Pigment epithelium–derived factor exerts antioxidative effects in granulosa cells  Hadas Bar-Joseph, Ph.D., Ido Ben-Ami, M.D., Ph.D., Raphael Ron-El, M.D., Ruth Shalgi, Ph.D., Dana Chuderland, Ph.D.  Fertility and Sterility  Volume 102, Issue 3, Pages 891-898.e3 (September 2014) DOI: 10.1016/j.fertnstert.2014.06.012 Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Effects of H2O2 on LH-15 cell viability and level of pigment epithelium-derived factor (PEDF) mRNA. LH-15 cells were exposed to increasing doses of H2O2 for 24 hours. (A) Cell viability was measured with the use of methylthiazolyl tetrazolium assay. Bars represent five independent experiments. (B) Changes in PEDF mRNA level were measured by quantitative polymerase chain reaction analysis with a specific primer for PEDF; calibrated with hypoxanthine-guanine phosphoribosyltransferase. Bars represent three independent experiments. The ratio between each treatment and the control is plotted as mean ± SD (∗P<.05; ∗∗P<.01; significantly different from control value). Fertility and Sterility 2014 102, 891-898.e3DOI: (10.1016/j.fertnstert.2014.06.012) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 2 PEDF enhances LH-15 cell viability under oxidative stress conditions. LH-15 cells were exposed to increasing doses of H2O2 with or without recombinant PEDF (1 nmol/L or 5 nmol/L) for 24 hours. (A) Cell viability was measured with the use of MTT assay. Bars represent three independent experiments. (B) Cells were lysed and their proteins analyzed by Western blotting with anti-BAX antibody, with the use of actin as a loading control. The intensity of bands was analyzed with the use of Image-J software. (C) Graphic representation of BAX Western blotting. Bars represent densitometry analysis of three independent experiments. The ratio between each treatment and control is plotted as mean ± SD (∗P<.05; significantly different from control value). Abbreviations as in Figure 1. Fertility and Sterility 2014 102, 891-898.e3DOI: (10.1016/j.fertnstert.2014.06.012) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 3 The expression of PEDF receptor PNPLA2 in the ovary. (A) Representative immunohistochemistry of mouse ovarian sections labeled with anti-PNPLA2 antibody (B, C) and a control sample of secondary antibody only (A). (B) Autoradiographs of representative polymerase chain reaction analyses (×35 cycles), demonstrating expression of PNPLA2 mRNA in (a) human primary granulosa cell culture, mouse ovaries, and LH-15 cells. (b) Control: GAPDH primers. (C) Western blot analysis of human primary granulosa cell culture, mouse ovaries, and LH-15 cells with the use of anti-PNPLA2 (PEDF-R) antibody. Abbreviations as in Figure 1. Fertility and Sterility 2014 102, 891-898.e3DOI: (10.1016/j.fertnstert.2014.06.012) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 4 (A–D) PEDF activates AKT signaling pathway in granulosa cells. (A, B) LH-15 cells or (C, D) human primary granulosa cell culture were serum starved and treated with rPEDF (1 nmol/L) for various durations. Cells were lysed and their proteins analyzed with the use of Western blotting using antiphosphorylated AKT (pho-AKT) and antigeneral AKT (gen-AKT) antibodies. Bars represent densitometry analysis of three independent experiments. The intensity of bands was analyzed with the use of Image-J software, and the ratio between each protein and its control is plotted as mean ± SD (∗P<.05; significantly different from control value). (E, F) PEDF activates AKT signaling pathway in human granulosa cells under oxidative stress conditions. Human primary granulosa cell culture was exposed to H2O2 for 24 hours with or without recombinant PEDF (1 nmol/L). Cells were lysed and their proteins analyzed with the use of Western blotting with anti–pho-AKT and anti–gen-AKT antibodies. (E) Representative Western blot analysis micrograph. (F) Bars represent densitometry analysis of three independent experiments. The intensity of bands was analyzed with the use of the Image-J software, and the ratio between each treatment and the control is plotted as mean ± SD (∗P<.05; ∗∗P<.01; significantly different from control value). Abbreviations as in Figure 1. Fertility and Sterility 2014 102, 891-898.e3DOI: (10.1016/j.fertnstert.2014.06.012) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Pigment epithelium–derived factor (PEDF) reverses the adverse effect of H2O2 on SOD-1 and GPX-1 mRNA. LH-15 cells were exposed to H2O2 for 24 hours with or without recombinant PEDF (1 nmol/L). Changes in the level of (A) SOD-1 and (B) GPX-1 mRNA were measured with the use of quantitative polymerase chain reaction analysis with specific primers; calibrated with hypoxanthine-guanine phosphoribosyltransferase. Bars represent three independent experiments. The ratio between each treatment and the control is plotted as mean ± SD, (∗P<.05; ∗∗P<.01; significantly different from control values). Fertility and Sterility 2014 102, 891-898.e3DOI: (10.1016/j.fertnstert.2014.06.012) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions