Morroniside suppresses hydrogen peroxide-stimulated autophagy and apoptosis in rat ovarian granulosa cells through the PI3K/AKT/mTOR pathway

Previous evidences have indicated that granulosa cells play a critical role in follicular growth. Hydrogen peroxide (H2O2)-induced oxidative stress has been associated with ovarian granulosa cell apoptosis and ovarian function. Recently, a study highlighted the protective role of morroniside against H2O2-induced damage. In this study, we aimed to investigate the effects of morroniside on H2O2-stimulated rat ovarian granulosa cells and its underlying molecular mechanisms. Our results showed that H2O2 treatment suppressed cell survival and increased apoptosis in rat granulosa cells, while treatment with morroniside markedly increased H2O2-induced granulosa cell survival in a dose-dependent manner (0, 10, 50 and 100 µM). Moreover, treatment with 50 µM morroniside impeded H2O2-induced cell apoptosis. An elevation in intracellular ROS, MDA, SOD, GSH-Px, and CAT level was observed in H2O2-induced granulosa cells; however, this effect was abrogated by morroniside treatment. Further studies suggested that administration of morroniside inhibited H2O2-induced granulosa cell apoptosis and caspase-3 activity. In addition, after morroniside treatment of H2O2-stimulated granulosa cells, autophagy-related protein (LC3-II/LC3-I ratio) and beclin-1 expression was decreased and p62 level was increased. Interestingly, we found that morroniside treatment activated the PI3K/AKT/mTOR pathway in H2O2-stimulated granulosa cells. Finally, we showed that treatment with PI3K and mTOR inhibitors reversed the protective effects of morroniside on H2O2-induced granulosa cells. Taken together, our data suggest that treatment with morroniside decreased apoptosis, autophagy, and oxidative stress in rat granulosa cells through the PI3K/AKT/mTOR pathway.

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Saturated FFAs, Palmitic Acid and Stearic Acid, Induce Apoptosis in Human Granulosa Cells

Endocrinology ◽

10.1210/endo.142.8.8293 ◽

2001 ◽

Vol 142 (8) ◽

pp. 3590-3597 ◽

Cited By ~ 115

Author(s):

Yi-Ming Mu ◽

Toshihiko Yanase ◽

Yoshihiro Nishi ◽

Atsushi Tanaka ◽

Masayuki Saito ◽

...

Keyword(s):

Insulin Resistance ◽

Arachidonic Acid ◽

Stearic Acid ◽

Palmitic Acid ◽

Cell Survival ◽

Granulosa Cell ◽

Granulosa Cells ◽

Critical Role ◽

Dependent Manner ◽

Human Granulosa Cells

Abstract Obesity is associated with insulin resistance and some reproductive abnormalities. Circulating FFAs are often elevated in obese subjects and are also closely linked to insulin resistance. In this study, we demonstrated that saturated FFAs, such as palmitic acid and stearic acid, markedly suppressed the granulosa cell survival in a time- and dose-dependent manner. Polyunsaturated FFA, arachidonic acid, had no effect on the cell survival, even at supraphysiological concentrations. The suppressive effect of saturated FFAs on cell survival was caused by apoptosis, as evidenced by DNA ladder formation and annexin V-EGFP/propidium iodide staining of the cells. The apoptotic effects of palmitic acid and stearic acid were unrelated to the increase of ceramide generation or nitric oxide production and were also completely blocked by Triacsin C, an inhibitor of acylcoenzyme A synthetase. In addition, acylcoenzyme A, pamitoylcoenzyme A, and stearylcoenzyme A markedly suppressed granulosa cell survival, whereas arachidonoylcoenzyme A had no such effect, and this finding was consistent with the effect of the respective FFA form. Surprisingly, arachidonic acid instead showed a protective effect on palmitic acid- and stearic acid-induced cell apoptosis. A Western blot analysis showed the apoptosis of the granulosa cells induced by palmitic acid to be accompanied by the down-regulation of an apoptosis inhibitor, Bcl-2, and the up-regulation of an apoptosis effector, Bax. These results indicate that saturated FFAs induce apoptosis in human granulosa cells caused by the metabolism of the respective acylcoenzyme A form, and the actual composition of circulating FFAs may thus play a critical role in the apoptotic events of human granulosa cells. These effects of FFAs on granulosa cell survival may be a possible mechanism for reproductive abnormalities, such as amenorrhea, which is frequently observed in obese women.

