Effect of follicle size and atresia grade on mitochondrial membrane potential and steroidogenic acute regulatory protein expression in bovine granulosa cells

Zygote ◽  
2018 ◽  
Vol 26 (6) ◽  
pp. 476-484
Author(s):  
Angela Ostuni ◽  
Maria Pina Faruolo ◽  
Carmen Sileo ◽  
Agata Petillo ◽  
Raffaele Boni
Keyword(s):  
Membrane Potential ◽  
Protein Expression ◽  
Mitochondrial Membrane Potential ◽  
Granulosa Cells ◽  
Mitochondrial Membrane ◽  
Tunel Assay ◽  
Aromatase Activity ◽  
Star Protein ◽  
Follicle Atresia ◽  
Steroidogenic Acute Regulatory

SummaryDuring follicular development, granulosa cells undergo functional and structural changes affecting their steroidogenic activity. Oestrogen synthesis mainly occurs in the endoplasmic reticulum and relies on aromatase activity to convert androgens that arise from theca cells. In the present study, indicators of mitochondria-related steroidogenic capacity, as steroidogenic acute regulatory (StAR) protein expression and mitochondrial membrane potential (MMP), have been evaluated in bovine granulosa cells (GCs) and related to follicle growth and atresia. Atresia was estimated by morphological examination of follicle walls and cumulus–oocyte complexes (COC) and assessed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay for apoptosis detection. Bovine ovarian follicles were macroscopically classified according to their atresia grade and grouped into small, medium or large follicles. After follicle opening, the COCs were morphologically classified for follicle atresia and the GCs were collected. Granulosa cells were fixed for immunofluorescence (IF) and TUNEL assay, frozen for western blotting (WB) or freshly maintained for MMP analyses. StAR protein expression was assessed using both IF and WB analyses. The follicle atresia grade could be efficiently discriminated based on either follicle wall or COC morphological evaluations. Granulosa cells collected from small non-atretic follicles showed a higher (P <0.01) MMP and WB-based StAR protein expression than small atretic follicles. For IF analysis, StAR protein expression in large atretic follicles was higher (P <0.05) than that in large non-atretic follicles. These results suggest a role played by mitochondria in GC steroidogenic activity, which declines in healthy follicles along with their growth. In large follicles, steroidogenic activity increases with atresia and is possibly associated with progesterone production.

scholarly journals Artemisinin attenuated oxidative stress and apoptosis by inhibiting autophagy in MPP+-treated SH-SY5Y cells

2021 ◽  
Vol 28 (1) ◽  
Author(s):  
Junqiang Yan ◽  
Hongxia Ma ◽  
Xiaoyi Lai ◽  
Jiannan Wu ◽  
Anran Liu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Membrane Potential ◽  
Protein Expression ◽  
Mitochondrial Membrane Potential ◽  
Western Blotting ◽  
Cell Apoptosis ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Neuroprotective Effect ◽  
Neuroprotective Effects

Abstract Background Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease. The oxidative stress is an important component of the pathogenesis of PD. Artemisinin (ART) has antioxidant and neuroprotective effects. The purpose of this study is to explore the neuroprotective effect of ART on 1-methyl-4-phenyliodine iodide (MPP +)-treated SH-SY5Y cells and underlying mechanism. Methods We used MPP+-treated SH-SY5Y cells to study the neuroprotective effect of ART. Cell viability was measured by MTT assay after incubating the cells with MPP+ and/or ART for 24 h. DCFH-DA was used to detect the level of intracellular reactive oxygen species (ROS), and WST-8 was used to detect the level of superoxide dismutase (SOD). The level of intracellular reduced glutathione (GSH) was detected with 5,5΄-dithiobis-(2-nitrobenzoic acid), and the level of malondialdehyde (MDA) was assessed based on the reaction of MDA and thiobarbituric acid. A mitochondrial membrane potential detection kit (JC-1) was used to detect changes in the mitochondrial membrane potential (MMP), and an Annexin V-FITC cell apoptosis kit was used to detect cell apoptosis. The expression levels of caspase-3, cleaved caspase-3 and the autophagy-related proteins LC3, beclin-1, and p62 were detected by Western blotting. In addition, to verify the change in autophagy, we used immunofluorescence to detect the expression of LC3 and p62. Results No significant cytotoxicity was observed at ART concentrations up to 40 μM. ART could significantly increase the viability of SH-SY5Y cells treated with MPP+ and reduce oxidative stress damage and apoptosis. In addition, the Western blotting and immunofluorescence results showed that MPP+ treatment could increase the protein expression of beclin1 and LC3II/LC3I and decrease the protein expression of p62, indicating that MPP+ treatment could induce autophagy. Simultaneous treatment with ART and MPP+ could decrease the protein expression of beclin1 and LC3II/LC3I and increase the protein expression of p62, indicating that ART could decrease the level of autophagy induced by MPP+. Conclusion Our results indicate that ART has a protective effect on MPP+-treated SH-SY5Y cells by the antioxidant, antiapoptotic activities and inhibition of autophagy. Our findings may provide new hope for the prevention and treatment of PD.

