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

Muller, Bethany; Lewis, Niamh; Adeniyi, Tope; Leese, Henry J.; Brison, Daniel R.; Sturmey, Roger G.

doi:10.1038/s41598-019-53066-9

Cited by 40 publications

(28 citation statements)

References 67 publications

(87 reference statements)

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“…The measured OCR results are comparable to the results from our previous work and others using various stages of bovine oocytes and embryos, where the OCRs were measured between 5 to 30 fmol/s [11,12,32,[66][67][68]. Furthermore, the OCR results for COCs measured by others from a variety of sources (mouse, bovine, and human) [9,69,70] also show a comparable range (3 to 60 fmol/s) as our measured OCR results. Supplementary Materials Video S6 shows the captured movie of heatmap presented in Figure 7.…”

Section: Oxygen Flux and Consumption Rate Analysissupporting

confidence: 90%

Real-Time Analysis of Oxygen Gradient in Oocyte Respiration Using a High-Density Microelectrode Array

et al. 2021

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Physiological events related to oxygen concentration gradients provide valuable information to determine the state of metabolizing biological cells. The existing oxygen sensing methods (i.e., optical photoluminescence, magnetic resonance, and scanning electrochemical) are well-established and optimized for existing in vitro analyses. However, such methods also present various limitations in resolution, real-time sensing performance, complexity, and costs. An electrochemical imaging system with an integrated microelectrode array (MEA) would offer attractive means of measuring oxygen consumption rate (OCR) based on the cell’s two-dimensional (2D) oxygen concentration gradient. This paper presents an application of an electrochemical sensor platform with a custom-designed complementary-metal-oxide-semiconductor (CMOS)-based microchip and its Pt-coated surface MEA. The high-density MEA provides 16,064 individual electrochemical pixels that cover a 3.6 mm × 3.6 mm area. Utilizing the three-electrode configuration, the system is capable of imaging low oxygen concentration (18.3 µM, 0.58 mg/L, or 13.8 mmHg) at 27.5 µm spatial resolution and up to 4 Hz temporal resolution. In vitro oxygen imaging experiments were performed to analyze bovine cumulus-oocytes-complexes cells OCR and oxygen flux density. The integration of a microfluidic system allows proper bio-sample handling and delivery to the MEA surface for imaging. Finally, the imaging results are processed and presented as two-dimensional (2D) heatmaps, representing the dissolved oxygen concentration in the immediate proximity of the MEA. This paper provides the results of real-time 2D imaging of OCR of live cells/tissues to gain spatial and temporal dynamics of target cell metabolism.

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Section: Oxygen Flux and Consumption Rate Analysissupporting

confidence: 90%

Real-Time Analysis of Oxygen Gradient in Oocyte Respiration Using a High-Density Microelectrode Array

et al. 2021

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“…Mitochondrial ROS production is primarily regulated by the ETC and oxygen consumption 17 . Majority of consumed oxygen is utilized by the mitochondria for ATP production 24 . However, proton leak through the membrane due to damage of the mitochondrial membrane and ETC, further increase oxygen consumption (uncoupled respiration) and exaggerate ROS production.…”

Section: Discussionmentioning

confidence: 99%

Perfusion reduces bispecific antibody aggregation via mitigating mitochondrial dysfunction-induced glutathione oxidation and ER stress in CHO cells

Sinharoy

Aziz

Majewska

et al. 2020

Sci Rep

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One major challenge observed for the expression of therapeutic bispecific antibodies (BisAbs) is high product aggregates. Aggregates increase the risk of immune responses in patients and therefore must be removed at the expense of purification yields. BisAbs contain engineered disulfide bonds, which have been demonstrated to form product aggregates, if mispaired. However, the underlying intracellular mechanisms leading to product aggregate formation remain unknown. We demonstrate that impaired glutathione regulation underlies BisAb aggregation formation in a CHO cell process. Aggregate formation was evaluated for the same clonal CHO cell line producing a BisAb using fed-batch and perfusion processes. The perfusion process produced significantly lower BisAb aggregates compared to the fed-batch process. Perfusion bioreactors attenuated mitochondrial dysfunction and ER stress resulting in a favorable intracellular redox environment as indicated by improved reduced to oxidized glutathione ratio. Conversely, mitochondrial dysfunction-induced glutathione oxidation and ER stress disrupted the intracellular redox homeostasis, leading to product aggregation in the fed-batch process. Combined, our results demonstrate that mitochondrial dysfunction and ER stress impaired glutathione regulation leading to higher product aggregates in the fed-batch process. This is the first study to utilize perfusion bioreactors as a tool to demonstrate the intracellular mechanisms underlying product aggregation formation.

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“…Detailed protocols and their justification can be found at (accessed from June 2020 to May 2021). Additionally, detailed protocols were previously published [ 70 , 71 , 72 ].…”

Section: Methodsmentioning

confidence: 99%

Anabolic Steroids-Driven Regulation of Porcine Ovarian Putative Stem Cells Favors the Onset of Their Neoplastic Transformation

Gorczyca

Wartalski

Wiater

et al. 2021

IJMS

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Nandrolone (Ndn) and boldenone (Bdn), the synthetic testosterone analogues with strong anabolic effects, despite being recognized as potentially carcinogenic compounds, are commonly abused by athletes and bodybuilders, which includes women, worldwide. This study tested the hypothesis that different doses of Ndn and Bdn can initiate neoplastic transformation of porcine ovarian putative stem cells (poPSCs). Immunomagnetically isolated poPSCs were expanded ex vivo in the presence of Ndn or Bdn, for 7 and 14 days. Results show that pharmacological doses of both Ndn and Bdn, already after 7 days of poPSCs culture, caused a significant increase of selected, stemness-related markers of cancer cells: CD44 and CD133. Notably, Ndn also negatively affected poPSCs growth not only by suppressing their proliferation and mitochondrial respiration but also by inducing apoptosis. This observation shows, for the first time, that chronic exposure to Ndn or Bdn represents a precondition that might enhance risk of poPSCs neoplastic transformation. These studies carried out to accomplish detailed molecular characterization of the ex vivo expanded poPSCs and their potentially cancerous derivatives (PCDs) might be helpful to determine their suitability as nuclear donor cells (NDCs) for further investigations focused on cloning by somatic cell nuclear transfer (SCNT). Such investigations might also be indispensable to estimate the capabilities of nuclear genomes inherited from poPSCs and their PCDs to be epigenetically reprogrammed (dedifferentiated) in cloned pig embryos generated by SCNT. This might open up new possibilities for biomedical research aimed at more comprehensively recognizing genetic and epigenetic mechanisms underlying not only tumorigenesis but also reversal/retardation of pro-tumorigenic intracellular events.

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

Cited by 40 publications

References 67 publications

Real-Time Analysis of Oxygen Gradient in Oocyte Respiration Using a High-Density Microelectrode Array

Real-Time Analysis of Oxygen Gradient in Oocyte Respiration Using a High-Density Microelectrode Array

Perfusion reduces bispecific antibody aggregation via mitigating mitochondrial dysfunction-induced glutathione oxidation and ER stress in CHO cells

Anabolic Steroids-Driven Regulation of Porcine Ovarian Putative Stem Cells Favors the Onset of Their Neoplastic Transformation

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