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Di-(2-ethylhexyl) phthalate (DEHP) inhibits steroidogenesis and induces mitochondria-ROS mediated apoptosis in rat ovarian granulosa cells

Toxicology Research ◽

10.1039/c8tx00263k ◽

2019 ◽

Vol 8 (3) ◽

pp. 381-394 ◽

Cited By ~ 11

Author(s):

Anima Tripathi ◽

Vivek Pandey ◽

Alakh N. Sahu ◽

Alok Singh ◽

Pawan K. Dubey

Keyword(s):

Oxidative Stress ◽

Granulosa Cell ◽

Granulosa Cells ◽

Ovarian Granulosa Cells ◽

Ethylhexyl Phthalate

Increased oxidative stress (OS) due to ubiquitous exposure to di-(2-ethylhexyl) phthalate (DEHP) can affect the quality of oocytes by inducing apoptosis and hampering granulosa cell mediated steroidogenesis.

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Effect of in vitro copper supplementation on granulosa cell estradiol synthesis and associated genes

Indian Journal of Animal Research ◽

10.18805/ijar.b-3396 ◽

2018 ◽

Author(s):

Ravi, P.S.P. Gupta, S. Nandi, S. Mondal, Kumar Soni ◽

P.S.P. Gupta ◽

S. Nandi ◽

S. Mondal, J.R. Ippala, Avantika Mor, A Mondal ◽

J.R. Ippala ◽

...

Keyword(s):

Cell Survival ◽

Mrna Expression ◽

Granulosa Cell ◽

Granulosa Cells ◽

Culture Medium ◽

Estrus Synchronization ◽

Ovarian Granulosa Cells ◽

Effect Of Copper ◽

Estradiol Synthesis

The study was conducted by supplementing cupric chloride dihydrate to modulate the estradiol synthesis in granulosa cells with a hypothesis of possible use of copper to potentiate or partially replace the hormones for estrus induction / estrus synchronization in future studies. In present study copper at three doses (0.1, 0.5 and 1 mM level in culture medium) were tested to deserve see their effects on in vitro granulosa cell survival, estradiol synthesis and their associated genes of ovarian granulosa cells of goat.There was no effect of copper on the ovarian granulosa cell survival rate. There was a considerable increase in the estradiol level per ml culture medium basis by 6th day of in vitro culture with the second dose of copper i.e. 0.5 mM, but the increase was non-significant (P greator than 0.05). There was no significant effect of copper on estradiol synthesis when expressed on per 30000 cell basis. Effect of copper (0.1 mM and 0.5 mM) on the mRNA expression of genes of aromatase (CYP19A1) and cyclin D2 (CCND2) was estimated. Copper had significantly (P less than 0.05) increased the mRNA expression of CCND2 and CYP19A1in ovarian granulosa cells with only one of the two doses tested i.e. 0.5 mM. Hence, copper can be considered as a potential mineral to supplement along with hormones in estrus induction or estrus synchronization protocols to minimize the use of hormones.

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Downregulating lncRNA NEAT1 induces proliferation and represses apoptosis of ovarian granulosa cells in polycystic ovary syndrome via microRNA-381/IGF1 axis

Journal of Biomedical Science ◽

10.1186/s12929-021-00749-z ◽

2021 ◽

Vol 28 (1) ◽

Author(s):

Jingran Zhen ◽

Jiangli Li ◽

Xia Li ◽

Xue Wang ◽

Yaling Xiao ◽

...

Keyword(s):