Encircling granulosa cells protects against di-(2-ethylhexyl)phthalate-induced apoptosis in rat oocytes cultured in vitro

Zygote ◽  
2019 ◽  
Vol 27 (4) ◽  
pp. 203-213 ◽  
Author(s):  
Anima Tripathi ◽  
Vivek Pandey ◽  
A.N. Sahu ◽  
Alok K. Singh ◽  
Pawan K. Dubey
Keyword(s):  
Oxidative Stress ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Granulosa Cells ◽  
Mitochondrial Membrane ◽  
Dependent Manner ◽  
Apoptotic Markers ◽  
Induced Apoptosis ◽  
Ethylhexyl Phthalate

SummaryThe present study investigated if the presence of encircling granulosa cells protected against di(2-ethylhexyl)phthalate (DEHP)-induced oxidative stress in rat oocytes cultured in vitro. Denuded oocytes and cumulus–oocyte complexes (COCs) were treated with or without various doses of DEHP (0.0, 25.0, 50.0, 100, 200, 400 and 800 μM) in vitro. Morphological apoptotic changes, levels of oxidative stress and reactive oxygen species (ROS), mitochondrial membrane potential, and expression levels of apoptotic markers (Bcl2, Bax, cytochrome c) were analyzed. Our results showed that DEHP induced morphological apoptotic changes in a dose-dependent manner in denuded oocytes cultured in vitro. The effective dose of DEHP (400 µg) significantly (P>0.05) increased oxidative stress by elevating ROS levels and the mitochondrial membrane potential with higher mRNA expression and protein levels of apoptotic markers (Bax, cytochrome c). Encircling granulosa cells protected oocytes from DEHP-induced morphological changes, increased oxidative stress and ROS levels, as well as increased expression of apoptotic markers. Taken together our data suggested that encircling granulosa cells protected oocytes against DEHP-induced apoptosis and that the presence of granulosa cells could act positively towards the survival of oocytes under in vitro culture conditions and may be helpful during assisted reproductive technique programmes.

scholarly journals Progesterone Maintains Basal Intracellular Adenosine Triphosphate Levels and Viability of Spontaneously Immortalized Granulosa Cells by Promoting an Interaction between 14-3-3σ and ATP Synthaseβ/Precursor through a Protein Kinase G-Dependent Mechanism

Endocrinology ◽  
2007 ◽  
Vol 148 (5) ◽  
pp. 2037-2044 ◽  
Author(s):  
John J. Peluso ◽  
Xiufang Liu ◽  
Jonathan Romak
Keyword(s):  
Membrane Potential ◽  
Protein Kinase ◽  
Mitochondrial Membrane Potential ◽  
Granulosa Cells ◽  
Mitochondrial Membrane ◽  
Apoptotic Cell ◽  
Protein Kinase G ◽  
Disulfonic Acid ◽  
Causative Role ◽  
Atp Content

