scholarly journals Application of extracellular flux analysis for determining mitochondrial function in mammalian oocytes and early embryos

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Bethany Muller ◽  
Niamh Lewis ◽  
Tope Adeniyi ◽  
Henry J. Leese ◽  
Daniel R. Brison ◽  
...  
Keyword(s):  
Real Time ◽  
Oocyte Maturation ◽  
Small Groups ◽  
Mitochondrial Function ◽  
Mitochondrial Activity ◽  
Flux Analysis ◽  
Mammalian Oocyte ◽  
Extracellular Flux ◽  
Early Embryos ◽  
The Impact

AbstractMitochondria provide the major source of ATP for mammalian oocyte maturation and early embryo development. Oxygen Consumption Rate (OCR) is an established measure of mitochondrial function. OCR by mammalian oocytes and embryos has generally been restricted to overall uptake and detailed understanding of the components of OCR dedicated to specific molecular events remains lacking. Here, extracellular flux analysis (EFA) was applied to small groups of bovine, equine, mouse and human oocytes and bovine early embryos to measure OCR and its components. Using EFA, we report the changes in mitochondrial activity during the processes of oocyte maturation, fertilisation, and pre-implantation development to blastocyst stage in response to physiological demands in mammalian embryos. Crucially, we describe the real time partitioning of overall OCR to spare capacity, proton leak, non-mitochondrial and coupled respiration – showing that while activity changes over the course of development in response to physiological demand, the overall efficiency is unchanged. EFA is shown to be able to measure mitochondrial function in small groups of mammalian oocytes and embryos in a manner which is robust, rapid and easy to use. EFA is non-invasive and allows real-time determination of the impact of compounds on OCR, facilitating an assessment of the components of mitochondrial activity. This provides proof-of-concept for EFA as an accessible system with which to study mammalian oocyte and embryo metabolism.

scholarly journals Application of extracellular flux analysis for determining mitochondrial function in mammalian oocytes and early embryos

2019 ◽  
Author(s):  
Bethany Muller ◽  
Niamh Lewis ◽  
Tope Adeniyi ◽  
Henry J Leese ◽  
Daniel Brison ◽  
...  
Keyword(s):  
Real Time ◽  
Oocyte Maturation ◽  
Small Groups ◽  
Mitochondrial Function ◽  
Mitochondrial Activity ◽  
Flux Analysis ◽  
Spare Capacity ◽  
Extracellular Flux ◽  
Early Embryos ◽  
The Impact

1.AbstractBackgroundMitochondria provide the major source of ATP for mammalian oocyte maturation and early embryo development. Oxygen Consumption Rate (OCR) is an established measure of mitochondrial function. OCR by mammalian oocytes and embryos has generally been restricted to overall uptake and detailed understanding of the components of OCR dedicated to specific molecular events remains lacking.ResultsHere, extracellular flux analysis (EFA) was applied to small groups of bovine, equine, mouse and human oocytes and bovine early embryos to measure OCR. Using EFA, we report the changes in mitochondrial activity during the processes of oocyte maturation, fertilization, and pre-implantation development to blastocyst stage in response to physiological demands in mammalian embryos. Crucially, we describe the real time partitioning of overall OCR to spare capacity, proton leak, non-mitochondrial and coupled respiration – showing that while there are alterations in activity over the course of development to respond to physiological demand, the overall efficiency is unchanged.ConclusionEFA is shown to be able to measure mitochondrial function in small groups of mammalian oocytes and embryos in a manner which is robust, rapid and easy to use. EFA is non-invasive and allows real-time determination of the impact of compounds on OCR, facilitating an assessment of the parameters of mitochondrial activity. This provides proof-of-concept for EFA as an accessible system with which to study oocyte and embryo metabolism.

scholarly journals The Acute Extracellular Flux (XF) Assay to Assess Compound Effects on Mitochondrial Function

