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Activation of Nrf2 might reduce oxidative stress in human granulosa cells
Accepted Manuscript Activation of Nrf2 might reduce oxidative stress in human granulosa cells Nana Akino, Osamu Wada-Hiraike, Hiromi Terao, Harunori Honjoh, Wataru Isono, Houju Fu, Mana Hirano, Yuichiro Miyamoto, Michihiro Tanikawa, Miyuki Harada, Tetsuya Hirata, Yasushi Hirota, Kaori Koga, Katsutoshi Oda, Kei Kawana, Tomoyuki Fujii, Yutaka Osuga PII:
S0303-7207(17)30522-1
DOI:
10.1016/j.mce.2017.10.002
Reference:
MCE 10097
To appear in:
Molecular and Cellular Endocrinology
Received Date: 17 April 2017 Revised Date:
8 September 2017
Accepted Date: 2 October 2017
Please cite this article as: Akino, N., Wada-Hiraike, O., Terao, H., Honjoh, H., Isono, W., Fu, H., Hirano, M., Miyamoto, Y., Tanikawa, M., Harada, M., Hirata, T., Hirota, Y., Koga, K., Oda, K., Kawana, K., Fujii, T., Osuga, Y., Activation of Nrf2 might reduce oxidative stress in human granulosa cells, Molecular and Cellular Endocrinology (2017), doi: 10.1016/j.mce.2017.10.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Author names 1 Nana Akino, 1, *Osamu Wada-Hiraike, 1Hiromi Terao, 1Harunori Honjoh, 1, 2Wataru Isono,1Houju Fu, 1Mana Hirano, 1Yuichiro Miyamoto, 1Michihiro Tanikawa, 1Miyuki Harada, 1 Tetsuya Hirata, 1Yasushi Hirota, 1Kaori Koga, 1Katsutoshi Oda, 1, ¶Kei Kawana, 1Tomoyuki Fujii, and 1Yutaka Osuga
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Affiliations 1 Department of Obstetrics and Gynecology, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 8655 Japan. 2 Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 8655 Japan. current address: Nihon University School of Medicine Department of Obsterics & Gynecology, 30-1 Ohyaguchi Kamicho, Itabashi-ku, Tokyo 173 8610 Japan
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Corresponding author Osamu Wada-Hiraike M.D.,Ph.D. E-mail address: [email protected] Department of Obstetrics and Gynecology, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 8655 Japan Telephone: +0081 3 3815 5411 Fax: +0081 3 3816 2017 Grants or fellowships supporting the writing of the paper This work was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan Agency for Medical Research and Development, Ministry of Health, Labour and Welfare, and The Japanese Foundation for Research and Promotion of Endoscopy Grant.
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Full title Activation of Nrf2 might reduce oxidative stress in human granulosa cells
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Disclosure summary The authors declare no possible conflicts of interest to disclose
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Abstract 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. Keywords Nrf2/Keap1; oxidative stress; granulosa cells; Dimethylfumarates; IVF-ET; infertility treatment
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Abbreviations oxidative stress (OS), reactive oxygen species (ROS), in vitro fertilization and embryo transfer (IVF-ET), granulosa cells (GCs), nuclear factor-E2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), antioxidant response element (ARE), superoxide dismutase (SOD), 8-Oxoguanine DNA glycosylase (OGG1), Dimethylfumarate (DMF), multiple sclerosis (MS), United States Food and Drug Administration (FDA), phosphate-buffered saline (PBS), fetal bovine serum (FBS), dimethylsulfoxide (DMSO), small interfering RNA (siRNA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), carboxymethyl-H2-dichlorofluorescein diacetate (CM-H2DCFDA), 8 Hydroxyguanosine (8-OHdG), 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI), steroidogenic acute regulatory protein (StAR), aromatase (P450arom), 17β-hydroxysteroid dehydrogenase 1 (17β-HSD1), 3β-hydroxysteroid dehydrogenase 1 (3β-HSD1), cholesterol side-chain cleavage enzyme (P450scc), fumaric acid ester (FAE), European Medicines Agency (EMA)
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Introduction It is widely known that oxidative stress (OS) is an inevitable threat to the human body[1]. OS is a state characterized by an imbalance between reactive oxygen species (ROS) and antioxidant scavenger enzymes[2]. The production of ROS and antioxidants is balanced in a healthy body normally, but once imbalance occurs, the cells are threatened by damaging of lipids, proteins, and nucleic acids[3]. This is known to be closely associated with aging and age-related diseases, such as inflammatory diseases, cancer and reproduction[4, 5]. It is documented that excess OS plays a key role in the pathogenesis of subfertility in both males and females[6]. The changes of lifestyles in developed countries are leading to more women tending to delay childbearing, and the number of couples undergoing infertility treatment is increasing by the year[7]. The concentration of ROS-associated molecules in the follicular fluid, which is known to increase by aging[8-10], is considered to affect the oocyte maturation, embryo quality and outcome of in vitro fertilization and embryo transfer (IVF-ET)[11]. Furthermore, OS has been shown to induce oocyte degeneration and apoptosis through disturbing the meiotic spindle. Therefore, one can imagine that exposure of oocytes to excess level of ROS might result in unfavorable IVF-ET outcome. Granulosa cells (GCs) are cells surrounding the oocyte and responsible for normal folliculogenesis[12]. Although adequate amount of ROS is known to be necessary for ovulation[13], excess generation of ROS in the human GCs of women with polycystic ovarian syndrome adversely affected IVF success rates[12]. From these facts, management of OS may lead to better infertility treatment outcomes. Here we focused on nuclear factor-E2-related factor 2 (Nrf2) / Kelch-like ECH-associated protein 1 (Keap1)-antioxidant response element (ARE) signaling pathway[14, 15], which is one of the most important defense mechanisms against oxidative stress[16]. During normoxia, Nrf2, a transcription factor with a high sensitivity to oxidative stress, is held in the cytoplasm and maintained at low levels by an inhibitory protein; Keap1[17]. Oxidative stress causes Nrf2 to dissociate from Keap1 and to subsequently translocate into the nucleus, which results in its binding to specific DNA sequence ARE and the transcription of downstream target genes[14], and antioxidants including superoxide dismutase (SOD), catalase, and 8-Oxoguanine DNA glycosylase (OGG1) are elevated[18]. The appropriate functioning of the Nrf2/Keap1 pathway is indispensable to counteracting OS and protecting multiple organs and cells[14]. Dimethylfumarate (DMF) is an activator of Nrf2, and has been approved by the United States Food and Drug Administration (FDA) as a new first-line oral drug to treat relapsing forms of multiple sclerosis (MS)[14, 19]. The exact mechanism of DMF of action in MS is not fully understood [20], but studies show that DMF reduces ROS production, and antioxidants were elevated in neurons[21]. The therapeutic potential of DMF is investigated in OS and inflammatory related diseases, such as Alzheimer’s disease, Parkinson’s disease, chronic pulmonary disease, asthma, diabetes, and rheumatoid arthritis[22]. Therefore, we hypothesized that activation of Nrf2 by DMF may lead to alleviated OS in human GCs, and this will lead to better outcomes in infertility treatment.
