001 for all) below the control level. In contrast, GGPP alone and FPP alone had no significant effect on mRNA expression of any of the above-mentioned genes, except for a decrease by 25% (P < 0.05) in Hsd3b1 transcripts in the presence of GGPP. The addition of substrates of isoprenylation to resveratrol-treated cultures did not restore the resveratrol-induced inhibitory effect on mRNA expression of any of these genes (Fig. 3, B–D).

As shown in Fig. 4A, resveratrol did not affect progesterone production, whereas GGPP and FPP alone induced a modest increase in progesterone level by 26% (P < 0.01) and 20% (P < 0.05), respectively. The addition of GGPP and FPP to resveratrol-treated cultures did not alter progesterone production. Androstenedione and androsterone levels in the spent media from the same experiments are shown in Fig. 4, B and C. Resveratrol decreased androstenedione and androsterone production, respectively, by 83% (P < 0.001) and 60% (P < 0.001), whereas GGPP and FPP alone had no significant effect on androgen levels. Furthermore, the addition of both GGPP and FPP to resveratrol-treated cultures did not abrogate resveratrol-induced inhibition of androgen production.

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).

Effects of inhibitors of sirtuins

To determine whether the resveratrol-induced inhibitory effect on theca cell steroidogenesis is mediated through substrate-specific activation of silent mating type information regulation-2 homolog (SIRT)1, additional experiments were performed to determine the effects of specific inhibitors of sirtuins, nicotinamide, and sirtinol on the key genes regulating ovarian steroidogenesis. Theca-interstitial cells were cultured for 48 h in the absence (control) or presence of resveratrol (10 μm), nicotinamide (1 mm), and/or sirtinol (10 μm). Both nicotinamide and sirtinol inhibited Cyp17a1 mRNA expression, respectively, by 38% (P < 0.01) and 63% (P < 0.001), and neither of these agents reversed resveratrol-induced inhibition of Cyp17a1 mRNA expression. In a similar fashion, nicotinamide and sirtinol decreased androstenedione production, respectively, by 29% (P < 0.05) and 42% (P < 0.001), whereas the addition of these compounds to resveratrol-treated cultures did not reverse inhibitory effects induced by resveratrol alone (data not shown).

Effects of Akt/PKB signaling pathway on steroidogenesis

Because resveratrol-induced inhibition of rat theca cell steroidogenesis is independent of mechanisms involving both isoprenylation and activation of sirtuins, further experiments were carried out to analyze the signaling cascade involved in this inhibition. Because the Akt/PKB pathway has been previously shown to mediate LH-induced stimulation of both androgen production and Cyp17a1 mRNA expression in bovine theca cells (31), further experiments were performed to evaluate whether Akt activity is involved in rat theca cell steroidogenesis. Theca-interstitial cells were cultured for 48 h in the absence (control) or presence of Akt inhibitor (1 μm). As shown in Fig. 5A, Akt inhibitor induced a 2.2-fold increase in progesterone production (P < 0.001) and inhibited both androstenedione and androsterone levels by 64% (P < 0.001) and 20% (P < 0.05), respectively.

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.

Furthermore, PCR analysis was carried out to evaluate the effect of inhibition of Akt activity on Cyp17a1 mRNA levels. Akt inhibitor dramatically decreased Cyp17a1 mRNA expression by 98% (P < 0.01) compared with control (Fig. 5B).

Effects of resveratrol on Akt/PKB phosphorylation

To determine whether the resveratrol-induced inhibitory effect on steroidogenesis is mediated through inhibition of Akt/PKB signaling pathway, theca-interstitial cells were cultured for 30 min in the absence (control) or presence of resveratrol (10 μm). Subsequently, phospho-Akt and total-Akt contents were examined using Western blotting. In the same set of experiments, insulin (0.1 μg/ml) and Akt inhibitor (1 μm) were included, respectively, as positive and negative controls. As shown in Fig. 6, resveratrol decreased Akt/PKB phosphorylation by 28% (P < 0.01) compared with control.

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.

Discussion

In the present study, we have demonstrated that in cultures of rat theca-interstitial cells resveratrol: 1) does not alter progesterone levels; 2) inhibits androgen production in a concentration-dependent fashion; 3) decreases mRNA expression of several genes regulating steroidogenesis, especially Cyp17a1; 4) the resveratrol-induced inhibitory effect on steroidogenesis is independent of mechanisms involving isoprenylation or activation of sirtuins; 5) the Akt/PKB signaling pathway is involved in theca-interstitial cell steroidogenesis; and 6) resveratrol decreases Akt/PKB phosphorylation.

To our knowledge, this is the first study evaluating the role of resveratrol on theca-interstitial cell steroidogenesis. The primary finding of the present study pertains to the demonstration that resveratrol selectively inhibits androgen production and mRNA expression of the key steroidogenic genes, especially Cyp17a1, the crucial gene regulating the androgen biosynthesis pathway. The present findings are consistent with previous studies demonstrating the inhibitory actions of this phytoestrogen on steroidogenesis on other androgen-producing cells. Supornsilchai et al. (18) showed that resveratrol suppresses steroidogenesis by rat adrenocortical cells by inhibiting cytochrome P450 c21-hydroxylase. In a similar fashion, Svechnikov et al. (19) demonstrated that resveratrol attenuated human chorionic gonadotropin-activated steroidogenesis in primary cultures of rat Leydig cells through suppression of the expression of Star and cytochrome P450c17. Thus, exposure of steroid-producing cells to resveratrol may decrease production of steroid hormones.

In the present study, resveratrol inhibited ovarian steroidogenesis by reducing Cyp17a1 mRNA expression and hence may induce accumulation of progesterone and decrease in androgen biosynthesis. However, in contrast to the previously observed stimulatory effect of resveratrol on granulosa cell progesterone production (32–34), no changes in progesterone levels were observed in interstitial-theca cells in response to resveratrol. We propose that the lack of effect of resveratrol on progesterone production, compared with a large effect on androgens, may be related to a net effect of resveratrol-induced decrease of mRNA expression of the genes involved in steroid production, such as Star, Cyp11a1, and Hsd3b1, as well as a reduced conversion of progesterone to androgens due to a profound inhibition of Cyp17a1 mRNA expression.

Isoprenoids are lipids that can be found in all living organisms, playing widely different roles in physiological processes of plants and animals (35). In humans, the nonsterol isoprenoids, FPP and GGPP, are products of mevalonate pathway. Isoprenoids, by the process of isoprenylation, may attach to the carboxyl terminus of proteins, facilitating the membrane attachment and function of members of the small G protein superfamily (36). This posttranslational modification has been shown to be involved in several cellular functions, such as proliferation, apoptosis, oxidative stress, and steroidogenesis (27, 28, 37). Our previous rat in vitro study demonstrated that resveratrol inhibits the mevalonate pathway by reducing 3-hydroxy-3-methylglutaryl coenzyme A reductase expression and activity, the rate-limiting enzyme of the mevalonate pathway, leading to a decrease of cholesterol, FPP, and GGPP levels. Furthermore, the inhibitory effect of resveratrol on theca cell proliferation was partly reversed by the addition of mevalonic acid, FPP, and GGPP (30). In contrast, in the present study, the addition of either FPP or GGPP to resveratrol-treated cells did not reverse the resveratrol-induced inhibition of theca cell steroidogenesis. Thus, these findings suggest that different mechanisms mediate resveratrol-induced effects on theca cell proliferation and steroidogenesis.

Silent Information Regulator 2 family proteins (sirtuins) are a highly conserved family of redox-sensitive, nicotinamide adenine dinucleotide⁺-dependent deacetylases that regulate gene expression by controlling the acetylation status of lysine residues on histones, nonhistone substrates, transcription factors, and transcriptional coactivators (38). In mammals, the first and most extensively studied sirtuin gene identified was SIRT1 coding for SIRT1 enzyme, located in the nucleus and cytoplasm (39). Previous reports have shown that resveratrol is a SIRT1 activator, exerting beneficial effects on glucose and lipid metabolism and extending lifespan in flies, worms, and rodents (40, 41). In the present study, we hypothesized that resveratrol might reduce ovarian steroidogenesis via activation of sirtuins in theca-interstitial cells. However, the blockade of sirtuins with SIRT1 inhibitors, such as sirtinol and nicotinamide, did not reverse resveratrol-induced inhibition of androgen production and Cyp17a1 mRNA expression, indicating that the resveratrol-induced inhibitory effect on theca cell steroidogenesis is not mediated through activation of sirtuins. We speculate that this pathway might be differentially activated by resveratrol regarding specific organs, suggesting a tissue-dependent effect of resveratrol on sirtuin activation.

