Suppression of p66Shc prevents hyperandrogenism-induced ovarian oxidative stress and fibrosis

Abstract

Background: Rats with hyperandrogen-induced polycystic ovary syndrome (PCOS) have been shown to develop ovarian oxidative stress (OS) and fibrosis. The Sirt1 agonist, resveratrol, can reduce OS through inhibiting p66Shc in other models of OS.

Methods: We created a rat PCOS model with increased OS levels following treatment with one of the two androgens, dehydroepiandrosterone (DHEA) and dihydrotestosterone (DHT). The PCOS related features were determined by measurement of malondialdehyde (MDA) and superoxide dismutase (SOD) levels or by examining the reactive oxygen species (ROS) levels using the DCF-DA probe. The potential mechanisms by which p66Shc/Sirt1 mediates ovarian fibrosis were explored by western blotting, quantitative reverse transcription-PCR, immunofluorescence staining, and immunohistochemistry.

Results: Hyperandrogen dramatically augmented OS and activation of fibrotic factors in the ovary. Our data demonstrated that treatment with resveratrol enhanced Sirt1 and decreased ovarian OS as well as inhibited phosphorylation of p66Shc both in vivo and in vitro. The treatment suppressed fibrotic factor activation and improved ovarian morphology. Lentivirus- or siRNA-mediated p66Shc knockdown resulted in a dramatic enhancement of Sirt1 expression, down-regulation of ROS and suppression of fibrotic factors in granulosa cells. Moreover, p66Shc overexpression markedly increased the expression of fibrotic factors. Additionally, silencing Sirt1 induced a dramatic increase in p66Shc and enhanced activation of fibrotic factors.

Conclusions: p66Shc may be a direct target of Sirt1 for inducing ROS and thus promoting fibrosis. Further exploration of the mechanisms of p66Shc in both fibrosis and OS may provide novel therapeutic strategies that will facilitate the improvement in PCOS symptoms and reproductive functions.

Keywords: Fibrotic factors; Granulosa cells; PCOS; Reactive oxygen species; p66Shc.

Fig. 1

Ovarian morphology is improved after treatment with resveratrol in dehydroepiandrosterone-exposed rats. Rats received DHEA for induction of polycystic ovarian syndrome together with or without resveratrol treatment. a Rat body weights were measured on the day of sacrifice (day 36). b Average weight of both ovaries was measured. c Photographs of the morphology of the ovaries from each treatment group were shown. d Ovarian and follicular morphology was assessed by H&E staining (5×). The percentage of each follicle was shown on the right. n = 7 in each group. Three independent experiments were performed with similar results. Data are shown as the mean ± SD. ^##p ≤ 0.01, ^###p ≤ 0.001 vs. Blank; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 vs. DHEA treatment. DHEA, dehydroepiandrosterone; Res, resveratrol; PAF, preantral and early antral follicle; AF, antral follicle; CF, cystic follicles; CL, corpus luteum

Fig. 2

Resveratrol suppresses DHEA-induced ovarian fibrosis and oxidative stress. a Collagen in ovarian slices was revealed by Sirius Red and Masson staining (10×). Images are representative of three independent experiments with similar results. Serum (b) and ovarian (c) malondialdehyde (MDA) levels were analyzed using an enzymatic colorimetric method. Serum (d) and ovarian (e) superoxide dismutase (SOD) activity was analyzed using an enzymatic colorimetric method. n = 7 in each group. Three independent experiments were performed with similar results. Data are shown as the mean ± SEM. *p ≤ 0.05, **p ≤ 0.01. DHEA, dehydroepiandrosterone

Fig. 3

Resveratrol inhibits p66Shc phosphorylation and fibrotic factors activation in rat ovaries. Rats received DHEA for the induction of polycystic ovarian syndrome, together with or without resveratrol treatment. a Sirt1, α-SMA, collagen I A1, CTGF, and p-p66Shc expression in rat ovaries (60×) was analyzed by double immunofluorescence staining. Images are representative of three independent experiments with similar results. b The expression of fibrotic factors and p-p66Shc in ovaries was assessed by western blot. The panel on the right shows the quantitative analysis. c The mRNA levels of AR was analyzed by real-time PCR. d Relative expression of p66Shc and fibrotic factors in rat ovaries was determined by real-time PCR. n = 7 in each group. Three independent experiments were performed with similar results. Data are shown as mean ± SD & mean ± SEM. *p ≤ 0.05, **p ≤ 0.01 vs. Blank; ^#p ≤ 0.05, ^##p ≤ 0.01 vs. DHEA treatment. DHEA dehydroepiandrosterone, AR androgen receptor, CTGF connective tissue growth factor, Sirt1 sirtuin 1, p-p66Shc phosphorylated 66-kDa Src homology 2 domain-containing protein

Fig. 4

Dihydrotestosterone promotes generation of ROS and TGF-β in GCs. a The expression of FSHR and LHR in GCs and TCs was measured by immunofluorescence staining (Alexa Fluor 488) and the nuclei were stained with DAPI (40×). Images are representative of three independent experiments with similar results. b GCs treated with various concentrations of DHT for 24 h followed by immunoblot analysis of the AR and TGF-β proteins. c GCs were pre-treated with DHT (500 nM) for 24 h, followed by treatment with flutamide (Flu) at various concentrations (0, 20, 50, 100, 200, 500 μM) for 24 h. The AR and TGF-β protein levels were measured by western blot. d ROS generation in GCs following various treatments was measured using the DCF-DA probe. DCF-DA fluorescence (green fluorescence) was measured by confocal microscopy (40×). Images are representative of three independent experiments with similar results. Quantification of the fluorescence is shown. Three independent experiments were performed with similar results. Data are shown as the mean ± SD. *p ≤ 0.05. DHT dihydrotestosterone, AR androgen receptor, TGF-β transforming growth factor-beta, FSHR follicle-stimulating hormone receptor, LHR luteinizing hormone receptor, GCs granulosa cells, TCs theca cells, ROS reactive oxygen species

