Abstract
Mitochondria 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.
Highlights
Mitochondria provide the major source of ATP for mammalian oocyte maturation and early embryo development
While this research has provided a mature set of data describing embryo metabolism, the assays used are technically complex and require dedicated equipment sensitive enough to measure very small quantities of biological material
We describe the first use of extracellular flux analysis (EFA) as an accessible system which can measure oxygen consumption and its components in small groups of mammalian oocytes and early embryos
Summary
Mitochondria provide the major source of ATP for mammalian oocyte maturation and early embryo development. Using EFA, we report the changes in mitochondrial activity during the processes of oocyte maturation, fertilisation, and preimplantation development to blastocyst stage in response to physiological demands in mammalian embryos. A major limiting factor has been the lack of appropriate technology since current methods are time-consuming and technically demanding This has restricted their applicability to Assisted Reproductive Technology (ART) clinics where they might potentially be used to support selection of healthy embryos or as a tool for high-throughput screening or research to facilitate understanding of this critical element of embryo function. EFA permits the real-time measurement of metabolism and the systematic evaluation of components of cellular oxygen consumption We have applied this method for the measurement of OCR by oocytes from a range of mammalian species, including the human. We have explored the components of OCR on bovine embryos using EFA and discovered that mitochondrial function changes post fertilisation and between the cleavage and blastocyst stages
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