Beneficial effect of resveratrol on bovine oocyte maturation and subsequent embryonic development after in vitro fertilization  Feng Wang, Ph.D., XiuZhi Tian, Ph.D., Lu Zhang, Ph.D., ChangJiu He, Ph.D., PengYun Ji, M.S., Yu Li, M.S., DunXian Tan, Ph.D., GuoShi Liu, Ph.D.  Fertility and Sterility  Volume 101, Issue 2, Pages 577-586.e1 (February 2014) DOI: 10.1016/j.fertnstert.2013.10.041 Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Effect of resveratrol on the secretion of steroid hormones and the development of fertilized bovine oocytes in maturation medium. (A) Concentrations of progesterone (P) (ng·mL−1) in the groups treated with different concentrations of resveratrol. (B) Concentrations of 17β-estradiol (E2) (pg·mL−1) in the groups treated with different concentrations of resveratrol. (C) The levels of expression of progesterone and E2 synthetic enzyme genes in the groups treated with different concentrations of resveratrol. (D) Survival rate and polar body rate (PB1). (E) Cumulus cell extension specific gene (PTX3) expression. (F) Cleavage rate, eight-cell rate, and blastocyst rate. (G) Hatched blastocyst rate. (H) Mean number of cells/blastocyst. (I) Epifluorescence photomicrographs of in vitro produced bovine blastocysts that developed in maturation medium supplemented with resveratrol at 0 (control), 0.1, 1.0, or 10.0 μM. (J) Blastocysts were stained with propidium iodide to determine the mean number of cells/blastocyst of blastocysts that developed in maturation medium supplemented with resveratrol at 0 (control), 0.1, 1.0, or 10.0 μM. Different superscripted letters in the same column (a, b) indicate a statistically significant difference (P<.05). Fertility and Sterility 2014 101, 577-586.e1DOI: (10.1016/j.fertnstert.2013.10.041) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effects of resveratrol on intracellular GSH and ROS levels and the expression of the antioxidant enzyme genes CAT, GPx4, and SOD1 during bovine oocyte maturation. (A) Effects of resveratrol on intracellular GSH and ROS levels. (B) Effects of resveratrol on the expression of antioxidant enzyme genes (CAT, GPx4, and SOD1). (C) Epifluorescence photomicrographs of in vitro matured bovine oocytes that were stained with 2′7′-dichlorodihydrofluorescein diacetate (H2DCFDA) to detect reactive oxygen species (ROS). The metaphase II (MII) oocytes were developed in maturation medium supplemented with resveratrol at 0 (control), 0.1, 1.0, or 10.0 μM. (D) Epifluorescence photomicrographs of in vitro matured bovine oocytes that were stained with CellTracker Blue to determine the level of intracellular glutathione (GSH). The MII oocytes were developed in maturation medium supplemented with resveratrol at 0 (control), 0.1, 1.0, or 10.0 μM. Different superscripted letters in the same column indicate a statistically significant difference (P<.05). Fertility and Sterility 2014 101, 577-586.e1DOI: (10.1016/j.fertnstert.2013.10.041) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Effect of resveratrol on the expression of genes related to oocyte maturation. (A) Oocyte maturation-related genes (c-mos, ERK2, and MAPK1). (B) Cell cycle-related genes CDC2/CyCB2. Different superscripted letters (a–d) in each column indicate statistically significant differences (P<.05). Fertility and Sterility 2014 101, 577-586.e1DOI: (10.1016/j.fertnstert.2013.10.041) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Levels of expression of SIRT1 in bovine granulosa cells, cumulus cells, oocytes, and blastocysts determined using quantitative RT-PCR, immunofluorescence, and Western blot analysis. (A) Immunofluorescence images of SIRT1 in bovine COCs and blastocysts. The cellular localization of SIRT1 was identified using confocal analysis. Bovine COCs were incubated with the SIRT1 antibody followed by an Alexa-488-conjugated donkey anti-goat IgG (red). (B) Level of expression of the SIRT1 gene evaluated using with Western blot analysis in the following cells: [1] granulosa cells (GCs); [2] cumulus cells in the GV stage (CCs, GV); [3] cumulus cells in the MII stage in the control group (CCs, MII-C); [4] cumulus cells in the MII stage in the resveratrol-treated group (CCs, MII-R); [5] oocytes in the GV stage (OO, GV); [6] oocytes in the MII stage in the control group (OO, MII-C); [7] oocytes in the MII stage in the resveratrol-treated group (OO, MII-R); [8] blastocysts. (C) Relative levels of SIRT1 mRNA in cumulus cells (CCs) treated with or without different concentrations of resveratrol. (D) Relative levels of SIRT1 mRNA in oocytes (OO) treated with or without different concentrations of resveratrol. (E) Relative protein levels of SIRT1 in cumulus cells (CCs) in the GV or MII stage treated with or without 1.0 μM resveratrol. (F) Relative protein levels of SIRT1 in oocytes (OO) in GV or MII stage treated with or without 1.0 μM resveratrol. Different superscripted letters (a–d) in each column indicate statistically significant differences (P<.05). Fertility and Sterility 2014 101, 577-586.e1DOI: (10.1016/j.fertnstert.2013.10.041) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions