1. Resveratrol inhibits the mevalonate pathway and potentiates the antiproliferative effects of simvastatin in rat theca-interstitial cells 
Donna H. Wong, B.S., Jesus A. Villanueva, D.V.M., Ph.D., Amanda B. Cress, B.S., Anna Sokalska, M.D., Ph.D., Israel Ortega, M.D., Antoni J. Duleba, M.D. 
Fertility and Sterility 
Volume 96, Issue 5, Pages (November 2011)
DOI: /j.fertnstert
Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
(A) Effect of resveratrol and simvastatin on proliferation. Ovarian theca-interstitial cells were cultured without (control) or with resveratrol (30 and 50 μM) and/or simvastatin (1 and 10 μM) for 48 hours in chemically defined media. Proliferation was evaluated by determination of DNA synthesis (by radiolabeled thymidine incorporation). Each bar represents means ± SEM from two independent experiments (each with n = 8); means without a common letter are significantly different (P<.001). (B) Effect of resveratrol (30 and 50 μM) on proliferation in the absence or presence of MA (100 μM), FPP (30 μM), or GGPP (30 μM). Ovarian theca-interstitial cells were stimulated with MA, FPP, or GGPP for 2 hours and then incubated with resveratrol for 24 hours in chemically defined media. Proliferation was assessed by determination of DNA synthesis (by radiolabeled thymidine incorporation). Each bar represents means ± SEM from two independent experiments (each with n = 8); means without a common letter are significantly different (P<.001).
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

3. Figure 2
(A) Effect of resveratrol and simvastatin on HMG-CoA reductase (HMGCR) gene expression after 24 or 48 hours of treatment. Ovarian theca-interstitial cells were incubated without (control) or with simvastatin (10 μM) and/or different concentrations of resveratrol (30–100 μM) for 24 or 48 hours. HMGCR mRNA expression was analyzed by quantitative real-time PCR and normalized to the endogenous reference hypoxanthine phosphoribosyltransferase. Data for the 24- and 48-hour treatments were analyzed separately. Each bar represents means ± SEM from three independent experiments (each with n = 4); means without a common letter are significantly different (P<.05). (B) Effect of resveratrol and simvastatin on HMGCR protein expression after 48 hours of treatment. Ovarian theca-interstitial cells were incubated without (control) or with resveratrol (50 μM) and/or simvastatin (10 μM) for 48 hours. HMGCR protein level was determined by Western blot analysis and normalized to β-actin (A). One of three representative blots is shown (B). Each bar represents means ± SEM (n = 4); means without a common letter are significantly different (P<.009). (C) Effect of resveratrol and simvastatin on HMGCR activity. Ovarian theca-interstitial cells were fractionated to obtain microsomes and microsomal HMGCR activity was determined by the conversion of [3-14C]-HMG-CoA to [14C]-mevalonic acid lactone, as described in Materials and Methods. Each bar represents means ± SEM from three independent experiments (each with n = 3); means without a common letter are significantly different (P<.02).
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
(A) Effect of resveratrol and simvastatin alone on cholesterol synthesis. Ovarian theca-interstitial cells were cultured without (control) or with resveratrol (30–100 μM) or simvastatin (0.1–10 μM) in the presence of [14C]-acetate for 24 hours in chemically defined media. Cholesterol synthesis was evaluated by the incorporation of [14C]-acetate into [14C]-cholesterol. Each bar represents means ± SEM from three independent experiments (each with n = 4); means without a common letter are significantly different (P<.001). (B) Effect of a combination of resveratrol and simvastatin on cholesterol synthesis. Ovarian theca-interstitial cells were incubated with [14C]-acetate in the presence of resveratrol (50 μM) and simvastatin (0.1 μM) for 24 hours in chemically defined media. Cholesterol synthesis was determined by incorporation of radiolabeled [14C]-acetate into [14C]-cholesterol. Each bar represents means ± SEM from three independent experiments (each with n = 4); means without a common letter are significantly different (P<.001).
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

5. Figure 4
Proposed diagram for the inhibition of HMG-CoA reductase by resveratrol (at the level of HMG-CoA reductase expression and activity) and by statins (at the level of competitive inhibition of HMG-CoA reductase activity). Farnesyl pyrophosphate and geranylgeranyl pyrophosphate are the downstream products of mevalonic acid. Products of the mevalonate pathway, such as cholesterol, inhibit HMGCR expression (36, 37). Inhibition of the mevalonate pathway also reduces the prenylation of proteins and hence the proliferation of cells.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions