Nrf2 is a redox sensitive transcription factor regulating the expression of antioxidant genes as defense mechanism against various stressors. The aim of this study is to investigate the potential role of noncoding miRNAs as endogenous and quercetin as exogenous regulators of Nrf2 pathway in bovine granulosa cells. For this cultured granulosa cells were used for modulation of miRNAs (miR-28, 153 and miR-708) targeting the bovine Nrf2 and supplementation of quercentin to investigate the regulatory mechanisms of the Nrf2 antioxidant system. Moreover, cultured cells were treated with hydrogen peroxide to induce oxidative stress in those cells. Our results showed that, oxidative stress activated the expression of Nrf2 as a defense mechanism, while suppressing the expression of those miRNAs. Overexpression of those miRNAs resulted in downregulation of Nrf2 expression resulted in higher ROS accumulation, reduced mitochondrial activity and cellular proliferation. Quercetin supplementation showed its protective role against oxidative stress induced by H2O2 by inducing the expression of antioxidant enzymes. In conclusion, this study highlighted the involvement of miR-153, miR-28 and miR-708 in regulatory network of Nrf2 mediated antioxidant system in bovine granulosa cells function. Furthermore, quercetin at a low dose played a protective role in bovine granulosa cells against oxidative stress damage.

During in vitro embryo production, preimplantation embryos are constantly exposed to multitudes of environmental stressors originating from conditions at various stages during the culture processes. Exposure to high atmospheric oxygen concentration within the in vitro culture environment induces oxidative stress, which results in the production of excessive reactive oxygen species (ROS) (

High atmospheric oxygen level during in vitro embryo culture system is reported to induce oxidative stress, leading to an increased intracellular reactive oxygen species (ROS) accumulation and lower the quality and development of the embryos. The Nrf2 is a redox sensitive transcription factor, which regulates the antioxidant machinery against oxidative stress in bovine pre-implantation embryos (Amin et al. 2014Mol. Reprod. Dev. 81, 497-513). Quercetin is a plant-derived flavonoid found in fruits and vegetables with antioxidant, anti-inflammatory, and anti-apoptotic properties. The effect of these antioxidants on the development and quality of bovine pre-implantation embryos with respect to the activation of the Nrf2 signalling pathways is not yet discovered. Here, we aimed to investigate the effect of quercetin supplementation on the activation of the Nrf2 signalling pathway and the subsequent impact on the quality of bovine embryos. For this, presumptive bovine zygotes were cultured in SOFaa embryo culture media supplemented with or without 10 µM quercetin (482 and 485 zygotes, respectively) and incubated under high oxygen level (20%) conditions. Day 7 and Day 8 blastocysts were analysed for the accumulation intracellular ROS and mitochondrial activity using H2DCFDA and MitoTracker® Red, respectively. Thereafter, blastocysts from both groups were subjected to total RNA isolation using Norgen total RNA purification plus kit (BioTek, Winooski, VT, USA). The relative abundance of Nrf2 and its downstream antioxidant genes (NQO1, PRDX1, SOD1, and CAT) was quantified using quantitative PCR. Moreover, the protein level of Nrf2 was detected using immunofluorescence staining. Data from 4 independent biological replicates were statistically analysed using a 2-tailed Student’s t-test. A significant difference in the mean value among treatments was determined at P=0.05. Results showed that quercetin supplementation in embryo culture medium significantly increased the blastocyst total cell number both at Day 7 (125±7.98 v. 100±3.74; P=0.007) and Day 8 (128±4.49 v. 95±3.15; P<0.0001). However, no difference was observed in the quercetin-treated group compared with control with respect to the cleavage rate (85.89±1.11v. 83.86±1.27; P=0.2) as well as the blastocyst rate at Day 7 (24.57±4.94 v. 27.23±1.68; P=0.64) and Day 8 (41.17±3.70 v. 38.87±0.85; P=0.58). Interestingly, quercetin-supplemented blastocysts showed a significant reduction in intracellular ROS level and increment in the mitochondrial activity compared with untreated counterparts. Supplementation of quercetin activated the Nrf2 transcriptional factor both at the mRNA and protein level, which subsequently activated the transcription of the aforementioned downstream antioxidant genes. In conclusion, supplementation of quercetin to embryo culture media protects embryos against oxidative stress by activating the Nrf2-mediated oxidative stress response mechanism, which leads to reduced accumulation of ROS and elevated mitochondrial activity.