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ACCEPTED MANUSCRIPT Materials and methods
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Tissue samples and immunohistochemistry The ovarian tissues used in this study were obtained from 12 female patients with regular menstrual cycles who took no hormonal drugs and underwent hysterectomy for uterine cervical cancer or endometrial cancer. These ovarian tissues were first identified by two pathologists as pathologically normal tissues. The female patients were 32~38 years old at the time of operation, and the operations were mostly performed during the proliferative phase of the menstrual cycle. The study was approved by the Institutional Review Board of the University of Tokyo Hospital, and written informed consent was obtained in each instance. Immunohistochemistry was performed by standard procedures as described previously (19). Paraffin sections (4 µm) were dewaxed in xylene and rehydrated through graded ethanol to water. Antigens were retrieved by boiling in 10 mM citrate buffer (pH 6.0) for 10 min in a microwave. The cooled sections were incubated in DAKO REAL peroxidase-blocking solution (DAKO, Carpinteria, CA) for 30 min to quench the endogenous peroxidase. To block nonspecific binding, sections were incubated in phosphate-buffered saline (PBS) containing 3% BSA and 0.5% Nonidet P-40 for 10 min at room temperature. Sections were then incubated with the anti-Nrf2 rabbit polyclonal antibody (16396-1-AP, Proteintech Group, Illinois, USA) or the anti-Keap1 rabbit polyclonal antibody (ab66620, Abcam Ltd., Cambridge, UK) in DAKO REAL antibody diluent (DAKO; 1:100) overnight at 4°C. Negative controls were incubated with PBS instead of the antibody. After washing with PBS, the sections were covered with DAB solution (DAKO) at room temperature, followed by Mayer’s hematoxylin (Wako Pure Chemical, Tokyo, Japan). Lastly, the sections were dehydrated through an ethanol series and xylene and mounted.
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Isolation and culture of primary human GCs Human luteinized GCs in follicular aspirates of preovulatory follicles were obtained from 171 patients (aged 26~46 years old) who underwent transvaginal oocyte retrieval for in vitro fertilization at the University of Tokyo Hospital and Kiba Park Clinic. This study was approved by the Institutional Review Board of the University of Tokyo, and written informed consent for the research use of human GCs was obtained from each patient. The method used to purify primary GCs was as described previously [23]. Briefly, follicular fluids were centrifuged at 1500 rpm for 10 min, resuspended in PBS with 0.2% hyaluronidase, and incubated at 37 °C for 30 min. The suspension was layered over Ficoll-Paque (GE Healthcare, Buckinghamshire, UK) and centrifuged at 700 g for 30 min. GCs were collected from the interphase and washed with PBS. GCs were cultured with DMEM/ F12 (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS; BioWest, Nuaille, France) and antibiotics (100 U/mL penicillin, 0.1 mg/mL streptomycin, and 250 ng/mL amphotericin B; Sigma Aldrich, Darmstadt, Germany) in 48-well (for quantitative real-time PCR) or 12-well plates (for Western blotting) at a density of 4 × 105 cells/mL and allowed to adhere at 37 °C in a humidified atmosphere containing 5% CO2. The GCs were ready to use 24 hours after attaching to the dishes, and the medium was freshly changed at this point to remove leukocytes from the GCs.
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Treatment of GCs To evaluate the effect of ROS on Nrf2 expression, varying concentrations of H2O2 (200, 400, 600 4
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Knockdown of Nrf2 using small interfering RNA (siRNA) Chemically synthesized 25-nucleotide stealth Nrf2 RNAi duplex oligonucleotides (set of 3) (GenBank accession no. BC 011558.1) were obtained from Invitrogen. Stealth RNAi negative control (Invitrogen) was used as a control small interfering RNA (siRNA). The seeded GCs were transfected with 40 nM of siRNA for 48 hours in Opti-MEM (Invitrogen) using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s protocol. After incubation with Nrf2 siRNA, the GCs were covered with serum-free medium, and were subjected to quantitative real-time PCR or Western Blotting.
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Reverse transcription and real-time comparative quantitative PCR Synthesis of cDNA templates from human GCs was performed using the SuperPrep Cell Lysis & RT Kit for qPCR (TOYOBO, Tokyo, Japan). To quantitate mRNA levels, real-time PCR was performed on a Light Cycler (Roche, Diagnostic GmBH, Mannheim, Germany), according to the manufacturer’s instructions. All samples of GCs were analyzed in triplicate or quadruplicate. Human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal standard for RNA loading. To obtain the relative gene expression data (fold change), the comparative 2-∆∆Ct method was used. The primer sets used are described in supplemental data 1.
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Western blotting GCs were lysed in lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing phosphatase inhibitors (Nacalai Tesque, Kyoto, Japan) and protease inhibitors (Roche), and insoluble material was removed by centrifugation at 14000 m/sec, for 12 minutes at 4°C. The supernatants were recovered, and the protein concentrations were measured using Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA). Equivalent amounts of lysate protein (10 µg) were subjected to Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad) and electrophoretically transferred onto Trans-Blot Turbo Transfer Packs (Bio-Rad) using Trans-Blot Turbo Transfer System (Bio-Rad). After blocking with 10% fat-free powdered milk in PBS at room temperature for 1 hour, the membranes were blotted overnight at 4°C with primary antibodies, including anti-Nrf2 (1:100; 16396-1-AP, Proteintech Group), anti-Keap1 (1:1000; ab66620, Abcam Ltd.), anti-OGG1 (1:1000; ab135940, Abcam Ltd.), anti-catalase (1:200; ab16731, Abcam Ltd.) and anti- SOD1 (1:1000; ab13499, Abcam Ltd.). Then, the blots were incubated with the appropriate secondary antibodies (anti-rabbit IgG, 7074S, 1:3000; anti-mouse IgG, 7076S; 1:3000, Cell Signaling) at room temperature for 1 hour and developed using ECL Plus Western blotting detection reagents (GE Healthcare). The images were scanned by the luminescent image analyzer Image Quant LAS 4000 mini (GE Healthcare). The expression of target proteins was internally normalized to the optical density of β-actin (1:2000; A2228, Sigma Aldrich) by the Image J software (http://rsb.info.nih.gov/ij/).
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Fluorescence microscopy GCs were isolated as described above and cultured on 4-well glass Millicell EZ slides (Merck Millipore, Darmstadt, Germany) at a density of 2 × 105 cells/mL. The cells were fixed with PBS containing 4% paraformaldehyde. After blocking, the cells were sequentially incubated with anti-Nrf2 (1:100; 16396-1-AP, Proteintech Group) and anti-8 Hydroxyguanosine (8-OHdG; 1:100; 1b48508, Abcam Ltd.) antibodies. Secondary antibodies were Alexa fluor 488-conjugated donkey anti-rabbit IgG (1:100; A-21206, Invitrogen) and Alexa fluor 558-conjugated goat anti-mouse IgG (1:100; A-11004, Invitrogen). The slides were briefly counterstained with 300nM of 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI; D1306, Thermofisher) and analyzed under a confocal fluorescence microscope (Carl-Zeiss LSM700 ZEN, Jena, Germany).
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Detection of intracellular ROS The intracellular levels of ROS were measured by loading the GCs with the fluoroprobe carboxymethyl-H2-dichlorofluorescein diacetate (CM-H2DCFDA; Invitrogen). Briefly, the CM-H2DCFDA probe was reconstituted in DMSO prior to loading. The GCs were incubated with 10 µM CM-H2DCFDA in PBS for 20 min at 37°C, counterstained with 10 µM of Hoechst 33342 (Thermofisher, Massachusetts, USA) in PBS for 5 min to visualize all cells, and then immediately observed under a confocal fluorescence microscope (Carl-Zeiss), with an excitation wavelength of 495 nm and an emission wavelength of 527 nm. The fluorescence intensity was analyzed and quantified using LSM700 ZEN. Quantitative data of fluorescence intensity were standardized by dividing each value by the average value of the control group in each experiment. The results are representative of three independent cultures.
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Statistical analysis Data were analyzed by Statistical analyses were performed using the JMP Pro 11 software (SAS institute Inc., Cary, NC, USA). All results are shown as means ± SD. For comparison of multiple values, ANOVA was used followed by Dunnett's test, with a significance of p values less than 0.05.
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ACCEPTED MANUSCRIPT Results Expression of Nrf2 and Keap1 in human ovaries Firstly, we aimed to show the presence of Nrf2 and Keap1 protein in ovarian tissue by immunohistochemistry. The Nrf2 and Keap1 proteins were predominantly detected in the cytoplasm of GCs at various stages of follicles (Fig. 1, 2 A-F).