Granulosa Cell ◽

Granulosa Cells ◽

Cell Apoptosis ◽

Polycystic Ovary ◽

Pathological Changes ◽

Ovary Syndrome ◽

Ovarian Granulosa Cells ◽

Ovarian Granulosa Cell ◽

Igf1 Expression

Abstract Objective Researchers have revealed the combined functions of long noncoding RNAs (lncRNAs) and microRNA (miRNAs) in polycystic ovary syndrome (PCOS). This study aimed to understand the role of nuclear-enriched abundant transcript 1 (NEAT1) and miR-381 involving insulin-like growth factor 1 (IGF1) in PCOS. Methods PCOS rat model was established by dehydroepiandrosterone induction. NEAT1, miR-381 and IGF1 expression in ovarian granulosa cells of PCOS patients and ovarian tissues of PCOS rats were tested. Bioinformatics website and dual luciferase reporter gene assay were utilized to verify the relationship between NEAT1 and miR-381 and that between miR-381 and IGF1. Levels of sex hormone, pathological changes and ovarian granulosa cell apoptosis in ovarian tissues of PCOS rats were detected. Ovarian granulosa cell proliferation and apoptosis were analyzed in vitro. Results NEAT1 and IGF1 expression increased while miR-381 expression decreased in the ovarian granulosa cells of patients with PCOS and the ovarian tissues of PCOS rats. In in vivo experiments, interference with NEAT1 improved the levels of sex hormones, alleviated pathological changes and suppressed ovarian granulosa cell apoptosis in the ovarian tissues of PCOS rats. In in vitro cell experiments, interference with NEAT1 suppressed apoptosis and enhanced cell proliferation of ovarian granulosa cells. NEAT1 interference-mediated effect would be reversed by up-regulating miR-381. NEAT1 acted as a ceRNA to adsorb miR-381 to target IGF1. Overexpression of IGF1 reversed the inhibitory effect of miR-381 on ovarian granulosa cell apoptosis. Conclusion Interference with NEAT1 increases miR-381 and reduces IGF1 levels, effectively improving the levels of sex hormones and reducing the pathological damage of ovarian tissue in rats with PCOS.

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Oxidized Oils and Oxidized Proteins Induce Apoptosis in Granulosa Cells by Increasing Oxidative Stress in Ovaries of Laying Hens

Oxidative Medicine and Cellular Longevity ◽

10.1155/2020/2685310 ◽

2020 ◽

Vol 2020 ◽

pp. 1-11

Author(s):

Ling Zhou ◽

Xuemei Ding ◽

Jianping Wang ◽

Shiping Bai ◽

Qiufeng Zeng ◽

...

Keyword(s):

Oxidative Stress ◽

Granulosa Cell ◽

Granulosa Cells ◽

Cell Apoptosis ◽

Egg Production ◽

Laying Hens ◽

Corn Oil ◽

Animal Feed ◽

Corn Gluten Meal ◽

Corn Gluten

The storage and preparation of corn for animal feed inevitably lead to lipid and protein peroxidation. Granulosa cells play an important role in follicular development in the ovaries, and hen laying productivity is likely to be dependent on follicle health and number. We hypothesized that oxidized oil and protein induce apoptosis via oxidative stress in laying hen granulosa cells. A sample of 360 38-week-old Lohmann commercial laying hens was used in a 2×2 factorial design for 8 weeks. Dietary treatments included dietary oil (fresh corn oil (FO) or oxidized corn oil (OO)) and corn gluten meal (fresh corn gluten meal (FP) or oxidized corn gluten meal (OP)). Productivity, ovarian histology, granulosa cell apoptosis, and indicators of oxidative stress were evaluated in all groups. Both dietary OO and OP decreased egg production and the average daily feed intake (ADFI) of laying hens. Flow cytometry, TUNEL, and real-time PCR revealed that both dietary OO and OP induced granulosa cell apoptosis in prehierarchical and hierarchical follicles. Furthermore, dietary OO and OP caused oxidative stress in prehierarchical and hierarchical follicles, as indicated by the downregulation of antioxidant-related-gene expression. Moreover, forkhead box O1 (FoxO1), extracellular regulated protein kinase (ERK), and c-Jun NH2 kinase (JNK) are involved in potential apoptosis regulation pathways in the granulosa cells of laying hens fed OO and OP, as indicated by the upregulation of FoxO1 expression and downregulation of ERK/JNK expression. These results indicate that OO and OP induce granulosa cell apoptosis via oxidative stress, and the combined use of OO and OP aggravates the adverse effects of oxidative stress in laying hens.

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Critical Role of FoxO1 in Granulosa Cell Apoptosis Caused by Oxidative Stress and Protective Effects of Grape Seed Procyanidin B2

Oxidative Medicine and Cellular Longevity ◽

10.1155/2016/6147345 ◽

2016 ◽

Vol 2016 ◽

pp. 1-16 ◽

Cited By ~ 13

Author(s):

Jia-Qing Zhang ◽

Bin-Wen Gao ◽

Jing Wang ◽

Qiao-Ling Ren ◽

Jun-Feng Chen ◽

...