Steroid hormone biosynthesis in theca-interstitial cells is regulated by trophic hormone activation of the protein kinase A signaling pathway (42). However, several signaling cascades regulating steroid production via pathways other than the protein kinase A pathway have been reported (12, 43). Akt, also known as PKB, is a serine-threonine protein kinase that drives cells through several biological functions, such as cell proliferation, survival, glucidic metabolism, and gene expression, in response to growth factors and other extracellular stimuli (44). However, little information is available regarding the role that this pathway plays in theca cell steroidogenesis. Fukuda et al. (31) demonstrated that activation of the Akt/PKB signaling pathway is involved in androgen production and Cyp17a1 mRNA expression in bovine theca cells. Furthermore, the addition of LH, a hormone that serves as a stimulus for theca cell steroidogenesis, increases both androgen production and Cyp17a1 mRNA expression through Akt phosphorylation (31). In the present study, the blockade of Akt/PKB signaling pathway with a selective Akt inhibitor (124018 Akt Inhibitor VIII, Isozyme-Selective, Akti-1/2), reduced LH-induced androgen production and Cyp17a1 gene expression and increased progesterone levels, indicating that activation of Akt/PKB pathway is required for LH-stimulated theca cell steroidogenesis acting at the key step of the conversion of progestins into androgens. Additionally, we demonstrated that resveratrol decreased Akt phosphorylation, suggesting that resveratrol-induced inhibition of theca cell steroidogenesis may be due, at least in part, to an inhibitory effect on Akt/PKB phosphorylation.

The present findings may be of potential therapeutic interest with regard to conditions associated with thecal hyperplasia and increased androgen production, such as that seen in PCOS. The total amount of androgens secreted by the ovary depends on two main factors: the number of androgen-producing cells and the steroidogenic capacity of individual cells. Resveratrol may alter ovarian steroidogenesis by reducing both steroidogenic capacity as well as decreasing the growth and hence the number of theca-interstitial cells. The present findings indicate that resveratrol decreases androgen production in a concentration-dependent fashion by selectively inhibiting Cyp17a1 mRNA expression, the key gene regulating androgen biosynthesis, which has been shown to be overexpressed in PCOS patients. On the other hand, our previous reports have shown that resveratrol also induces an inhibition of cell growth, decreasing the number of viable cells and increasing apoptosis of theca-interstitial cells. Thus, we speculate that resveratrol may be of clinical relevance to PCOS due to its antisteroidogenic and antiproliferative effects on theca-interstitial cells.

A clinically important issue is whether the concentrations of resveratrol used in this study correspond to a reasonable dose for human use. In the present study, the inhibitory effect of resveratrol on rat theca-interstitial cell steroidogenesis was detected at concentrations ranging from 1 to 10 μm; these concentrations are comparable with those used in other in vitro studies (18, 19). Present observations that the resveratrol concentration required to inhibit steroidogenesis is in the micromolar range may be of clinical relevance, because the bioavailability of resveratrol in human and in rodent models is in the micromolar range (45, 46). Furthermore, because resveratrol is highly lipophilic, its concentrations in tissues are significantly greater than in serum. Recent study evaluating patients with colorectal cancer revealed that resveratrol administered for 8 d at a dose of 1 g/d resulted in very high concentrations of resveratrol in tumor tissues (>600 μm) (47). These observations suggest that resveratrol, administered at pharmacological concentrations, may be effective to induce an inhibitory effect on theca-interstitial cell steroidogenesis.

In summary, our results suggest that resveratrol reduces androgen production and Cyp17a1 mRNA gene expression, at least in part, via inhibition of Akt/PKB phosphorylation in theca-interstitial cells. These findings may be of potential clinical relevance to conditions associated with excessive androgen production, such as PCOS, whereby resveratrol might represent a novel therapeutic agent.