The present studies were designed to 1) describe changes in both the mitochondrial membrane potential and ATP content of spontaneously immortalized granulosa cells as they undergo apoptosis, 2) identify some of the downstream events that are activated by progesterone (P4), and 3) relate these downstream events to changes in mitochondrial function and apoptotic cell death. These studies revealed that in response to serum deprivation, the mitochondrial membrane potential initially hyperpolarizes and ATP content increases. That this increase in ATP is required for apoptosis was demonstrated by the finding that oligomycin inhibited the increase in ATP and apoptosis. Piridoxalphosphate-6-azopeyl-2′-4′-disulfonic acid, an inhibitor of purinergic receptors, which are activated by ATP, also inhibited apoptosis due to serum withdrawal. This study provides additional support for ATP’s causative role in apoptosis. Moreover, 8-Br-cGMP, a protein kinase G (PKG) activator, mimicked P4’s action, whereas a PKG antagonist, DT-3, attenuated P4’s suppressive effect on ATP and apoptosis. Finally, DT-3 treatment was shown to attenuate P4-regulated phosphorylation of 14-3-3σ and its binding partner, ATP synthaseβ/precursor and the amount of ATP synthaseβ/precursor that bound to 14-3-3σ. Based on these data, it is proposed that P4 prevents apoptosis in part by activating PKG, which in turn maintains the interaction between ATP synthaseβ/precursor and 14-3-3σ. In the absence of P4-induced PKG activity, we further propose that some ATP synthaseβ precursor dissociates from 14-3-3σ, resulting in its activation and incorporation into the ATP synthase complex, which ultimately results in an increase in ATP and apoptosis.

Effect of thyroid hormone on mitochondrial properties and oxidative stress in cells from patients with mtDNA defects

2009 ◽  
Vol 296 (2) ◽  
pp. C355-C362 ◽  
Author(s):  
Keir J. Menzies ◽  
Brian H. Robinson ◽  
David A. Hood
Keyword(s):  
Oxidative Stress ◽  
Membrane Potential ◽  
Thyroid Hormone ◽  
Protein Expression ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Biogenesis ◽  
Mitochondrial Membrane ◽  
Ros Production ◽  
Mitochondrial Mass ◽  
Mtdna Mutations

Mitochondrial (mt)DNA mutations contribute to various disease states characterized by low ATP production. In contrast, thyroid hormone [3,3′,5-triiodothyronine (T3)] induces mitochondrial biogenesis and enhances ATP generation within cells. To evaluate the role of T3-mediated mitochondrial biogenesis in patients with mtDNA mutations, three fibroblast cell lines with mtDNA mutations were evaluated, including two patients with Leigh's syndrome and one with hypertrophic cardiomyopathy. Compared with control cells, patient fibroblasts displayed similar levels of mitochondrial mass, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), mitochondrial transcription factor A (Tfam), and uncoupling protein 2 (UCP2) protein expression. However, patient cells exhibited a 1.6-fold elevation in ROS production, a 1.7-fold elevation in cytoplasmic Ca2+ levels, a 1.2-fold elevation in mitochondrial membrane potential, and 30% less complex V activity compared with control cells. Patient cells also displayed 20–25% reductions in both cytochrome c oxidase (COX) activity and MnSOD protein levels compared with control cells. After T3 treatment of patient cells, ROS production was decreased by 40%, cytoplasmic Ca2+ was reduced by 20%, COX activity was increased by 1.3-fold, and ATP levels were elevated by 1.6-fold, despite the absence of a change in mitochondrial mass. There were no significant alterations in the protein expression of PGC-1α, Tfam, or UCP2 in either T3-treated patient or control cells. However, T3 restored the mitochondrial membrane potential, complex V activity, and levels of MnSOD to normal values in patient cells and elevated MnSOD levels by 21% in control cells. These results suggest that T3 acts to reduce cellular oxidative stress, which may help attenuate ROS-mediated damage, along with improving mitochondrial function and energy status in cells with mtDNA defects.

Protective Effect of SCUBE1 on Oxidative Stress-induced Apoptosis in Human Ovarian Granulosa Cells

2021 ◽  
Author(s):  
Huijiao Fu ◽  
Xuzi Cai ◽  
Qiwen Liu ◽  
Wei Yang ◽  
xuefeng wang
Keyword(s):  
Membrane Potential ◽  
Western Blot ◽  
Cell Viability ◽  
Mitochondrial Membrane Potential ◽  
Granulosa Cells ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Cell Counting ◽  
Ovarian Granulosa Cells ◽  
Induced Apoptosis

Abstract Background: Apoptosis of ovarian granulosa cells (GCs) is a sign of follicular atresia. This study aimed to explore the role and mechanism of signal peptide, CUB domain, epidermal growth factor-like protein1 (SCUBE1) in protecting GCs from apoptosis induced by hydrogen peroxide (H2O2). Methods: Firstly, the expression of SCUBE1 on the ovaries of humans and mice was analyzed by qRT-PCR, western blot and immunohistochemistry. Subsequently, the H2O2 treated GCs were pretreated with SCUBE1 recombinant protein, and their cell viability and proliferation were detected by Cell Counting Kit-8 (CCK-8) assay. The levels of reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) in the cells were determined by DCFH-DA and rhodamine 123, respectively. The percentage of apoptotic cells was analyzed by flow cytometry after staining with Annexin V/PI. The expression levels of pathway related proteins, such as Bcl-2, Bax, p53, caspase-3, were determined by western blot analysis. Finally, the pathogenicity of SCUBE1 (c.1169C>G, p.P390R) were analyzed based on the software.Results: SCUBE1 was expressed in women of all ages and had the highest expression level in the ovaries in multiple organs and tissues of KM mouse. In vitro cell experiments show that SCUBE1 pretreatment reduced H2O2-induced apoptosis and improved cell viability. SCUBE1 also blocked the production of ROS in cells and improved mitochondrial membrane potential. After SCUBE1 pretreatment, anti-apoptotic protein Bcl-2 expression was upregulated, whereas the expression of the pro-apoptotic proteins Bax, Bax/Bcl-2, Caspase-3, and p53 were downregulated. Analysis of the impact of SCUBE1 (c.1169C >G, p.P390R) mutation from the aspect of mutation pathogenicity; protein stability; and gene haplotype insufficiency, indicated that the p.P390R mutation is significantly pathogenic.Conclusions: This is the first time that the potential role of SCUBE1 in protecting GCs from H2O2-induced damage through the mitochondrial pathways, attributing to POI, is studied. SCUBE1 (c.1169C >G, p.P390R) mutation has significant pathogenicity but the specific harm needs to be confirmed by further studies. Trial registration: Not applicable.

NAD+ deficiency and mitochondrial dysfunction in granulosa cells of women with polycystic ovary syndrome‡

2021 ◽  
Author(s):  
Yujiao Wang ◽  
Qingling Yang ◽  
Huan Wang ◽  
Jing Zhu ◽  
Luping Cong ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Polycystic Ovary Syndrome ◽  
Mitochondrial Dysfunction ◽  
Mitochondrial Membrane Potential ◽  
Granulosa Cells ◽  
Mitochondrial Membrane ◽  
Polycystic Ovary ◽  
Tumor Cell Line ◽  
Ovary Syndrome ◽  
Atp Generation

Abstract Polycystic ovary syndrome (PCOS) is a prevalent heterogeneous endocrine disorder characterized by ovulation dysfunction, androgen excess, ovarian polycystic changes, insulin resistance, and infertility. Although underlying mechanisms for PCOS are still unknown, inflammation and mitochondrial dysfunction in granulosa cells (GCs) of PCOS patients have been reported. Here, we found that Nicotinamide Adenine Dinucleotide (NAD+) levels in GCs of PCOS patients was significantly decreased when compared with controls. Also, we found that higher expression of inflammation factors, increased reactive oxygen species (ROS) accumulation, lower adenosine triphosphate (ATP) generation, and decreased mitochondrial membrane potential, as well as abnormal mitochondrial dynamics in GCs of PCOS patients. In addition, the NAD+ levels were decreased after activation of inflammation in human granulosa-like tumor cell line (KGN) treated by Lipopolysaccharide (LPS). However, supplementation of nicotinamide riboside (NR), a NAD+ precursor, could largely restore the NAD+ content, reduce ROS levels and improve mitochondrial function demonstrated by increased mitochondrial membrane potential and ATP generation in LPS-treated KGN cells. Our data suggested that inflammation decreased NAD+ levels in GCs of PCOS patients, while supplementation of NR could restore NAD+ levels and alleviated mitochondrial dysfunction in GCs of PCOS patients.