2014 ◽  
Vol 20 (3) ◽  
pp. 422-429 ◽  
Author(s):  
Ruolan Wang ◽  
Steven J. Novick ◽  
James B. Mangum ◽  
Kennedy Queen ◽  
David A. Ferrick ◽  
...  
Keyword(s):  
Mitochondrial Function ◽  
Mitochondrial Activity ◽  
Screening Methods ◽  
Drug Induced ◽  
Whole Cells ◽  
Drug Candidates ◽  
Adverse Health Outcomes ◽  
Extracellular Flux ◽  
Screening Platform ◽  
The Impact

Numerous investigations have linked mitochondrial dysfunction to adverse health outcomes and drug-induced toxicity. The pharmaceutical industry is challenged with identifying mitochondrial liabilities earlier in drug development and thereby reducing late-stage attrition. Consequently, there is a demand for reliable, higher-throughput screening methods for assessing the impact of drug candidates on mitochondrial function. The extracellular flux (XF) assay described here is a plate-based method in which galactose-conditioned HepG2 cells were acutely exposed to test compounds, then real-time changes in the oxygen consumption rate and extracellular acidification rate were simultaneously measured using a Seahorse Bioscience XF-96 analyzer. The acute XF assay was validated using marketed drugs known to modulate mitochondrial function, and data analysis was automated using a spline curve fitting model developed at GlaxoSmithKline. We demonstrate that the acute XF assay is a robust, sensitive screening platform for evaluating drug-induced effects on mitochondrial activity in whole cells.

Extracellular Flux Analysis to Investigate the Impact of NF-κB on in Colorectal Carcinoma (CRC)

2021 ◽  
pp. 293-303
Author(s):  
Daria Capece ◽  
Daniela Verzella ◽  
Federica Begalli ◽  
Jason Bennett ◽  
Daniel D’Andrea ◽  
...  
Keyword(s):  
Colorectal Carcinoma ◽  
Flux Analysis ◽  
Extracellular Flux ◽  
The Impact

scholarly journals The spatio-temporal dynamics of mitochondrial membrane potential during oocyte maturation

2019 ◽  
Vol 25 (11) ◽  
pp. 695-705 ◽  
Author(s):  
Usama AL-Zubaidi ◽  
Jun Liu ◽  
Ozgur Cinar ◽  
Rebecca L Robker ◽  
Deepak Adhikari ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Oocyte Maturation ◽  
Mitochondrial Function ◽  
Mitochondrial Membrane ◽  
Local Level ◽  
Meiotic Spindle ◽  
Mitochondrial Activity ◽  
Wide Range ◽  
Spatio Temporal

Abstract Mitochondria are highly dynamic organelles and their distribution, structure and activity affect a wide range of cellular functions. Mitochondrial membrane potential (∆Ψm) is an indicator of mitochondrial activity and plays a major role in ATP production, redox balance, signaling and metabolism. Despite the absolute reliance of oocyte and early embryo development on mitochondrial function, there is little known about the spatial and temporal aspects of ΔΨm during oocyte maturation. The one exception is that previous findings using a ΔΨm indicator, JC-1, report that mitochondria in the cortex show a preferentially increased ΔΨm, relative to the rest of the cytoplasm. Using live-cell imaging and a new ratiometric approach for measuring ΔΨm in mouse oocytes, we find that ΔΨm increases through the time course of oocyte maturation and that mitochondria in the vicinity of the first meiotic spindle show an increase in ΔΨm, compared to other regions of the cytoplasm. We find no evidence for an elevated ΔΨm in the oocyte cortex. These findings suggest that mitochondrial activity is adaptive and responsive to the events of oocyte maturation at both a global and local level. In conclusion, we have provided a new approach to reliably measure ΔΨm that has shed new light onto the spatio-temporal regulation of mitochondrial function in oocytes and early embryos.

scholarly journals Assessment of the impact of mitochondrial genotype upon drug-induced mitochondrial dysfunction in platelets derived from healthy volunteers