Keyword(s):

Oxidative Stress ◽

Granulosa Cell ◽

Protein Level ◽

Cell Apoptosis ◽

Molecular Mechanisms ◽

Critical Role ◽

Grape Seed ◽

Protective Effects ◽

Apoptosis Process ◽

First Time

Reactive oxygen species (ROS) are closely related to the follicular granulosa cell apoptosis. Grape seed procyanidin B2 (GSPB2) has been reported to possess potent antioxidant activity. However, the GSPB2-mediated protective effects and the underlying molecular mechanisms in granulosa cell apoptosis process remain unknown. In this study, we showed for the first time that GSPB2 treatment decreased FoxO1 protein level, improved granulosa cell viability, upregulated LC3-II protein level, and reduced granulosa cell apoptosis rate. Under a condition of oxidative stress, GSPB2 reversed FoxO1 nuclear localization and increased its level in cytoplasm. In addition, FoxO1 knockdown inhibited the protective effects of GSPB2 induced. Our findings suggest that FoxO1 plays a pivotal role in regulating autophagy in granulosa cells, GSPB2 exerts a potent and beneficial role in reducing granulosa cell apoptosis and inducing autophagy process, and targeting FoxO1 could be significant in fighting against oxidative stress-reduced female reproductive system diseases.

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Malathion-Induced Granulosa Cell Apoptosis in Caprine Antral Follicles: An Ultrastructural and Flow Cytometric Analysis

Microscopy and Microanalysis ◽

10.1017/s1431927614013452 ◽

2014 ◽

Vol 20 (6) ◽

pp. 1861-1868 ◽

Cited By ~ 15

Author(s):

Jitender K. Bhardwaj ◽

Priyanka Saraf

Keyword(s):

Oxidative Stress ◽

Lipid Peroxidation ◽

Granulosa Cell ◽

Granulosa Cells ◽

Cell Apoptosis ◽

Ovarian Function ◽

Flow Cytometric Analysis ◽

Thiobarbituric Acid ◽

Antral Follicles ◽

Tbars Assay

AbstractOrganophosphate pesticides (OPs) like malathion interfere with normal ovarian function resulting in an increased incidence of atresia and granulosa cell apoptosis that plays a consequential role in the loss of ovarian follicles or follicular atresia. The aim of present study was to assess malathion-induced (100 nM) reproductive stress, ultrastructural damage and changes in apoptosis frequency in ovarian granulosa cells of antral follicles. Transmission electron microscopy (TEM) was employed for ultrastructural characterization, oxidative stress was evaluated using thiobarbituric acid reactive substances (TBARS) assay to measure lipid peroxidation, and apoptosis was quantified via flow cytometry. By TEM, apoptosis was identified by the presence of an indented nuclear membrane with blebbing, pyknotic crescent-shaped fragmented nuclei, increased vacuolization, degenerating mitochondria, and lipid droplets. The results indicate a significant increase in malondialdehyde (MDA) level (nmols/g wet tissue) at a 100 nM dose of malathion i.e. 7.57±0.033*, 8.53±0.12*, and 12.87±0.78** at 4, 6, or 8 h, respectively, as compared with controls (6.07±0.033, p<0.01*, p<0.05**) showing a positive correlation between malathion-induced lipid peroxidation and percentage of granulosa cell apoptosis (r=1; p<0.01). The parallel use of these three methods enabled us to determine the role of malathion in inducing apoptosis as a consequence of cytogenetic damage and oxidative stress generated in granulosa cells of antral follicles.

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miR-21 is involved in norepinephrine-mediated rat granulosa cell apoptosis by targeting SMAD7

Journal of Molecular Endocrinology ◽

10.1530/jme-16-0248 ◽

2017 ◽

Vol 58 (4) ◽

pp. 199-210 ◽

Cited By ~ 7

Author(s):

Li Zhang ◽

Jie Gao ◽

Sheng Cui

Keyword(s):

