Effects of resveratrol on growth and function of rat ovarian granulosa cells

Abstract

Objective: To evaluate the effects of resveratrol on growth and function of granulosa cells. Previously, we demonstrated that resveratrol exerts profound proapoptotic effects on theca-interstitial cells.

Design: In vitro study.

Setting: Research laboratory.

Animal(s): Immature Sprague-Dawley female rats.

Intervention(s): Granulosa cells were cultured in the absence or presence of resveratrol.

Main outcome measure(s): DNA synthesis was determined by thymidine incorporation assay, apoptosis by activity of caspases 3/7, cell morphology by immunocytochemistry, steroidogenesis by mass spectrometry, antimüllerian hormone (AMH), and vascular endothelial growth factor (VEGF) expression by polymerase chain reaction and Western blot.

Result(s): Resveratrol induced a biphasic effect on DNA synthesis, whereby a lower concentration stimulated thymidine incorporation and higher concentrations inhibited it. Additionally, resveratrol slightly increased the cell number and modestly decreased the activity of caspases 3/7 with no effect on cell morphology or progesterone production. However, resveratrol decreased aromatization and VEGF expression, whereas AMH expression remained unaltered.

Conclusion(s): Resveratrol, by exerting cytostatic but not cytotoxic effects, together with antiangiogenic actions mediated by decreased VEGF in granulosa cells, may alter the ratio of theca-to-granulosa cells and decrease vascular permeability, and therefore may be of potential therapeutic use in conditions associated with highly vascularized theca-interstitial hyperplasia and abnormal angiogenesis, such as those seen in women with polycystic ovary syndrome.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Figures

Fig. 1

(A) Effect of resveratrol (10–50 μM) on proliferation. Granulosa cells were cultured in chemically defined media supplemented with FSH (30 ng/ml) for 24 and 48 h in the absence (control) or in the presence of resveratrol. Proliferation was evaluated by determination of DNA synthesis (by thymidine incorporation). Results are presented as a percentage of control. Each bar represents mean ± SEM from two independent experiments (each with N=8). Means with no superscripts in common are significantly different (P

Fig 2

Effect of resveratrol (10–50 μM) on cell morphology. Granulosa cells were cultured for 24 h (A) and 48 h (B) in the absence (control) or in the presence of resveratrol, then fixed, stained and visualized under a fluorescent microscope (40x magnification) as described in Material and Methods. Nuclear staining with 4′,6-diamidino-2-phenylindole and F-actin was used to observe morphological changes. Control (a), Resveratrol 10 μM (b), Resveratrol 30 μM (c), Resveratrol 50 μM (d).

Fig. 3

(A, B) Effect of resveratrol (10–30 μM) on progesterone (A) and estradiol (B) production. Granulosa cells were cultured for 24 and 48 h in chemically defined media supplemented with FSH (30 ng/ml) and testosterone (0.5 μM) in the absence (control) or in the presence of resveratrol. Progesterone and estrogen levels were determined using liquid chromatography-mass spectrometry. 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

Fig. 4

(A) Effect of resveratrol (10–30 μM) on AMH mRNA expression. Granulosa cells were cultured for 24 and 48 h in chemically defined media supplemented with FSH (30 ng/ml) and testosterone (0.5 μM) 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