Monitoring oocyte/embryo respiration using electrochemical-based oxygen sensors

Abstract

Current commercially available instruments for monitoring mitochondrial respiration are incapable of single cell measurements. Therefore, we developed a three-electrode, Clark-type biosensor suitable for mitochondrial respirometry in single oocytes and embryos. The biosensor was embedded in a PMMA (polymethyl methacrylate) micro-chamber to allow investigation of single oocytes/embryos immersed in up to 100 µL of respiration buffer. The micro-chamber was completely sealed to avoid oxygen exchange between the inside of the chamber and the atmosphere, while being maintained at a temperature of 38.5 ˚C to preserve cell viability. Using amperometry, the oxygen consumption of cells inside the micro-chamber was measured as a change in output current and converted to femto-mol (fmol) oxygen consumed per second based on calibrations with known buffer oxygen concentrations. The sensor measured basal cell respiration supported by endogenous substrates, respiration associated with proton leak induced by inhibition of the adenosine triphosphate (ATP) synthase (complex V) with oligomycin, and the maximal noncoupled respiratory capacity revealed by Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) titration. Some potential applications of this oxygen sensor system include evaluating effects of metabolic therapies on oocyte bioenergetics, and monitoring mitochondrial function throughout oocyte maturation and blastocyst development to predict embryo viability to compliment assisted reproductive technologies.

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

“…Using equine, mouse and human oocytes, the suitability of EFA to determine OCR in a variety of species was confirmed. The data were reassuringly close to those values obtained by previous methods; for example in the human, viable oocytes have been reported to consume 0.37 pmol/min 23 ; in the mouse, 0.21 pmol/min 33 ; and equine oocytes 3 pmol/min 22 ; values close to our own. In the present work, mouse, human and equine oocytes showed higher basal respiration than bovine oocytes, however, the mouse and human oocytes used had failed to fertilise, suggesting they may not have been of optimal quality.…”

“…Mitochondrial oxidative phosphorylation, measured as the oxygen consumption rate (OCR) 14 , is the largest contributor to cellular ATP demand during preimplantation development 12,15,16 . A number of techniques have been used to measure OCR in oocytes and embryos, including the Cartesian diver 17 , microspectrophotometry 18 , pyrene ultramicrofluorescence 10,11 , scanning electron microscopy 19,20 and micro-respirometry 21,22 . Combined, these approaches have been instrumental in defining the overall metabolism of oocytes and early embryos, and have yielded remarkably consistent data.…”

A multi-sensor system for measuring bovine embryo metabolism

“…To determine the calibration curve and linearity of the DO sensor, the dissolved oxygen concentration was changed by adding 0.1 M Na 2 SO 3 to the saturated solution in incremental steps with continuous stirring to produce different oxygen concentrations for generating the calibration data (Obeidat et al, 2018). All O 2 concentration measurements were made at 38.5°C and validated using a calibrated Oakton DO6+ dissolved oxygen meter.…”

Design of a multi-sensor platform for integrating extracellular acidification rate with multi-metabolite flux measurement for small biological samples

“…The OCR, GCR, and LPR were calculated based on a model in our previously published papers (Obeidat et al, 2018a; Obeidat et al, 2018b) and included in S.8 in the Supplementary Material.…”

“…Additionally, electrochemical sensors can enable label-free, real-time intracellular and extracellular measurements without perturbing the system under investigation (Wang et al, 2012). There have been a number of reported studies that used electrochemical methods for measuring DO (Obeidat et al, 2016; Obeidat et al, 2018a), glucose (Pemberton et al, 2009; Obeidat et al, 2018b), lactate (Rawson et al, 2009; Obeidat et al, 2018b), and pH (McConnell et al, 1992; Liao et al, 1998).…”