PKCδ contributes to oxidative stress-induced apoptosis in porcine ovarian granulosa cells via activating JNK

Oxidative stress-induced apoptosis of granulosa cells (GCs) is believed to be an important cause of follicular atresia. Our previous work showed that the c-Jun N-terminal kinase (also known as JNK) might promote apoptosis in GCs during oxidative stress. The aim of this study was to investigate the upstream signaling required for JNK-mediated GCs apoptosis during oxidative stress. Since PKC delta, and ASK1 have been suggested to regulate JNK activity in some types of cells, we hypothesized that PKC delta and ASK1 might contribute to JNK-dependent apoptosis in GCs suffering oxidative stimulation. To test this assumption, porcine GCs obtained from healthy follicles were treated with H2O2 alone, or together with inhibitors against PKC delta and JNK, and then collected for cell viability assay, TUNEL staining, immunoprecipitation, western blotting, or JNK activity detection in vitro. The current results showed that the cell viability loss, DNA fragmentation, morphological shrinkage, and nuclear condensation in H2O2-treated porcine GCs was correlated with enhanced activation of JNK. Although ASK1 was supposed to be a JNK activator, we found no definite role of ASK1 in JNK-induced GCs apoptosis during oxidative stress. Further investigations revealed that H2O2-mediated PKC delta activation was required for the apoptotic death of porcine GCs. Particularly, the pro-apoptotic effects of PKC delta on porcine GCs might be achieved by activating the mitochondrial pathway. Importantly, we found that p-PKC delta acts as an upstream activator of JNK in H2O2-treated porcine GCs. However, JNK has no regulatory effect on PKC delta activity. Taken together, our findings provided a novel model of GCs apoptosis involving the activation of PKC delta/JNK/mitochondrial apoptosis axis during oxidative stress. (C) 2019 Published by Elsevier Inc.