NRF2-mediated Signaling is a Master Regulator of Transcription Factors in Bovine Granulosa Cells under Oxidative Stress Condition

Transcription factors (TFs) are known to be involved in regulating the expression of several classes of genes during folliculogenesis. However, the regulatory role of TFs during oxidative stress (OS) is not fully understood. The current study was aimed to investigate the regulation of the TFs in bovine granulosa cells (bGCs) during exposure to OS induced by H 2 O 2 in vitro. For this, bGCs derived from ovarian follicles were cultured in vitro till their confluency and then treated with H 2 O 2 for 40 min. Twenty-four hours later, cells were subjected to various phenotypic and gene expression analyses for genes related to TFs, endoplasmic reticulum stress, apoptosis, cell proliferation, and differentiation markers. The bGCs exhibited higher reactive oxygen species accumulation, DNA fragmentation, and endoplasmic reticulum stress accompanied by reduction of mitochondrial activity after exposure to OS. In addition, higher lipid accumulation and lower cell proliferation were noticed in H 2 O 2 -challenged cells. The mRNA level of TFs including NRF2 , E2F1 , KLF6 , KLF9 , FOS , SREBF1 , SREBF2 , and NOTCH1 was increased in H 2 O 2 -treated cells compared with non-treated controls. However, the expression level of KLF4 and its downstream gene, CCNB1 , were downregulated in the H 2 O 2 -challenged group. Moreover, targeted inhibition of NRF2 using small interference RNA resulted in reduced expression of KLF9 , FOS , SREBF2 , and NOTCH1 genes, while the expression of KLF4 was upregulated. Taken together, bovine granulosa cells exposed to OS exhibited differential expression of various transcription factors, which are mediated by the NRF2 signaling pathway.