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OS stimulates the expression of Nrf2, Catalase, SOD1 and OGG1 Nrf2 plays a role in salvaging cells from ROS. To explore the function of Nrf2 in GCs, cultured GCs were exposed to various concentrations of H2O2 (200, 400, 600 µM) for 48 hours to investigate how OS affects the expression of Nrf2 and antioxidant products, including catalase, SOD1, and OGG1. We could not observe apparent apoptosis in this condition, and elevation of mRNA levels of Nrf2, catalase, SOD1, and OGG1 with OS were determined by quantitative real-time RT-PCR. OS significantly elevated the mRNA levels of Nrf2, catalase, SOD1 and OGG1 after exposure to 600 µM of H2O2, and 400 µM of H2O2 to SOD1 and OGG1. Simultaneously, elevated OS resulted in the increased expression of Nrf2, catalase, SOD1, and OGG1 proteins in Western blotting analysis. Conversely, the protein level of Keap1 was decreased with OS exposure (Fig. 3).
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Knockdown of endogenous Nrf2 reduced antioxidants in GCs Next, we aimed to determine the depletion of Nrf2 and the generation of antioxidant product levels in GCs. We first confirmed whether decreased expression of Nrf2 could affect the cell viability, and this was judged by MTS assays and Trypan blue dye exclusion test. However, we were unable to find significant effect of Nrf2 depletion both on cell viability and cell number (data not shown). We found that siRNA-mediated depletion of endogenous Nrf2 resulted in significant decrease of mRNA levels of antioxidants, such as catalase, SOD1, and OGG1 in all three Nrf2 siRNA (#1~3). Protein levels of catalase, SOD1 and OGG1 were also decreased with siRNA-mediated depletion of endogenous Nrf2 (Fig. 4). It is well known that ROS levels affect the luteal function and we have shown that the level of NRF2 could affect the steroidogenesis function of granulosa cells[24], but we were unable to reach the conclusive data after downregulation of endogenous Nrf2.
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DMF elevates the expression of Nrf2, catalase, SOD1 and OGG1 DMF is known to activate the Nrf2/Keap1 pathway in cells, resulting in elevation of antioxidants, such as catalase, SOD1 and OGG1. Therefore GCs were treated with DMF (50 or 100 µM) for 48 hours and the mRNA levels and protein levels of Nrf2, catalase, SOD1 and OGG1 were investigated. We found that 100 µM of DMF exposure to GCs resulted in significant elevation of the mRNA levels of Nrf2, catalase, SOD1 and OGG1. Simultaneously, DMF treatment resulted in the increased expression of Nrf2, catalase, SOD1, and OGG1 proteins in Western blotting analysis. The protein levels of Keap1 was conversely decreased by DMF treatment (Fig. 5). Activation of Nrf2 reduces 8-OHdG generation in GCs It is known that accumulation of oxidative stress to cells result in elevation of 8-OHdG, which is one of the major products of DNA oxidation. GCs were treated with either DMF (Fig. 6 E~H) or 7
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DMF reduces intracelluar ROS generation in GCs Endogenous levels of intracellular ROS were measured by CM-H2DCFDA fluorescence, and we found that DMF treatment of GCs reduced ROS generation in the GCs. Conversely, H2O2 treatment of GCs resulted in enhanced levels of ROS generation, and the DCF staining is clearly shown in nucleus (Fig. 7, A~I). Addition of DMF to GCs treated with H2O2 reduced the ROS levels and DCF fluorescence was increased both in nucleus and cytosol (Fig. 7, J~L), showing that DMF treatment may alleviate stress in severe oxidative environments. The fluorescence intensity was subsequently analyzed and quantified, and the fluorescence intensity of GCs treated with DMF was approximately reduced to 1/3-fold than that of the control GCs (Fig. 7, M).
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Discussion Studies on ROS and antioxidant levels in human GCs are limited, and the relation of Nrf2/Keap1 pathway in GCs is not documented, to our knowledge. The results of immunohistochemistry showed presence of Nrf2 and Keap1 protein in the cytoplasm of GCs at various stages of follicles, suggesting the association of the Nrf2/Keap1 theory in human GCs. It is widely known that the production of antioxidants increases under the condition of OS, and catalase and SOD1 are representative biological molecules that are known to maintain cellular homeostasis by neutralizing excess ROS levels[3]. We have previously reported that OS stimulated the expression of catalase and SOD1 in human GCs[24], Considering recent studies that manipulation of ROS levels through ROS scavenging systems might control follicular development and/or survival, and Nrf2 expression showed simultaneous increase in the present study, we proposed the clue that Nrf2 can be a potent survival factor of follicular maintenance. 8-OHdG is one of the most commonly formed DNA lesions produced in response to OS, and is considered as a cellular marker for both OS and oxidative DNA damage [25]. OGG1 is the main enzyme involved in the removal of 8-OHdG from DNA[26], and thus suggested to be involved in protection against DNA damage[27, 28]. It is known that the human OGG1 promoter contains a Nrf2 binding site and Nrf2 leads to OGG1 transcription [29], Notably, Nrf2 is primarily regarded as a transcription factor, but catalase and SOD1 were reported mainly in peroxisomes [30] and the cytosol [31], respectively. It is also known that the human SOD1 promoter contains a Nrf2 binding site, and we also identified that steroidogenic acute regulatory protein (StAR), a key factor that positively regulates luteinization, was increased after upregulation of Nrf2 (data not shown). In view of our findings, we would like to state that Nrf2 might primarily function as a transcription factor in ovarian follicle and the activation of Nrf2/Keap1 pathway results in the generation of antioxidants and cytoprotection. Actually, we first doubt that the expression level of Nrf2 might have effects on ovarian follicle steroidogenesis because number of reports suggest that the regulation of oxidative stress is correlated with luteal function, but mRNA expression of folliculogenesis-associated molecules including aromatase (P450arom), 17β-hydroxysteroid dehydrogenase 1 (17β-HSD1) and luteinization-associated molecules, 3β-hydroxysteroid dehydrogenase 1 (3β-HSD1), and the cholesterol side-chain cleavage enzyme (P450scc) showed no significant changes in both upregulation and downregulation of Nrf2 level, and further investigation will be required regarding Nrf2/Keap1 pathway and function of folliculogenesis and luteinization. Since the discovery of Nrf2/Keap1 pathway, many studies have focused on the ‘good’ factors of the pathway in protecting us from OS related diseases[32]. However, the relation between Nrf2/Keap1 pathway and enhanced resistance of various cancer cells to chemotherapy including ovarian cancer was discovered[33], and the ‘dark’ side of the pathway drew high attention[34-38]. The exact mechanism of this pathway is not fully solved yet[16], but the mutation of Keap1 in cancer cells results in persistent induction of Nrf2[36, 37], and provides growth advantages to the cancer cells. This negates the concern in using Nrf2 activators in prevention of OS in normal cells, since the induction of the Nrf2-dependent response is transient because the negative regulator Keap1 is only inhibited temporarily[36, 37], and recent studies support that the activation of Nrf2/Keap1 pathway by Nrf2 activators in normal cells acts to prevent OS, and shows cytoprotective functions[39]. Fumaric acid esters (FAE) have been used since 1959 as treatment for psoriasis[40], a chronic
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inflammatory skin condition that can markedly reduce life quality[41]. Tecfidera (BG12) is an oral formulation of the FAE, containing the active DMF, which was recently FDA approved for first-line monotherapy in early stage of MS[20]. Although the precise mechanism of DMF in MS in unknown, DMF is thought to exert neuroprotective effects by activating the Nrf2/Keap1 pathway[42], and reducing ROS production[21]. The therapeutic potential of DMF is investigated in OS and inflammatory related diseases, like Alzheimer’s disease, Parkinson’s disease, chronic pulmonary disease, asthma, vascular calcification, diabetes, and rheumatoid arthritis[22, 43-49]. Considering our results that activation of Nrf2 by DMF elevates levels of antioxidants, DMF apparently reduced OS in human GCs. A review on the effects and safety of oral FAE for psoriasis show that FAE are superior to placebo in treatment of psoriasis, and no serious adverse effects were seen[41], so it is concluded that FAE has been safely used to treat psoriasis safely for over 50 years. MS is on the rise among young woman[50], and the number of patients taking Tecfidera/BG12 for treatment is increasing, the most prescribed oral treatment for MS now. Among the 13 approved disease-modifying therapies for MS, none are recommended for use while pregnant or breastfeeding[51], and the safety of Tecfidera (BG12) is a major concern among MS patients and physicians. The European Medicines Agency (EMA) and the FDA both state that prepregnancy washout of Tecfidera/BG12 is not necessary, and prescription of Tecfidera/BG12 will possibly increase to young woman of reproductive age. By studying the ovarian functions in these patients, the possibility of taking oral DMF to prevent aging of ovaries may not be a fantasy. If the safety of DMF is demonstrated using animal models, we might be able to propose the possibility that the ovarian aging could be postponed. As a result, we hope to obtain better pregnancy outcomes in aged woman, which will result in fewer patients undergoing infertility treatment due to advanced age.
