Pigment epithelium-derived factor exerts antioxidative effects in 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.