Fig. 5

Dihydrotestosterone promotes p66Shc phosphorylation resulting in mitochondrial dysfunction. Granulosa cells from naïve rats were treated with DHT, H₂O₂ or SOD. a p-p66Shc expression (Alexa Fluor 488) and mitochondria (MitoTracker Red) were revealed by confocal microscopy. b Quantification of the p-p66Shc fluorescence intensity is shown. c Quantification of MitoTrack Red intensity is shown. d Mitochondrial membrane potential was analyzed by the ratio of JC-1 monomers/polymers (60×). e Quantitative analysis of the ratios of the red to green fluorescence. Images are representative of three independent experiments with similar results. Data are shown as the mean ± SEM. *p ≤ 0.05. DHT, dihydrotestosterone; H₂O₂, hydrogen peroxide; SOD, superoxide dismutase; p-p66Shc, phosphorylated 66-kDa Src homology 2 domain-containing protein

Fig. 6

p66Shc silencing suppresses oxidative stress and fibrotic factor activation in granulosa cells. Granulosa cells were transfected with p66Shc siRNA followed by treatment with DHT, TGF-β1 or resveratrol. a Levels of p-p66Shc (Alexa Fluor 488) and TGF-β (Cy3) were measured with immunofluorescence (60×). Images are representative of three independent experiments with similar results. Quantification of the p-p66Shc (b) and TGF-β (c) fluorescence is shown. d ROS was assessed by the DCF-DA probe using confocal microscopy (40×). Images are representative of three independent experiments with similar results. e SOD enzymatic activity in granulosa cells was measured by colourimetric method. f The expression of fibrotic factors and AR was assessed by western blot assay. mRNA expression of p66Shc (g), Sirt1 (h), and TGF-β (i) was analyzed by real-time PCR. Three independent experiments were performed with similar results. Data are shown as the mean ± SD. *p ≤ 0.05. DHT dihydrotestosterone, TGF-β transforming growth factor-beta, p-p66Shc phosphorylated 66-kDa Src homology 2 domain-containing protein, ROS reactive oxygen species, SOD superoxide dismutase, Sirt1 sirtuin 1, AR androgen receptor

Fig. 7

p66Shc silencing inhibits fibrotic factors activation and improves mitochondrial dysfunction. p66Shc in granulosa cells was silenced by lentivirus or overexpressed by a plasmid vector. After 72 h, the cells were then treated with DHT or TGF-β1. a The expression of p66Shc, p-p66Shc, Sirt1, TGF-β and AR was analyzed by western blot assay. mRNA levels of p66Shc (b), Sirt1 (c), and α-SMA (d) was analyzed by real-time PCR. e The expression of p66Shc, p-p66Shc, Sirt1, and TGF-β was assessed by western blot assay. f The expression of p-p66Shc (Alexa Fluor 488) and α-SMA (Cy3) were analyzed using immunofluorescence staining (60×). Images are representative of three independent experiments with similar results. g p-p66Shc (Alexa Fluor 488) and mitochondria were revealed by immunofluorescence and MitoTracker Red staining, respectively using confocal microscopy. h Mitochondrial membrane potential was analyzed by JC-1 monomers/polymers (60×). Images are representative of three independent experiments with similar results. i Quantitative analysis of the ratios of the ratios red to green fluorescence is shown. Three independent experiments were performed with similar results. Data are shown as the mean ± SD. *p ≤ 0.05. DHT, dihydrotestosterone; TGF-β1, transforming growth factor-beta 1; p-p66Shc, phosphorylated 66-kDa Src homology 2 domain-containing protein; Sirt1, sirtuin 1; AR, androgen receptor; α-SMA, alpha-smooth muscle actin; GCs, granulosa cells

Fig. 8

Sirt1 suppresses fibrotic factors activation in a p66Shc-dependent manner. Sirt1 in granulosa cells was silenced using siRNA. After 48 h, the cells were treated with DHT, resveratrol, H₂O₂, or EX527. a p-p66Shc and Sirt1 were analyzed by double immunofluorescence staining (40×). Images are representative of three independent experiments with similar results. b Quantification of the p-p66Shc and Sirt1 fluorescence is shown. c ROS was measured using a DCF-DA probe, and assessed by confocal microscopy (40×). Images are representative of three independent experiments with similar results. d Quantification of DCF fluorescence is shown. e Granulosa cells were treated with various concentrations of EX527. The expression of p66Shc, p-p66Shc, Sirt1, TGF-β, and AR was analyzed by western blot assay. f The expression of p66Shc, Sirt1 and TGF-β was assessed by western blot assay in granulosa cells after treatment with DHT, resveratrol, or EX527. g mRNA levels of p66Shc, TGF-β, α-SMA, and CTGF were measured by real-time PCR. h The expression of p66Shc, Sirt1, TGF-β, CTGF, β-catenin, and AR in granulosa cells treated with DHT or H₂O₂ was analyzed by western blot after Sirt1 silencing. Three independent experiments were performed with similar results. Data are shown as the mean ± SD. *p ≤ 0.05. DHT dihydrotestosterone, TGF-β transforming growth factor-beta, p-p66Shc phosphorylated 66-kDa Src homology 2 domain-containing protein, Sirt1 sirtuin 1, AR androgen receptor, α-SMA alpha-smooth muscle actin, CTGF connective tissue growth factor, GCs granulosa cells, DCF dichlorodihydrofluorescein, ROS reactive oxygen species, EX527, a Sirt1 inhibitor