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Acknowledgement We sincerely thank the patients and Kiba Park Hospital for providing us with ovarian follicular fluids.
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Figure 1 Expression of Nrf2 in human GCs. Immunohistochemical detection of Nrf2 proteins in the human ovary. Representative data from 12 specimens are shown. (A) primary follicle; (B) secondary follicle; (C) antral follicle; (D) corpus luteum; (E) corpora albicans; (F) negative control. The cytoplasm of the GCs and oocytes were positively stained with anti-Nrf2 antibodies at various stages of follicular development. Intense immunostaining was detected in the active corpus luteum (D; check mark ✔). In contrast, no immunostaining was observed in the corpora albicans (E; star ★). The negative control was incubated without anti-Nrf2 antibody, and no positive signal was observed. Bars indicate 20 µm (A, F), 50 µm (B), 200 µm (C), 500 µm (D, E) in a high-power field.
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Figure 2 Expression of Keap1 in human GCs. Immunohistochemical detection of Keap1 proteins in the human ovary. Representative data from 12 specimens are shown. (A) primary follicle; (B) secondary follicle; (C) antral follicle; (D) corpus luteum; (E) corpora albicans; (F) negative control. The cytoplasm of the GCs and oocytes were positively stained with anti-Keap1 antibodies at various stages of follicular development. Intense immunostaining was detected in the active corpus luteum (D; check mark ✔). In contrast, no immunostaining was observed in the corpora albicans (E; star ★). The negative control was incubated without anti-Keap1 antibody, and no positive signal was observed. Bars indicate 20 µm (A, F), 50 µm (B), 200 µm (C), 500 µm (D, E) in a high-power field.
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Figure 3 Expression of Nrf2 and antioxidants in human luteinized GCs treated with H2O2. (A) Quantitative real-time RT-PCR analysis of Nrf2, catalase, SOD1, and OGG1 mRNA expression was performed after exposure to various concentrations (100, 200, and 400 µM) of H2O2 for 48 hours in human GCs from 43 patients. The mRNA expression of Nrf2, catalase, SOD1, and OGG1 was normalized to RNA loading for each sample using GAPDH mRNA as an internal standard. The results are shown as the mean ± SD (bars) of 4 independent experiments. * p < 0.05. (B) Western blot analysis showing the presence of Nrf2 and the effect of H2O2 exposure in human GCs from 18 patients. H2O2 exposure for 48 hours resulted in increased protein expression of Nrf2, catalase, SOD1, and OGG1. Conversely, H2O2 exposure resulted in decreased protein expression of Keap1. Western blot of β-actin serves as an internal control and representative images of Western blots are shown.
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Figure 4 Effect of Nrf2 expression on antioxidants in human GCs. (A) Effect of Nrf2 expression on the mRNA levels of catalase, SOD1, and OGG1 was investigated by quantitative real-time RT-PCR. Endogenous Nrf2 mRNA was depleted using 3 different types of siRNA (#1~3) for Nrf2 in human GCs from 36 patients, and the cells were collected 48 hours after knockdown. The mRNA level of the control siRNA group was arbitrarily set at 1.0, and that of the knockdown group was estimated relative to the control value. The results are shown as the mean ± SD (bars) of 6 independent experiments. * p < 0.05 vs. control. (B) Western blot analysis showing the effect of Nrf2 depletion and generation of antioxidants in 14
ACCEPTED MANUSCRIPT human GCs from 14 patients. Endogenous Nrf2 mRNA was depleted using siRNA for Nrf2 in human GCs, and the cells were collected 48 hours after knockdown. Knockdown of Nrf2 in GCs resulted in decreased protein expression of catalase, SOD1, and OGG1. Conversely, knockdown of Nrf2 resulted in increased protein expression of Keap1. Western blot of β-actin serves as an internal control and representative images of Western blots are shown.
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Figure 5 Expression of Nrf2 and antioxidants in human GCs treated with DMF. (A) Quantitative real-time RT-PCR analysis of Nrf2, catalase, SOD1, and OGG1 gene expression was performed after exposure to various concentrations of DMF for 48 hours in human GCs from 47 patients. The mRNA expression of Nrf2, catalase, SOD1, and OGG1 was normalized to RNA loading for each sample using GAPDH mRNA as an internal standard. Data were independently collected from four samples. The results are shown as the mean ± SD (bars) of 4 independent experiments. * p < 0.05. (B) Western blot analysis showing the presence of Nrf2 and the effect of DMF exposure in human GCs from 19 patients. DMF exposure for 48 hours resulted in increased protein expression of Nrf2, catalase, SOD1, and OGG1. Conversely, DMF exposure resulted in decreased protein expression of Keap1. Western blot of β-actin serves as an internal control and representative images of Western blots are shown.
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Figure 6 Nrf2 elevation reduces 8-OHdG in human GCs. The expression of 8-OHdG (red) and Nrf2 (green) was investigated by immunofluorescence analysis in human GCs from 12 patients. Representative images from 3 independent experiments are shown (A-D); cells treated with vehicle, (E-H); cells treated with 100 µM of DMF, (I-L); cells treated with 400 µM of H2O2 (positive control). DMF treatment results in an increased Nrf2 fluorescence expression (F) signal compared to the control (B), and decreased 8-OHdG fluorescence expression (E) signal compared to the control (A). Bars indicate 50 µm.
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Figure 7 Nrf2 is responsible for maintaining intracellular ROS levels. Intracellular ROS levels in human GCs from 15 patients were measured by CM-H2DCFDA fluorescence. Representative images are shown (A-C); cells treated with vehicle, (D-F); cells treated with 100 µM of DMF, (G-I); cells treated with 400 µM of H2O2 (positive control), (J~L); cells treated with combination of 400 µM of H2O2 and 100 µM of DMF. DMF treatment (D) results in a significantly decreased CM-H2DCFDA fluorescence signal compared to the control (A), and H2O2 treatment (H) significantly increased CM-H2DCFDA fluorescence signal. Combination of H2O2 and DMF treatment (J) decreased the CM-H2DCFDA fluorescence signal compared to only H2O2 treatment. Bars indicate 100 µm. (M) Quantitative data of fluorescence intensity were obtained and analyzed using Carl-Zeiss LSM700 ZEN software. Values were standardized by dividing each value by the average value of the control group in each experiment. The results are shown as the mean ± SD (bars) of three independent experiments. * p < 0.05.
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Highlights Activation of Nrf2 leads to alleviate oxidative stress in human granulosa cells. Activation of Nrf2 mainly results in the generation of antioxidants and
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cytoprotective factors.