2021 ◽  
Author(s):  
Amy L. Ball ◽  
Katarzyna M. Bloch ◽  
Lucille Rainbow ◽  
Xuan Liu ◽  
John Kenny ◽  
...  
Keyword(s):  
Mitochondrial Dysfunction ◽  
Healthy Volunteers ◽  
Adverse Drug Reactions ◽  
Mitochondrial Function ◽  
Flux Analysis ◽  
Drug Reactions ◽  
Drug Induced ◽  
Mtdna Variation ◽  
Mitochondrial Genotype ◽  
The Impact

AbstractMitochondrial DNA (mtDNA) is highly polymorphic and encodes 13 proteins which are critical to the production of ATP via oxidative phosphorylation. As mtDNA is maternally inherited and undergoes negligible recombination, acquired mutations have subdivided the human population into several discrete haplogroups. Mitochondrial haplogroup has been found to significantly alter mitochondrial function and impact susceptibility to adverse drug reactions. Despite these findings, there are currently limited models to assess the effect of mtDNA variation upon susceptibility to adverse drug reactions. Platelets offer a potential personalised model of this variation, as their anucleate nature offers a source of mtDNA without interference from the nuclear genome. This study, therefore, aimed to determine the effect of mtDNA variation upon mitochondrial function and drug-induced mitochondrial dysfunction in a platelet model. The mtDNA haplogroup of 383 healthy volunteers was determined using next-generation mtDNA sequencing (Illumina MiSeq). Subsequently, 30 of these volunteers from mitochondrial haplogroups H, J, T and U were recalled to donate fresh, whole blood from which platelets were isolated. Platelet mitochondrial function was tested at basal state and upon treatment with compounds associated with both mitochondrial dysfunction and adverse drug reactions, flutamide, 2-hydroxyflutamide and tolcapone (10–250 μM) using extracellular flux analysis. This study has demonstrated that freshly-isolated platelets are a practical, primary cell model, which is amenable to the study of drug-induced mitochondrial dysfunction. Specifically, platelets from donors of haplogroup J have been found to have increased susceptibility to the inhibition of complex I-driven respiration by 2-hydroxyflutamide. At a time when individual susceptibility to adverse drug reactions is not fully understood, this study provides evidence that inter-individual variation in mitochondrial genotype could be a factor in determining sensitivity to mitochondrial toxicants associated with costly adverse drug reactions.

Interferon-beta affects mitochondrial activity in CD4+lymphocytes: Implications for mechanism of action in multiple sclerosis

2014 ◽  
Vol 21 (10) ◽  
pp. 1262-1270 ◽  
Author(s):  
Aiden Haghikia ◽  
Simon Faissner ◽  
Derek Pappas ◽  
Bartosz Pula ◽  
Denis A Akkad ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
T Cells ◽  
Energy Metabolism ◽  
Mitochondrial Function ◽  
Interferon Beta ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Mitochondrial Activity ◽  
Mitochondrial Energy Metabolism ◽  
The Impact

Background:Whereas cellular immune function depends on energy supply and mitochondrial function, little is known on the impact of immunotherapies on cellular energy metabolism.Objective:The objective of this paper is to assess the effects of interferon-beta (IFN-β) on mitochondrial function of CD4+T cells.Methods:Intracellular adenosine triphosphate (iATP) in phytohemagglutinin (PHA)-stimulated CD4+cells of multiple sclerosis (MS) patients treated with IFN-β and controls were analyzed in a luciferase-based assay. Mitochondrial-transmembrane potential (ΔΨm) in IFN-β-treated peripheral blood mononuclear cells (PBMCs) was investigated by flow cytometry. Expression of genes involved in mitochondrial oxidative phosphorylation (OXPHOS) in CD4+cells of IFN-β-treated individuals and correlations between genetic variants in the key metabolism regulator PGC-1α and IFN-β response in MS were analyzed.Results:IFN-β-treated MS patients exhibited a dose-dependent reduction of iATP levels in CD4+T cells compared to controls ( p < 0.001). Mitochondrial effects were reflected by depolarization of ΔΨm. Expression data revealed changes in the transcription of OXPHOS-genes. iATP levels in IFN-β-responders were reduced compared to non-responders ( p < 0.05), and the major T allele of the SNP rs7665116 of PGC-1α correlated with iATP-levels.Conclusion:Reduced iATP-synthesis ex vivo and differential expression of OXPHOS-genes in CD4+T cells point to unknown IFN-β effects on mitochondrial energy metabolism, adding to potential pleiotropic mechanisms of action.

