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NRF2-mediated signaling is a master regulator of transcription factors in bovine granulosa cells under oxidative stress condition. Taqi MO et al. (2021) 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 H2O2 in vitro. For this, bGCs derived from ovarian follicles were cultured in vitro till their confluency and then treated with H2O2 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 H2O2-challenged cells. The mRNA level of TFs including NRF2, E2F1, KLF6, KLF9, FOS, SREBF1, SREBF2, and NOTCH1 was increased in H2O2-treated cells compared with non-treated controls. However, the expression level of KLF4 and its downstream gene, CCNB1, were downregulated in the H2O2-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.//////////////////Non-Esterified Fatty Acid-Induced Reactive Oxygen Species Mediated Granulosa Cells Apoptosis Is Regulated by Nrf2/p53 Signaling Pathway. Wang Y et al. (2020) Negative energy balance (NEB) during the perinatal period can affect dairy cow follicular development and reduce the fecundity. Non-esterified fatty acid (NEFA) concentration is elevated during NEB, and is known to be toxic for multiple cell types. In the ovary, NEB increased NEFA, and may influences follicular growth and development. However, the effect and mechanism of NEFA on granulosa cells (GCs) in vitro remains unknown. In this study, we found that NEFA dose-dependently induced apoptosis in primary cultured granulosa cells. Mechanistically, our data showed that NEFA significantly increased reactive oxygen species (ROS) levels, resulting in the activation of endoplasmic reticulum stress (ERS) and eventually cell apoptosis in GCs. Moreover, NEFA also increased the phosphorylation levels of ERK1/2 and p38MAPK pathways, upregulated the expression of p53 and potentially promoted its translocation to the nuclear, thus transcriptionally activated Bax, a downstream gene of this pathway. NEFA also promoted nuclear factor E2 (Nrf2) expression and its level in the nuclear. To elucidate the mechanism of NEFA action, N-acetyl-L-cysteine (NAC), a ROS scavenger was used to verify the role of ROS in NEFA induced apoptosis of GCs. NAC pretreatment reversed the NEFA-induced ERS-related protein and apoptosis-related protein levels. Meanwhile, NAC pretreatment also blocked the phosphorylation of ERK1/2 and p38 induced by NEFA, and the nucleation of Nrf2 and p53, suggesting that ROS plays a crucial role in regulating the NEFA-induced apoptosis of GCs. Together, these findings provide an improved understanding of the mechanisms underlying GCs apoptosis, which could potentially be useful for improving ovarian function.//////////////////
Activation of Nrf2/Keap1 pathway by oral Dimethylfumarate administration alleviates oxidative stress and age-associated infertility might be delayed in the mouse ovary. Akino N et al. (2019) Age-associated infertility is a problem worldwide, and management of oxidative stress is known to be essential. Nuclear factor-E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1)-antioxidant response element (ARE) signaling pathway works as an essential defense mechanism against oxidative stress, and an oral drug Dimethylfumarate (DMF) is known to activate the pathway. We tested the hypothesis that oral DMF could alleviate oxidative stress in the ovary, resulting in salvation of age-associated infertility in a mouse model of reproductive age, and we examined the effects of DMF administration. 20 mg/kg DMF was administrated to female mice from 32 to 48 weeks, and Nrf2 levels, antioxidant levels, ovarian reserve, DNA damage, and oxidative stress were examined. DMF administration resulted in elevated mRNA and protein levels of Nrf2, antioxidants, and telomere, and serum levels of Nrf2 and anti-mullerian hormone were also elevated. Results of TUNEL assay and Immunohistochemistry of mice ovarian tissues showed that DNA damage and oxidative stress were decreased by DMF administration, and significantly more oocytes were collected along with preservation of 60% more primordial follicles. Our data suggest that DMF administration activates the Nrf2/Keap1 pathway, elevate levels of antioxidants, and decrease DNA damage and oxidative stress, resulting in improved ovarian reserve in the mouse ovary.//////////////////
Expression and localization of nuclear factor erythroid 2-related factor 2 in the ovarian tissues of mice at different ages. Sindan N et al. (2018) The aim of the present study was to investigate the expression and localization of nuclear factor erythroid 2-related factor 2 (Nrf2) in the ovaries of mice in different age groups, and to explore the association between Nrf2 and premature ovarian aging. The present study identified the localization of Nrf2 protein by performing immunohistochemical assay of ovarian tissues obtained from mice in different age groups. The mRNA expression levels of Nrf2 were detected via reverse transcription-quantitative polymerase chain reaction, while the expression levels of Nrf2 protein and apoptosis-associated proteins, including Caspase3 and B-cell lymphoma 2 (Bcl-2), were evaluated by western blot analysis. The results revealed that Nrf2 protein was mainly localized in granulosa cells, as well as in the secondary follicles and antral follicles of oocytes. Nrf2 expression levels were significantly lower in mice aged 4 days compared with 12-week-old mice (P<0.05), and the level of Nrf2 was lower in mice aged 40 weeks compared with those aged 12 weeks (P<0.05). In addition, the expression of the apoptosis protein Caspase3 in the ovarian tissue of mice aged 3, 8 and 12 weeks remained markedly greater when compared with those aged 4 days and 40 weeks. Bcl-2, an anti-apoptotic protein, was also significantly expressed in the ovarian tissues of juvenile (4-day-old) mice when compared with mice aged >40 weeks (P<0.05). In conclusion, Nrf2 was highly expressed in the ovarian tissues of mice of childbearing age (8-12 weeks old) and may possibly be involved in ovarian regulatory functions. The results indicated that Nrf2 expression and localization may have important implications in the prevention of ovarian aging.//////////////////
Activation of Nrf2 might reduce oxidative stress in human granulosa cells. Akino N et al. (2017) Nuclear factor-E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1)-antioxidant response element (ARE) signaling pathway is one of the most important defense mechanisms against oxidative stress (OS). It is well documented that equilibration status of OS plays fundamental roles in human reproductive medicine, and the physiological role of Nrf2 in ovarian granulosa cells (GCs) has not been determined yet. Herein we aimed to study the function of Nrf2 in GCs. Human ovarian tissues were subjected to immunohistochemistry to localize Nrf2 and Keap1 and we detected the expression of Nrf2 and Keap1 in the human GCs. Human luteinized GCs were isolated and cultured, and hydrogen peroxide (H2O2) or Dimethylfumarates (DMF), an activator of Nrf2, were added to GCs to analyze the relationship between Nrf2 and antioxidants by quantitative RT-PCR. The mRNA levels of Nrf2, catalase, superoxide dismutase 1 (SOD1), and 8-Oxoguanine DNA glycosylase (OGG1) were elevated by H2O2, and DMF treatment showed similar but pronounced effects through activation of Nrf2. To determine the relationship of Nrf2 and the generation of antioxidants, siRNAs were used and quantitative RT-PCR were conducted. Decreased expression of Nrf2 resulted in a decreased level of these antioxidant mRNA. Intracellular levels of ROS were investigated by fluorescence of 8-hydroxy-2'-deoxyguanosine and fluorescent dye, 2',7'-dichlorodihydrofluorescein diacetate after H2O2 and/or DMF treatment, and DMF treatment quenched intracellular ROS generation by H2O2. These results show that activation of Nrf2 might lead to alleviate OS in human GCs, and this could provide novel insight to conquer the age-related fertility decline that is mainly attributed to the accumulation of aberrant OS.//////////////////
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