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Dimethylfumarates quenched intracellular reactive oxygen species generation.
S0303-7207(17)30522-1
DOI:
10.1016/j.mce.2017.10.002
Reference:
MCE 10097
To appear in:
Molecular and Cellular Endocrinology
Received Date: 17 April 2017 Revised Date:
8 September 2017
Accepted Date: 2 October 2017
Please cite this article as: Akino, N., Wada-Hiraike, O., Terao, H., Honjoh, H., Isono, W., Fu, H., Hirano, M., Miyamoto, Y., Tanikawa, M., Harada, M., Hirata, T., Hirota, Y., Koga, K., Oda, K., Kawana, K., Fujii, T., Osuga, Y., Activation of Nrf2 might reduce oxidative stress in human granulosa cells, Molecular and Cellular Endocrinology (2017), doi: 10.1016/j.mce.2017.10.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Author names 1 Nana Akino, 1, *Osamu Wada-Hiraike, 1Hiromi Terao, 1Harunori Honjoh, 1, 2Wataru Isono,1Houju Fu, 1Mana Hirano, 1Yuichiro Miyamoto, 1Michihiro Tanikawa, 1Miyuki Harada, 1 Tetsuya Hirata, 1Yasushi Hirota, 1Kaori Koga, 1Katsutoshi Oda, 1, ¶Kei Kawana, 1Tomoyuki Fujii, and 1Yutaka Osuga
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Affiliations 1 Department of Obstetrics and Gynecology, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 8655 Japan. 2 Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 8655 Japan. current address: Nihon University School of Medicine Department of Obsterics & Gynecology, 30-1 Ohyaguchi Kamicho, Itabashi-ku, Tokyo 173 8610 Japan
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Corresponding author Osamu Wada-Hiraike M.D.,Ph.D. E-mail address: [email protected] Department of Obstetrics and Gynecology, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 8655 Japan Telephone: +0081 3 3815 5411 Fax: +0081 3 3816 2017 Grants or fellowships supporting the writing of the paper This work was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan Agency for Medical Research and Development, Ministry of Health, Labour and Welfare, and The Japanese Foundation for Research and Promotion of Endoscopy Grant.
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Full title Activation of Nrf2 might reduce oxidative stress in human granulosa cells
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Disclosure summary The authors declare no possible conflicts of interest to disclose
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Abstract 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. Keywords Nrf2/Keap1; oxidative stress; granulosa cells; Dimethylfumarates; IVF-ET; infertility treatment
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Abbreviations oxidative stress (OS), reactive oxygen species (ROS), in vitro fertilization and embryo transfer (IVF-ET), granulosa cells (GCs), nuclear factor-E2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), antioxidant response element (ARE), superoxide dismutase (SOD), 8-Oxoguanine DNA glycosylase (OGG1), Dimethylfumarate (DMF), multiple sclerosis (MS), United States Food and Drug Administration (FDA), phosphate-buffered saline (PBS), fetal bovine serum (FBS), dimethylsulfoxide (DMSO), small interfering RNA (siRNA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), carboxymethyl-H2-dichlorofluorescein diacetate (CM-H2DCFDA), 8 Hydroxyguanosine (8-OHdG), 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI), steroidogenic acute regulatory protein (StAR), aromatase (P450arom), 17β-hydroxysteroid dehydrogenase 1 (17β-HSD1), 3β-hydroxysteroid dehydrogenase 1 (3β-HSD1), cholesterol side-chain cleavage enzyme (P450scc), fumaric acid ester (FAE), European Medicines Agency (EMA)
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Introduction It is widely known that oxidative stress (OS) is an inevitable threat to the human body[1]. OS is a state characterized by an imbalance between reactive oxygen species (ROS) and antioxidant scavenger enzymes[2]. The production of ROS and antioxidants is balanced in a healthy body normally, but once imbalance occurs, the cells are threatened by damaging of lipids, proteins, and nucleic acids[3]. This is known to be closely associated with aging and age-related diseases, such as inflammatory diseases, cancer and reproduction[4, 5]. It is documented that excess OS plays a key role in the pathogenesis of subfertility in both males and females[6]. The changes of lifestyles in developed countries are leading to more women tending to delay childbearing, and the number of couples undergoing infertility treatment is increasing by the year[7]. The concentration of ROS-associated molecules in the follicular fluid, which is known to increase by aging[8-10], is considered to affect the oocyte maturation, embryo quality and outcome of in vitro fertilization and embryo transfer (IVF-ET)[11]. Furthermore, OS has been shown to induce oocyte degeneration and apoptosis through disturbing the meiotic spindle. Therefore, one can imagine that exposure of oocytes to excess level of ROS might result in unfavorable IVF-ET outcome. Granulosa cells (GCs) are cells surrounding the oocyte and responsible for normal folliculogenesis[12]. Although adequate amount of ROS is known to be necessary for ovulation[13], excess generation of ROS in the human GCs of women with polycystic ovarian syndrome adversely affected IVF success rates[12]. From these facts, management of OS may lead to better infertility treatment outcomes. Here we focused on nuclear factor-E2-related factor 2 (Nrf2) / Kelch-like ECH-associated protein 1 (Keap1)-antioxidant response element (ARE) signaling pathway[14, 15], which is one of the most important defense mechanisms against oxidative stress[16]. During normoxia, Nrf2, a transcription factor with a high sensitivity to oxidative stress, is held in the cytoplasm and maintained at low levels by an inhibitory protein; Keap1[17]. Oxidative stress causes Nrf2 to dissociate from Keap1 and to subsequently translocate into the nucleus, which results in its binding to specific DNA sequence ARE and the transcription of downstream target genes[14], and antioxidants including superoxide dismutase (SOD), catalase, and 8-Oxoguanine DNA glycosylase (OGG1) are elevated[18]. The appropriate functioning of the Nrf2/Keap1 pathway is indispensable to counteracting OS and protecting multiple organs and cells[14]. Dimethylfumarate (DMF) is an activator of Nrf2, and has been approved by the United States Food and Drug Administration (FDA) as a new first-line oral drug to treat relapsing forms of multiple sclerosis (MS)[14, 19]. The exact mechanism of DMF of action in MS is not fully understood [20], but studies show that DMF reduces ROS production, and antioxidants were elevated in neurons[21]. The therapeutic potential of DMF is investigated in OS and inflammatory related diseases, such as Alzheimer’s disease, Parkinson’s disease, chronic pulmonary disease, asthma, diabetes, and rheumatoid arthritis[22]. Therefore, we hypothesized that activation of Nrf2 by DMF may lead to alleviated OS in human GCs, and this will lead to better outcomes in infertility treatment.
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Tissue samples and immunohistochemistry The ovarian tissues used in this study were obtained from 12 female patients with regular menstrual cycles who took no hormonal drugs and underwent hysterectomy for uterine cervical cancer or endometrial cancer. These ovarian tissues were first identified by two pathologists as pathologically normal tissues. The female patients were 32~38 years old at the time of operation, and the operations were mostly performed during the proliferative phase of the menstrual cycle. The study was approved by the Institutional Review Board of the University of Tokyo Hospital, and written informed consent was obtained in each instance. Immunohistochemistry was performed by standard procedures as described previously (19). Paraffin sections (4 µm) were dewaxed in xylene and rehydrated through graded ethanol to water. Antigens were retrieved by boiling in 10 mM citrate buffer (pH 6.0) for 10 min in a microwave. The cooled sections were incubated in DAKO REAL peroxidase-blocking solution (DAKO, Carpinteria, CA) for 30 min to quench the endogenous peroxidase. To block nonspecific binding, sections were incubated in phosphate-buffered saline (PBS) containing 3% BSA and 0.5% Nonidet P-40 for 10 min at room temperature. Sections were then incubated with the anti-Nrf2 rabbit polyclonal antibody (16396-1-AP, Proteintech Group, Illinois, USA) or the anti-Keap1 rabbit polyclonal antibody (ab66620, Abcam Ltd., Cambridge, UK) in DAKO REAL antibody diluent (DAKO; 1:100) overnight at 4°C. Negative controls were incubated with PBS instead of the antibody. After washing with PBS, the sections were covered with DAB solution (DAKO) at room temperature, followed by Mayer’s hematoxylin (Wako Pure Chemical, Tokyo, Japan). Lastly, the sections were dehydrated through an ethanol series and xylene and mounted.
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Isolation and culture of primary human GCs Human luteinized GCs in follicular aspirates of preovulatory follicles were obtained from 171 patients (aged 26~46 years old) who underwent transvaginal oocyte retrieval for in vitro fertilization at the University of Tokyo Hospital and Kiba Park Clinic. This study was approved by the Institutional Review Board of the University of Tokyo, and written informed consent for the research use of human GCs was obtained from each patient. The method used to purify primary GCs was as described previously [23]. Briefly, follicular fluids were centrifuged at 1500 rpm for 10 min, resuspended in PBS with 0.2% hyaluronidase, and incubated at 37 °C for 30 min. The suspension was layered over Ficoll-Paque (GE Healthcare, Buckinghamshire, UK) and centrifuged at 700 g for 30 min. GCs were collected from the interphase and washed with PBS. GCs were cultured with DMEM/ F12 (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS; BioWest, Nuaille, France) and antibiotics (100 U/mL penicillin, 0.1 mg/mL streptomycin, and 250 ng/mL amphotericin B; Sigma Aldrich, Darmstadt, Germany) in 48-well (for quantitative real-time PCR) or 12-well plates (for Western blotting) at a density of 4 × 105 cells/mL and allowed to adhere at 37 °C in a humidified atmosphere containing 5% CO2. The GCs were ready to use 24 hours after attaching to the dishes, and the medium was freshly changed at this point to remove leukocytes from the GCs.
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Treatment of GCs To evaluate the effect of ROS on Nrf2 expression, varying concentrations of H2O2 (200, 400, 600 4
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Knockdown of Nrf2 using small interfering RNA (siRNA) Chemically synthesized 25-nucleotide stealth Nrf2 RNAi duplex oligonucleotides (set of 3) (GenBank accession no. BC 011558.1) were obtained from Invitrogen. Stealth RNAi negative control (Invitrogen) was used as a control small interfering RNA (siRNA). The seeded GCs were transfected with 40 nM of siRNA for 48 hours in Opti-MEM (Invitrogen) using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s protocol. After incubation with Nrf2 siRNA, the GCs were covered with serum-free medium, and were subjected to quantitative real-time PCR or Western Blotting.
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Reverse transcription and real-time comparative quantitative PCR Synthesis of cDNA templates from human GCs was performed using the SuperPrep Cell Lysis & RT Kit for qPCR (TOYOBO, Tokyo, Japan). To quantitate mRNA levels, real-time PCR was performed on a Light Cycler (Roche, Diagnostic GmBH, Mannheim, Germany), according to the manufacturer’s instructions. All samples of GCs were analyzed in triplicate or quadruplicate. Human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal standard for RNA loading. To obtain the relative gene expression data (fold change), the comparative 2-∆∆Ct method was used. The primer sets used are described in supplemental data 1.
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Western blotting GCs were lysed in lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing phosphatase inhibitors (Nacalai Tesque, Kyoto, Japan) and protease inhibitors (Roche), and insoluble material was removed by centrifugation at 14000 m/sec, for 12 minutes at 4°C. The supernatants were recovered, and the protein concentrations were measured using Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA). Equivalent amounts of lysate protein (10 µg) were subjected to Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad) and electrophoretically transferred onto Trans-Blot Turbo Transfer Packs (Bio-Rad) using Trans-Blot Turbo Transfer System (Bio-Rad). After blocking with 10% fat-free powdered milk in PBS at room temperature for 1 hour, the membranes were blotted overnight at 4°C with primary antibodies, including anti-Nrf2 (1:100; 16396-1-AP, Proteintech Group), anti-Keap1 (1:1000; ab66620, Abcam Ltd.), anti-OGG1 (1:1000; ab135940, Abcam Ltd.), anti-catalase (1:200; ab16731, Abcam Ltd.) and anti- SOD1 (1:1000; ab13499, Abcam Ltd.). Then, the blots were incubated with the appropriate secondary antibodies (anti-rabbit IgG, 7074S, 1:3000; anti-mouse IgG, 7076S; 1:3000, Cell Signaling) at room temperature for 1 hour and developed using ECL Plus Western blotting detection reagents (GE Healthcare). The images were scanned by the luminescent image analyzer Image Quant LAS 4000 mini (GE Healthcare). The expression of target proteins was internally normalized to the optical density of β-actin (1:2000; A2228, Sigma Aldrich) by the Image J software (http://rsb.info.nih.gov/ij/).
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Fluorescence microscopy GCs were isolated as described above and cultured on 4-well glass Millicell EZ slides (Merck Millipore, Darmstadt, Germany) at a density of 2 × 105 cells/mL. The cells were fixed with PBS containing 4% paraformaldehyde. After blocking, the cells were sequentially incubated with anti-Nrf2 (1:100; 16396-1-AP, Proteintech Group) and anti-8 Hydroxyguanosine (8-OHdG; 1:100; 1b48508, Abcam Ltd.) antibodies. Secondary antibodies were Alexa fluor 488-conjugated donkey anti-rabbit IgG (1:100; A-21206, Invitrogen) and Alexa fluor 558-conjugated goat anti-mouse IgG (1:100; A-11004, Invitrogen). The slides were briefly counterstained with 300nM of 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI; D1306, Thermofisher) and analyzed under a confocal fluorescence microscope (Carl-Zeiss LSM700 ZEN, Jena, Germany).
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Detection of intracellular ROS The intracellular levels of ROS were measured by loading the GCs with the fluoroprobe carboxymethyl-H2-dichlorofluorescein diacetate (CM-H2DCFDA; Invitrogen). Briefly, the CM-H2DCFDA probe was reconstituted in DMSO prior to loading. The GCs were incubated with 10 µM CM-H2DCFDA in PBS for 20 min at 37°C, counterstained with 10 µM of Hoechst 33342 (Thermofisher, Massachusetts, USA) in PBS for 5 min to visualize all cells, and then immediately observed under a confocal fluorescence microscope (Carl-Zeiss), with an excitation wavelength of 495 nm and an emission wavelength of 527 nm. The fluorescence intensity was analyzed and quantified using LSM700 ZEN. Quantitative data of fluorescence intensity were standardized by dividing each value by the average value of the control group in each experiment. The results are representative of three independent cultures.
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Statistical analysis Data were analyzed by Statistical analyses were performed using the JMP Pro 11 software (SAS institute Inc., Cary, NC, USA). All results are shown as means ± SD. For comparison of multiple values, ANOVA was used followed by Dunnett's test, with a significance of p values less than 0.05.
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ACCEPTED MANUSCRIPT Results Expression of Nrf2 and Keap1 in human ovaries Firstly, we aimed to show the presence of Nrf2 and Keap1 protein in ovarian tissue by immunohistochemistry. The Nrf2 and Keap1 proteins were predominantly detected in the cytoplasm of GCs at various stages of follicles (Fig. 1, 2 A-F).
