Novel aspects of mitochondrial biology in early embryos Bethany Muller, BSc (Hons), MSc A thesis submitted for the degree of PhD The University of Hull and the University of York Hull York Medical School May 2019 Novel aspects of mitochondrial biology in early embryos Abstract Mitochondria play an essential role in early development through facilitating the production of energy required to undergo key physiological processes. Mitochondrial function in oocytes and embryos has been associated with viability and reproductive outcomes, and is increasingly becoming a focus as a target for assisted reproduction technology. A number of tools have been applied to measure mitochondrial activity, however each have limitations. Here, we apply Seahorse XFp to mammalian oocytes and embryos for the first time. Seahorse XFp allows real-time measurement of mitochondrial activity in an automated and high-throughput manner and, coupled with mitochondrial inhibitors, can be used to gain insight on the parameters of mitochondrial function. This tool was used to carry out an investigation on physiological embryo function, revealing values of OCR similar to those generated from established techniques. Expected trends of cumulus increasing OCR, increased capacity following in vitro fertilisation and an increase in OCR between cleavage and blastocyst stage were observed. Seahorse XFp was then applied alongside metabolic tools to measure glycolytic activity and amino acid turnover to investigate clinically relevant interventions. Application of cytoskeletal inhibitors as used during mitochondrial replacement therapy (MRT) resulted in no major changes to gross metabolic measures. However, amino acid metabolism did show alterations at both cleavage and blastocyst stages, demonstrating a legacy effect that warrants further work to support safety of MRT. Exposure to mitochondrial modulator CoQ10 during in vitro maturation has previously been shown to alter metabolic function in oocytes, and we expanded this finding to show that metabolic changes were observed in embryos. CoQ10 exposure during in vitro culture, on the other hand, was shown to require further optimization. These data contribute to the emerging data set regarding mitochondrial modulation in ART and importantly present a tool which has the potential to be widely applied in reproductive biology. 1 Novel aspects of mitochondrial biology in early embryos Thesis outputs Aspects of this work have been submitted for publication. Publications Submitted Muller, B., Lewis, N., Adeniyi, T., Leese, H.J., Brison, D. & Sturmey, R.G. (2019). Application of extracellular flux analysis for determining mitochondrial function in mammalian oocytes and early embryos. bioRxiv, http://dx.doi.org/10.1101/626333. ➔ Currently under review at Scientific Reports, Nature In preparation Muller, B. & Sturmey, R.G. The effect of cytoskeletal inhibitors as used in nuclear transfer technologies on embryo metabolism and viability. Muller, B. & Sturmey, R.G. The impact of Coenzyme Q10 on oocyte and embryo metabolism. Conference presentations Muller, B., Lewis, N., Aibibula, M. & Sturmey, R.G. (5-7 January 2017). Novel approach for measuring oxygen consumption of mammalian oocytes [Poster presentation]. Fertility 2017, Edinburgh International Conference Centre, UK. Muller, B. & Sturmey, R.G. (8-9 September 2017). Novel approach for measuring mitochondrial function in bovine oocytes and embryos [Oral presentation]. 33rd Scientific Meeting Association of Embryo Technology in Europe, Bath, UK. Muller, B. & Sturmey, R.G. (20 April 2018). The effect of cytoskeletal inhibitors on embryo metabolism and viability [Oral presentation]. Allam Lecture 2018, University of Hull, UK. 2 Novel aspects of mitochondrial biology in early embryos Table of Contents Abstract ................................................................................................................................................... 1 Thesis outputs ......................................................................................................................................... 2 Table of Contents .................................................................................................................................... 3 List of Tables ........................................................................................................................................... 8 List of Figures .......................................................................................................................................... 9 List of abbreviations .............................................................................................................................. 13 Acknowledgements ............................................................................................................................... 17 Author’s declaration ............................................................................................................................. 18 Chapter 1 – Introduction ....................................................................................................................... 19 1.1 Early embryo development in the cow ....................................................................................... 19 1.1.1 The oestrus cycle .................................................................................................................. 19 1.1.2 Gametogenesis..................................................................................................................... 21 1.1.3 Fertilization .......................................................................................................................... 23 1.1.4 Pre-implantation embryo development .............................................................................. 25 1.1.5 In vitro interventions ............................................................................................................ 27 1.1.6 Post-implantation embryo development ............................................................................ 29 1.1.7 The role of the bovine embryo in ART research .................................................................. 30 1.2 Cellular metabolism .................................................................................................................... 31 1.2.1 Metabolism: the whole picture ........................................................................................... 31 1.2.2 Glycolysis .............................................................................................................................. 32 1.2.3 Fatty acid metabolism .......................................................................................................... 33 1.2.4 TCA cycle .............................................................................................................................. 34 1.2.5 Amino acid metabolism ....................................................................................................... 34 1.2.6 Oxidative phosphorylation ................................................................................................... 35 1.2.7 Metabolism of the mammalian oocyte and embryo ........................................................... 37 1.3 Mitochondria in reproduction .................................................................................................... 40 1.3.1 Mitochondrial structure and function ................................................................................. 40 1.3.2 Mitochondria in reproduction ............................................................................................. 42 1.3.3 Mitochondria as a biomarker for embryo selection ............................................................ 43 1.3.4 Mitochondrial function as a research and screening tool ................................................... 47 1.3.5 Techniques for measuring mitochondrial function ............................................................. 48 3 Novel aspects of mitochondrial biology in early embryos 1.4 Mitochondria and assisted reproduction ................................................................................... 52 1.4.1 Mitochondrial replacement therapy (MRT) ......................................................................... 52 1.4.2 Mitochondrial supplementation .......................................................................................... 55 1.4.3 Antioxidant application ........................................................................................................ 56 1.5 Aims............................................................................................................................................. 58 Chapter 2 – Materials and methods ..................................................................................................... 59 2.1 Bovine in vitro production (IVP) .................................................................................................. 59 2.1.1 General principles ................................................................................................................ 59 2.1.2 Media used for IVP ............................................................................................................... 59 2.1.3 Tissue preparation ............................................................................................................... 63 2.1.4 Isolation and maturation of cumulus oocyte complexes (COCs)