Hadas Bar-Joseph1, Ido Ben-Ami2, Raphael Ron-El2, Ruth Shalgi1, Dana Chuderland3. 1. Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv, Tel Aviv, Israel. 2. IVF and Infertility Unit, Department of Obstetrics and Gynecology, Assaf Harofeh Medical Center, Zerifin Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. 3. Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv, Tel Aviv, Israel. Electronic address: chuderla@gmail.com.
Abstract
OBJECTIVE: To determine whether supplementing granulosa cells cultures with pigment epithelium-derived factor (PEDF) can protect them from oxidative stress. DESIGN: We used either granulosa cell line or human primary granulosa cell culture from women undergoing in vitro fertilization (IVF) treatments. SETTING: University research facilities. ANIMAL(S): Imprinting control region female mice. INTERVENTION(S): Recombinant PEDF (rPEDF) was added to cultures of either primary granulosa cell culture or granulosa cell line in the present or absence of H2O2 triggering. MAIN OUTCOME MEASURE(S): We followed cell viability with the use of methylthiazolyl tetrazolium assay and tracked PEDF mechanism of action with the use of Western blot analysis, measuring the level of SOD-1 and GPX-1 mRNA, protein level of BAX, and phosphorylation of AKT. RESULT(S): We found that granulosa cell viability and the level of PEDF mRNA were both significantly reduced, in a dose-dependent manner, after exposure to H2O2. The rate of H2O2-induced apoptosis was significantly attenuated in granulosa cells treated with rPEDF. We showed that granulosa cells, of both humans and rodents, express the PEDF receptor, PNPLA2; once stimulated by rPEDF, the cells exhibited phosphorylation of AKT. Finally, we showed that PEDF exerts its antioxidative activity through the AKT signaling pathway. CONCLUSION(S): This study demonstrates that PEDF represents a novel intrinsic antioxidant of granulosa cells.
OBJECTIVE: To determine whether supplementing granulosa cells cultures with pigment epithelium-derived factor (PEDF) can protect them from oxidative stress. DESIGN: We used either granulosa cell line or human primary granulosa cell culture from women undergoing in vitro fertilization (IVF) treatments. SETTING: University research facilities. ANIMAL(S): Imprinting control region female mice. INTERVENTION(S): Recombinant PEDF (rPEDF) was added to cultures of either primary granulosa cell culture or granulosa cell line in the present or absence of H2O2 triggering. MAIN OUTCOME MEASURE(S): We followed cell viability with the use of methylthiazolyl tetrazolium assay and tracked PEDF mechanism of action with the use of Western blot analysis, measuring the level of SOD-1 and GPX-1 mRNA, protein level of BAX, and phosphorylation of AKT. RESULT(S): We found that granulosa cell viability and the level of PEDF mRNA were both significantly reduced, in a dose-dependent manner, after exposure to H2O2. The rate of H2O2-induced apoptosis was significantly attenuated in granulosa cells treated with rPEDF. We showed that granulosa cells, of both humans and rodents, express the PEDF receptor, PNPLA2; once stimulated by rPEDF, the cells exhibited phosphorylation of AKT. Finally, we showed that PEDF exerts its antioxidative activity through the AKT signaling pathway. CONCLUSION(S): This study demonstrates that PEDF represents a novel intrinsic antioxidant of granulosa cells.
Authors: Khalid M Salama; Hesham M Abo Ragab; Mohammed F El Sherbiny; Ali A Morsi; Ibrahim I Souidan Journal: BMC Womens Health Date: 2017-11-13 Impact factor: 2.809