scholarly journals Temporal Manipulation of Mitochondrial Function by Virulent Francisella tularensis To Limit Inflammation and Control Cell Death

2018 ◽  
Vol 86 (8) ◽  
Author(s):  
Forrest Jessop ◽  
Benjamin Schwarz ◽  
Emily Heitmann ◽  
Robert Buntyn ◽  
Tara Wehrly ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Mitochondrial Function ◽  
Control Cell ◽  
Flux Analysis ◽  
Dependent Manner ◽  
Metabolic Shift ◽  
Loss Of Function ◽  
Content Type ◽  
Extracellular Flux ◽  
Bacterial Replication

ABSTRACT Francisella tularensis subsp. tularensis is a highly pathogenic intracellular bacterium that suppresses host inflammation by impairing the metabolic shift from oxidative phosphorylation to glycolysis. Decreased mitochondrial metabolism is central to initiating a metabolic shift to glycolysis and regulating inflammation, but F. tularensis subsp. tularensis manipulation of host mitochondrial function has not been explored. We demonstrate, using extracellular flux analysis, that F. tularensis subsp. tularensis infection initially improves host macrophage mitochondrial bioenergetics in a capsule-dependent manner. Enhancement of mitochondrial function by F. tularensis subsp. tularensis allowed for modest replication and inhibition of apoptosis early after infection. However, using live cell imaging, we found that F. tularensis subsp. tularensis facilitated the loss of mitochondrial function at later time points during infection in a capsule-independent fashion. This loss of function was paired with oncosis and rapid bacterial replication. Inhibition of oncosis reduced intracellular bacterial numbers, underscoring the requirement for this process during F. tularensis subsp. tularensis infection. These findings establish that temporal mitochondrial manipulation by F. tularensis subsp. tularensis is critical for maintenance of a noninflammatory environment and subsequently aids in optimal replication and dissemination of this pathogenic organism.

Essential shared and species-specific features of mammalian oocyte maturation-associated transcriptome changes impacting oocyte physiology

2021 ◽  
Author(s):  
Peter Z Schall ◽  
Keith E. Latham
Keyword(s):  
Oxidative Phosphorylation ◽  
Oocyte Maturation ◽  
Mitochondrial Function ◽  
Meta Analysis ◽  
Genetic Material ◽  
Data Sets ◽  
High Homology ◽  
Mammalian Oocyte ◽  
Maternal Mrnas ◽  
Species Specific

Oogenesis is a complex process resulting in the production of a truly remarkable cell-the oocyte. Oocytes execute many unique processes and functions such as meiotic segregation of maternal genetic material, and essential life-generating functions after fertilization including post-transcriptional support of essential homeostatic and metabolic processes, and activation and reprogramming of the embryonic genome. An essential goal for understanding female fertility and infertility in mammals is to discover critical features driving the production of quality oocytes, particularly the complex regulation of oocyte maternal mRNAs. We report here the first in-depth meta-analysis of oocyte maturation-associated transcriptome changes, using eight data sets encompassing 94 RNAseq libraries for human, rhesus monkey, mouse, and cow. A majority of maternal mRNAs are regulated in a species-restricted manner, highlighting considerable divergence in oocyte transcriptome handling during maturation. We identified 121 mRNAs changing in relative abundance similarly across all four species (92 of high homology), and 993 (670 high homology) mRNAs regulated similarly in at least three of the four species, corresponding to just 0.84% and 6.9% of mRNAs analyzed. Ingenuity Pathway Analysis (IPA) revealed an association of these shared mRNAs with many shared pathways and functions, most prominently oxidative phosphorylation and mitochondrial function. These shared functions were reinforced further by primate-specific and species-specific DEGs. Thus, correct down-regulation of mRNAs related to oxidative phosphorylation and mitochondrial function is a major shared feature of mammalian oocyte maturation.

circPTP4A2-miR-330-5p-PDK2 Signaling Facilitates In Vivo Survival of HuMSCs on SF-SIS Scaffolds and Improves The Repair of Damaged Endometrium

2021 ◽  
Author(s):  
Yuanyuan Zheng ◽  
Linhao Li ◽  
Xuewei Bi ◽  
Ruyue Xue
Keyword(s):  
Real Time ◽  
Small Intestinal Submucosa ◽  
Mitochondrial Metabolism ◽  
Human Umbilical Cord ◽  
Extracellular Flux ◽  
Cellular Oxygen ◽  
Small Intestinal ◽  
Intestinal Submucosa ◽  
The Impact

Abstract Background Human umbilical cord MSCs (HuMSC)-based therapy has shown promising results in the treatment of intrauterine adhesions (lUA). In this study, our aim was to construct a HuMSC-seeded silk fibroin small-intestinal submucosa (SF-SIS) scaffold and evaluate the impact of repairing the damaged endometrium in an lUA mouse model. Methods To identify the functional effect of HuMSCs-silk cellulose (SF)- small-intestinal submucosa (SIS) scaffolds on the repair of damaged endometrium, a mouse lUA model was established in this study. The uterine morphology and fibrosis were evaluated by hematoxylin - eosin (H&E) staining and Masson staining. CircRNA sequencing, real-time PCR and RNA fluorescence in situ hybridization were used to screen and verify the potential circRNAs that involved in the repair of damaged endometrium by HuMSCs. Real time integrated cellular oxygen consumption rate (OCR) was measured using the Seahorse XF24 Extracellular Flux Analyser. The potential down-stream miRNAs and proteins of circRNAs were analyzed dual-luciferase report and Western Blot. Results We found that HuMSCs-SF-SIS not only increased the number of glands, but also reduced the ulcer area in the IUA model. Furthermore, we demonstrated that circPTP4A2 was elevated in the HuMSCs seeded on the SF-SIS scaffolds and stabilized the mitochondrial metabolism through miR-330-5p-PDK2 signaling, which contributes to endometrial repair progression. Conclusion In this study, we demonstrated that circPTP4A2 was elevated in the HuMSCs seeded on the SF-SIS scaffolds and stabilized the mitochondrial metabolism through miR-330-5p-PDK2 signaling, which contributes to endometrial repair progression. These findings demonstrate that HuMSC-seeded SF-SIS scaffolds are an encouraging method for the treatment of lUA.

Bioenergetic differences between MCF-7 and T47D breast cancer cells and their regulation by oestradiol and tamoxifen

2014 ◽  
Vol 465 (1) ◽  
pp. 49-61 ◽  
Author(s):  
Brandie N. Radde ◽  
Margarita M. Ivanova ◽  
Huy Xuan Mai ◽  
Joshua K. Salabei ◽  
Bradford G. Hill ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Real Time ◽  
Oestrogen Receptor ◽  
Breast Cancer Cells ◽  
Flux Analysis ◽  
Intact Cells ◽  
Extracellular Flux ◽  
Oestrogen Receptor Α ◽  
Mcf 7

Oestrogen receptor α-expressing breast cancer cells show differences in basal bioenergetics profiles and bioenergetics responses to serum depletion, oestradiol and tamoxifen as measured in real time by extracellular flux analysis in intact cells.

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