Granulosa Cell ◽

Granulosa Cells ◽

Transforming Growth Factor Beta ◽

Cell Apoptosis ◽

Transforming Growth Factor ◽

Sympathetic Innervation ◽

Combined Treatment ◽

Sympathetic Nerves ◽

Dependent Manner ◽

Protein Levels

Substantive evidence has indicated that the sympathetic innervation contributes to the regulation and development of ovarian functions. Norepinephrine (NE) is one of the major neurotransmitters contained in the extrinsic ovarian sympathetic nerves and is thought to be a potent moderator of ovarian functions such as steroidogenesis and granulosa cell proliferation or apoptosis. However, the mechanisms of NE regulation of granulosa cell apoptosis in the rat ovary are rare. Real-time PCR and Western blot results show that NE regulates the expression of miR-21 in primary granulosa cells in a dose-dependent manner. Additionally, we found that miR-21 is involved in NE-mediated rat granulosa cells apoptosis and blocks granulosa cell apoptosis by targeting Smad7, a transforming growth factor-beta-inducible mediator of apoptosis in granulosa cells. In primary granulosa cells, a combined treatment of NE and TGF-β increased apoptosis and decreased miR-21 expression, but increased SMAD7 protein levels. We also demonstrated that NE regulates miR-21 by coupling to α1A-adrenergic receptor (α1A-AR). This is the first demonstration that NE controls the reproductive functions by modulating the expression of miR-21 and promoting TGF-β-induced granulosa cell apoptosis. Such NE-mediated effects could be potentially used for regulating the reproductive processes and for treating reproductive disorders.

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P0975EFFECT OF REGULATED INTRAMEMBRANE PROTEOLYSIS ON MEGALIN EXPRESSION DURING OXIDATIVE STRESS EXPOSURE

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa142.p0975 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Yoshifumi Kurosaki ◽

Futoshi Ikarashi ◽

Akemi Imoto ◽

Fumitaka Kawakami ◽

Masanori Yokoba ◽

...

Keyword(s):

Oxidative Stress ◽

Hydrogen Peroxide ◽

Western Blotting ◽

Critical Role ◽

Mrna Levels ◽

Dependent Manner ◽

Gamma Secretase ◽

Sprague Dawley ◽

Intramembrane Proteolysis ◽

Regulated Intramembrane Proteolysis

Abstract Background and Aims Megalin, an endocytic receptor in proximal tubular cells, plays a critical role in renal tubular protein reabsorption. We previously reported that oxidative stress induced the temporally increase in renal megalin expression through the PI3K/AKT signaling pathway, but that megalin elevation is normalized or decreased during long term exposure to oxidative stress (hydrogen peroxide). However, the underlying mechanisms are unclear. Studies have addressed that megalin is subjected to regulated intramembrane proteolysis (RIP). Intracellular megalin COOH-terminal fragment (MCTF) is produced by protein kinase C-regulated, metalloprotease-mediated ectodomain shedding and further cleavage by gamma-secretase to produce the soluble megalin intracellular domain (MICD). The MICD in turn translocates to the nucleus where it decreases expression of the Lrp2 gene encoding megalin. In the present study, we evaluated the effect of megalin RIP on the oxidative stress-regulated megalin expression. Method HK-2 cells were cultured with hydrogen peroxide (0.4 mmol/l) for 4.5 or 24 h, followed by treatment with gamma-secretase inhibitor, Compound E (5 mmol/L) or PKC activator, Phorbol 12-myristate 13-acetate (PMA, 0.5 mmol/L). Megalin expression was determined by performing western blotting or real-time PCR. The MCTF in medium was detected by western blotting. In animal experiments, Sprague-Dawley rats were randomly divided into two groups (n = 5): (i) STZ group (diabetic phenotype induced by streptozotocin administration) and (ii) sham group (vehicle). Urine was collected at two weeks after STZ administration, and the excretion of MCTF in urine was analyzed. Results Treatment of HK-2 cells with hydrogen peroxide (0.4 mmol/L) significantly increased megalin protein and mRNA levels at 4.5 h. Pretreatment of Compound E showed further increase in megalin expression in hydrogen peroxide-exposed cells. It was also found that presenilin-1 and -2, which are components of gamma-secretase, double knockdown with siRNA increased megalin expression in hydrogen peroxide treated-cells. On the other hand, PMA treatment inhibited the increase in both megalin protein and mRNA levels. In the cells treated with hydrogen peroxide for 24 h, megalin mRNA levels were normalized, but pretreatment of Compound E kept the elevation in megalin mRNA levels at 24 h after the treatment with hydrogen peroxide. Interestingly, megalin MCTF in the medium was increased by hydrogen peroxide treatment in a dose-dependent manner. Furthermore, megalin MCTF excretion in the urine of STZ-induced diabetes was significantly increased compared to sham rats. Conclusion These results suggested that oxidative stress-induced megalin upregulation was inhibited by RIP activation of megalin, suggesting that megalin RIP plays a role as a negative feedback system to oxidative stress-induced megalin upregulation. Furthermore, our data indicate that oxidative stress induces urinary excretions of MCTF in diabetic rats during the normoalbuminuric stage and potentially act as a marker of diabetic kidney disease.

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