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OS stimulates the expression of Nrf2, Catalase, SOD1 and OGG1 Nrf2 plays a role in salvaging cells from ROS. To explore the function of Nrf2 in GCs, cultured GCs were exposed to various concentrations of H2O2 (200, 400, 600 µM) for 48 hours to investigate how OS affects the expression of Nrf2 and antioxidant products, including catalase, SOD1, and OGG1. We could not observe apparent apoptosis in this condition, and elevation of mRNA levels of Nrf2, catalase, SOD1, and OGG1 with OS were determined by quantitative real-time RT-PCR. OS significantly elevated the mRNA levels of Nrf2, catalase, SOD1 and OGG1 after exposure to 600 µM of H2O2, and 400 µM of H2O2 to SOD1 and OGG1. Simultaneously, elevated OS resulted in the increased expression of Nrf2, catalase, SOD1, and OGG1 proteins in Western blotting analysis. Conversely, the protein level of Keap1 was decreased with OS exposure (Fig. 3).
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Knockdown of endogenous Nrf2 reduced antioxidants in GCs Next, we aimed to determine the depletion of Nrf2 and the generation of antioxidant product levels in GCs. We first confirmed whether decreased expression of Nrf2 could affect the cell viability, and this was judged by MTS assays and Trypan blue dye exclusion test. However, we were unable to find significant effect of Nrf2 depletion both on cell viability and cell number (data not shown). We found that siRNA-mediated depletion of endogenous Nrf2 resulted in significant decrease of mRNA levels of antioxidants, such as catalase, SOD1, and OGG1 in all three Nrf2 siRNA (#1~3). Protein levels of catalase, SOD1 and OGG1 were also decreased with siRNA-mediated depletion of endogenous Nrf2 (Fig. 4). It is well known that ROS levels affect the luteal function and we have shown that the level of NRF2 could affect the steroidogenesis function of granulosa cells[24], but we were unable to reach the conclusive data after downregulation of endogenous Nrf2.
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DMF elevates the expression of Nrf2, catalase, SOD1 and OGG1 DMF is known to activate the Nrf2/Keap1 pathway in cells, resulting in elevation of antioxidants, such as catalase, SOD1 and OGG1. Therefore GCs were treated with DMF (50 or 100 µM) for 48 hours and the mRNA levels and protein levels of Nrf2, catalase, SOD1 and OGG1 were investigated. We found that 100 µM of DMF exposure to GCs resulted in significant elevation of the mRNA levels of Nrf2, catalase, SOD1 and OGG1. Simultaneously, DMF treatment resulted in the increased expression of Nrf2, catalase, SOD1, and OGG1 proteins in Western blotting analysis. The protein levels of Keap1 was conversely decreased by DMF treatment (Fig. 5). Activation of Nrf2 reduces 8-OHdG generation in GCs It is known that accumulation of oxidative stress to cells result in elevation of 8-OHdG, which is one of the major products of DNA oxidation. GCs were treated with either DMF (Fig. 6 E~H) or 7
ACCEPTED MANUSCRIPT H2O2 (Fig. 6 I~L) and stained with anti-Nrf2 and anti-8-OHdG antibodies, and the fluorescence was detected. GCs treated with DMF showed higher levels of Nrf2 and lower levels of 8-OHdG compared to control GCs. H2O2 treated GCs showed elevated levels of 8-OHdG compared to control GCs. This shows that activation of Nrf2 through DMF appears to decrease oxidative stress in GCs.
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DMF reduces intracelluar ROS generation in GCs Endogenous levels of intracellular ROS were measured by CM-H2DCFDA fluorescence, and we found that DMF treatment of GCs reduced ROS generation in the GCs. Conversely, H2O2 treatment of GCs resulted in enhanced levels of ROS generation, and the DCF staining is clearly shown in nucleus (Fig. 7, A~I). Addition of DMF to GCs treated with H2O2 reduced the ROS levels and DCF fluorescence was increased both in nucleus and cytosol (Fig. 7, J~L), showing that DMF treatment may alleviate stress in severe oxidative environments. The fluorescence intensity was subsequently analyzed and quantified, and the fluorescence intensity of GCs treated with DMF was approximately reduced to 1/3-fold than that of the control GCs (Fig. 7, M).
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Discussion Studies on ROS and antioxidant levels in human GCs are limited, and the relation of Nrf2/Keap1 pathway in GCs is not documented, to our knowledge. The results of immunohistochemistry showed presence of Nrf2 and Keap1 protein in the cytoplasm of GCs at various stages of follicles, suggesting the association of the Nrf2/Keap1 theory in human GCs. It is widely known that the production of antioxidants increases under the condition of OS, and catalase and SOD1 are representative biological molecules that are known to maintain cellular homeostasis by neutralizing excess ROS levels[3]. We have previously reported that OS stimulated the expression of catalase and SOD1 in human GCs[24], Considering recent studies that manipulation of ROS levels through ROS scavenging systems might control follicular development and/or survival, and Nrf2 expression showed simultaneous increase in the present study, we proposed the clue that Nrf2 can be a potent survival factor of follicular maintenance. 8-OHdG is one of the most commonly formed DNA lesions produced in response to OS, and is considered as a cellular marker for both OS and oxidative DNA damage [25]. OGG1 is the main enzyme involved in the removal of 8-OHdG from DNA[26], and thus suggested to be involved in protection against DNA damage[27, 28]. It is known that the human OGG1 promoter contains a Nrf2 binding site and Nrf2 leads to OGG1 transcription [29], Notably, Nrf2 is primarily regarded as a transcription factor, but catalase and SOD1 were reported mainly in peroxisomes [30] and the cytosol [31], respectively. It is also known that the human SOD1 promoter contains a Nrf2 binding site, and we also identified that steroidogenic acute regulatory protein (StAR), a key factor that positively regulates luteinization, was increased after upregulation of Nrf2 (data not shown). In view of our findings, we would like to state that Nrf2 might primarily function as a transcription factor in ovarian follicle and the activation of Nrf2/Keap1 pathway results in the generation of antioxidants and cytoprotection. Actually, we first doubt that the expression level of Nrf2 might have effects on ovarian follicle steroidogenesis because number of reports suggest that the regulation of oxidative stress is correlated with luteal function, but mRNA expression of folliculogenesis-associated molecules including aromatase (P450arom), 17β-hydroxysteroid dehydrogenase 1 (17β-HSD1) and luteinization-associated molecules, 3β-hydroxysteroid dehydrogenase 1 (3β-HSD1), and the cholesterol side-chain cleavage enzyme (P450scc) showed no significant changes in both upregulation and downregulation of Nrf2 level, and further investigation will be required regarding Nrf2/Keap1 pathway and function of folliculogenesis and luteinization. Since the discovery of Nrf2/Keap1 pathway, many studies have focused on the ‘good’ factors of the pathway in protecting us from OS related diseases[32]. However, the relation between Nrf2/Keap1 pathway and enhanced resistance of various cancer cells to chemotherapy including ovarian cancer was discovered[33], and the ‘dark’ side of the pathway drew high attention[34-38]. The exact mechanism of this pathway is not fully solved yet[16], but the mutation of Keap1 in cancer cells results in persistent induction of Nrf2[36, 37], and provides growth advantages to the cancer cells. This negates the concern in using Nrf2 activators in prevention of OS in normal cells, since the induction of the Nrf2-dependent response is transient because the negative regulator Keap1 is only inhibited temporarily[36, 37], and recent studies support that the activation of Nrf2/Keap1 pathway by Nrf2 activators in normal cells acts to prevent OS, and shows cytoprotective functions[39]. Fumaric acid esters (FAE) have been used since 1959 as treatment for psoriasis[40], a chronic
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inflammatory skin condition that can markedly reduce life quality[41]. Tecfidera (BG12) is an oral formulation of the FAE, containing the active DMF, which was recently FDA approved for first-line monotherapy in early stage of MS[20]. Although the precise mechanism of DMF in MS in unknown, DMF is thought to exert neuroprotective effects by activating the Nrf2/Keap1 pathway[42], and reducing ROS production[21]. The therapeutic potential of DMF is investigated in OS and inflammatory related diseases, like Alzheimer’s disease, Parkinson’s disease, chronic pulmonary disease, asthma, vascular calcification, diabetes, and rheumatoid arthritis[22, 43-49]. Considering our results that activation of Nrf2 by DMF elevates levels of antioxidants, DMF apparently reduced OS in human GCs. A review on the effects and safety of oral FAE for psoriasis show that FAE are superior to placebo in treatment of psoriasis, and no serious adverse effects were seen[41], so it is concluded that FAE has been safely used to treat psoriasis safely for over 50 years. MS is on the rise among young woman[50], and the number of patients taking Tecfidera/BG12 for treatment is increasing, the most prescribed oral treatment for MS now. Among the 13 approved disease-modifying therapies for MS, none are recommended for use while pregnant or breastfeeding[51], and the safety of Tecfidera (BG12) is a major concern among MS patients and physicians. The European Medicines Agency (EMA) and the FDA both state that prepregnancy washout of Tecfidera/BG12 is not necessary, and prescription of Tecfidera/BG12 will possibly increase to young woman of reproductive age. By studying the ovarian functions in these patients, the possibility of taking oral DMF to prevent aging of ovaries may not be a fantasy. If the safety of DMF is demonstrated using animal models, we might be able to propose the possibility that the ovarian aging could be postponed. As a result, we hope to obtain better pregnancy outcomes in aged woman, which will result in fewer patients undergoing infertility treatment due to advanced age.