scholarly journals Effect of EPA on Hsp90 and GRα protein expression in multiple myeloma drug-resistant cells

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Shenghao Wu ◽  
Yuemiao Chen ◽  
Xueshuang Wang ◽  
Shanshan Weng ◽  
Wenjin Zhou ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Membrane Potential ◽  
Protein Expression ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Influential Factors ◽  
Glucocorticoid Resistance ◽  
Cell Counting ◽  
Ligand Docking

Abstract Background Approximately 20% of MM patients harbor glucocorticoid (GC) resistance and are not responsive to therapeutic effect. Chaperoneheat-shock proteins Hsp90 is needed for ligand docking, The imbalance of Hsp90/GRα (glucocorticoid receptor α) may be an important cause of GC resistance. Recent studies have indicated that EPA could repress cancer cell growth by regulating critical influential factors in progression of cancer, consisting of resistance to drugs, chemosensitivity. The aim of the present study was to test the cytotoxic effects of EPA alone or EPA + Dexamethasone in dexamethasone-resistant MM cell (MM.1R) and investigate whether DHA can induce apoptosis and reverse acquired glucocorticoid resistance in dexamethasone-resistant MM cell (MM.1R). Methods Cell Counting Kit-8 (CCK-8) was used to detect the proliferation of MM.1R cells after treating with EPA alone and EPA combined with DEX. Mitochondrial membrane potential was measured by flow cytometry and GRα and Hsp90 protein expression were assessed by western blot analysis. Results EPA alone was able to inhibit cell proliferation as evidenced by CCK-8 assay and the tumor growth was remarkably suppressed by EPA + Dexamethasone, Cell apoptosis after EPA treatment was obviously observed by Flow cytometry analysis of the mitochondrial membrane potential. Analysis of Hsp90 and GRα proteins in MM.1R cells incubated with EPA revealed down-regulation of Hsp90 and up-regulation of GRα. Accordingly, the Hsp90/GRα ratio was significantly decreased with the increase of EPA concentration. Conclusions EPA might be used as a new effective treatment for reversal of glucocorticoid-resistance in multiple myeloma.

scholarly journals Rab11a Is Overexpressed in Gastric Cancer and Regulates FAK/AKT Signaling

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Jiang Du ◽  
Lin Fu ◽  
Jie Hao ◽  
Xiumin Lin ◽  
Qianze Dong
Keyword(s):  
Gastric Cancer ◽  
Cell Proliferation ◽  
Membrane Potential ◽  
Protein Expression ◽  
Cyclin D1 ◽  
Mitochondrial Membrane Potential ◽  
Cell Lines ◽  
Mitochondrial Membrane ◽  
Akt Signaling ◽  
Gastric Cancers

Dysregulation of Rab11a has been implicated in the progression of several cancers. However, there have been no such studies for human gastric cancers. In the current study, we examined Rab11a protein expression and found it was upregulated in 49 of 108 gastric cancer tissues and correlated with local invasion, nodal metastasis, and advanced stage. Rab11a protein was higher in gastric cancer cell lines than normal gastric cell line. We transfected Rab11a plasmid and siRNA in both MGC803 and AGS cell lines. Rab11a overexpression increased the cell growth rate, colony numbers, and invasion ability in both MGC803 and AGS cell lines. Downregulation of Rab11a using siRNA decreased the cell proliferation rate, colony numbers, and inhibited invasion. Rab11a overexpression also conferred cisplatin resistance. Annexin V/PI staining showed that Rab11a overexpression suppressed cisplatin-induced apoptosis, while Rab11a depletion promoted cell apoptosis. We also showed that Rab11a overexpression maintained mitochondrial membrane potential. Western blot analysis revealed that Rab11a increased protein expression of MMP2, cyclin D1, Bcl-2, p-FAK, and p-AKT, while Rab11a depletion showed the opposite effects. Blockage of FAK using inhibitor downregulated Bcl-2, cyclin D1, MMP2, and p-AKT expression and abolished the effects of Rab11a on these proteins. In summary, our data demonstrated that Rab11a is upregulated in human gastric cancers. Rab11a facilitated cell proliferation and invasion, as well as cisplatin sensitivity and mitochondrial membrane potential, possibly via the FAK/AKT signaling pathway.

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