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Acknowledgement We sincerely thank the patients and Kiba Park Hospital for providing us with ovarian follicular fluids.
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Figure 1 Expression of Nrf2 in human GCs. Immunohistochemical detection of Nrf2 proteins in the human ovary. Representative data from 12 specimens are shown. (A) primary follicle; (B) secondary follicle; (C) antral follicle; (D) corpus luteum; (E) corpora albicans; (F) negative control. The cytoplasm of the GCs and oocytes were positively stained with anti-Nrf2 antibodies at various stages of follicular development. Intense immunostaining was detected in the active corpus luteum (D; check mark ✔). In contrast, no immunostaining was observed in the corpora albicans (E; star ★). The negative control was incubated without anti-Nrf2 antibody, and no positive signal was observed. Bars indicate 20 µm (A, F), 50 µm (B), 200 µm (C), 500 µm (D, E) in a high-power field.
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Figure 2 Expression of Keap1 in human GCs. Immunohistochemical detection of Keap1 proteins in the human ovary. Representative data from 12 specimens are shown. (A) primary follicle; (B) secondary follicle; (C) antral follicle; (D) corpus luteum; (E) corpora albicans; (F) negative control. The cytoplasm of the GCs and oocytes were positively stained with anti-Keap1 antibodies at various stages of follicular development. Intense immunostaining was detected in the active corpus luteum (D; check mark ✔). In contrast, no immunostaining was observed in the corpora albicans (E; star ★). The negative control was incubated without anti-Keap1 antibody, and no positive signal was observed. Bars indicate 20 µm (A, F), 50 µm (B), 200 µm (C), 500 µm (D, E) in a high-power field.
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Figure 3 Expression of Nrf2 and antioxidants in human luteinized GCs treated with H2O2. (A) Quantitative real-time RT-PCR analysis of Nrf2, catalase, SOD1, and OGG1 mRNA expression was performed after exposure to various concentrations (100, 200, and 400 µM) of H2O2 for 48 hours in human GCs from 43 patients. The mRNA expression of Nrf2, catalase, SOD1, and OGG1 was normalized to RNA loading for each sample using GAPDH mRNA as an internal standard. The results are shown as the mean ± SD (bars) of 4 independent experiments. * p < 0.05. (B) Western blot analysis showing the presence of Nrf2 and the effect of H2O2 exposure in human GCs from 18 patients. H2O2 exposure for 48 hours resulted in increased protein expression of Nrf2, catalase, SOD1, and OGG1. Conversely, H2O2 exposure resulted in decreased protein expression of Keap1. Western blot of β-actin serves as an internal control and representative images of Western blots are shown.
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Figure 4 Effect of Nrf2 expression on antioxidants in human GCs. (A) Effect of Nrf2 expression on the mRNA levels of catalase, SOD1, and OGG1 was investigated by quantitative real-time RT-PCR. Endogenous Nrf2 mRNA was depleted using 3 different types of siRNA (#1~3) for Nrf2 in human GCs from 36 patients, and the cells were collected 48 hours after knockdown. The mRNA level of the control siRNA group was arbitrarily set at 1.0, and that of the knockdown group was estimated relative to the control value. The results are shown as the mean ± SD (bars) of 6 independent experiments. * p < 0.05 vs. control. (B) Western blot analysis showing the effect of Nrf2 depletion and generation of antioxidants in 14
ACCEPTED MANUSCRIPT human GCs from 14 patients. Endogenous Nrf2 mRNA was depleted using siRNA for Nrf2 in human GCs, and the cells were collected 48 hours after knockdown. Knockdown of Nrf2 in GCs resulted in decreased protein expression of catalase, SOD1, and OGG1. Conversely, knockdown of Nrf2 resulted in increased protein expression of Keap1. Western blot of β-actin serves as an internal control and representative images of Western blots are shown.
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Figure 5 Expression of Nrf2 and antioxidants in human GCs treated with DMF. (A) Quantitative real-time RT-PCR analysis of Nrf2, catalase, SOD1, and OGG1 gene expression was performed after exposure to various concentrations of DMF for 48 hours in human GCs from 47 patients. The mRNA expression of Nrf2, catalase, SOD1, and OGG1 was normalized to RNA loading for each sample using GAPDH mRNA as an internal standard. Data were independently collected from four samples. The results are shown as the mean ± SD (bars) of 4 independent experiments. * p < 0.05. (B) Western blot analysis showing the presence of Nrf2 and the effect of DMF exposure in human GCs from 19 patients. DMF exposure for 48 hours resulted in increased protein expression of Nrf2, catalase, SOD1, and OGG1. Conversely, DMF exposure resulted in decreased protein expression of Keap1. Western blot of β-actin serves as an internal control and representative images of Western blots are shown.
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Figure 6 Nrf2 elevation reduces 8-OHdG in human GCs. The expression of 8-OHdG (red) and Nrf2 (green) was investigated by immunofluorescence analysis in human GCs from 12 patients. Representative images from 3 independent experiments are shown (A-D); cells treated with vehicle, (E-H); cells treated with 100 µM of DMF, (I-L); cells treated with 400 µM of H2O2 (positive control). DMF treatment results in an increased Nrf2 fluorescence expression (F) signal compared to the control (B), and decreased 8-OHdG fluorescence expression (E) signal compared to the control (A). Bars indicate 50 µm.
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Figure 7 Nrf2 is responsible for maintaining intracellular ROS levels. Intracellular ROS levels in human GCs from 15 patients were measured by CM-H2DCFDA fluorescence. Representative images are shown (A-C); cells treated with vehicle, (D-F); cells treated with 100 µM of DMF, (G-I); cells treated with 400 µM of H2O2 (positive control), (J~L); cells treated with combination of 400 µM of H2O2 and 100 µM of DMF. DMF treatment (D) results in a significantly decreased CM-H2DCFDA fluorescence signal compared to the control (A), and H2O2 treatment (H) significantly increased CM-H2DCFDA fluorescence signal. Combination of H2O2 and DMF treatment (J) decreased the CM-H2DCFDA fluorescence signal compared to only H2O2 treatment. Bars indicate 100 µm. (M) Quantitative data of fluorescence intensity were obtained and analyzed using Carl-Zeiss LSM700 ZEN software. Values were standardized by dividing each value by the average value of the control group in each experiment. The results are shown as the mean ± SD (bars) of three independent experiments. * p < 0.05.
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Highlights Activation of Nrf2 leads to alleviate oxidative stress in human granulosa cells. Activation of Nrf2 mainly results in the generation of antioxidants and
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cytoprotective factors.
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Dimethylfumarates quenched intracellular reactive oxygen species generation.