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Label-Free Multiphoton Microscopy of Lipid Droplets in Oocytes, Eggs and Early Embryos

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Label-Free Multiphoton Microscopy of Lipid Droplets in Oocytes, Eggs and Early Embryos Label-Free Multiphoton Microscopy of Lipid Droplets in Oocytes, Eggs and Early Embryos Josephine Bradley Thesis submitted for the degree of Doctor of Philosophy (Ph.D.) 2016 CARDIFF UNIVERSITY Author’s Declaration I declare that the work comprising this thesis was carried out only through my own investigation, and the views expressed are my own. No portion of this work has been submitted or is currently in canditature for any other degree or award at this, or any other university or place of learning. ICSI was carried out by Dr. Randa Sanusi. Initial imaging of embryo stages was performed using fixed embryos provided by Dr. Judith Eckert. All other experimental work was performed by myself. Josephine Bradley July 2016 i “Anything’s possible, if you’ve got enough nerve” – J.K. Rowling For Milly, without your unyielding strength and courage, I could not have completed this thesis. ii Acknowledgements Thank you to my supervisors, Karl and Paola, who have given me invaluable support, kindness and guidance throughout my project. Two utterly brilliant brains that I have had the pleasure of working with. Thank you to Pete, for all of his support and motivational speeches! Thank you to Iestyn, without whom I would never have been able to complete this project or thesis. Thank you to Wolfgang and Francesco, for the use of your software. Thank you to Randa and Judith for your experimental help. Thank you to all of the friends I have made along the way in the lab, whose support and friendship will always be incredibly valued. A special mention to Mela, my fellow Enabler for making lab and office life ‘tip-top’! To my Sister Wives, my rocks, my army- thank you for your eternal love and support. We have all proven this year, more than any other, that we can get through the toughest of times and come out smiling. To Smiles, and my Ron and Harry (Chandler and Ross), your friendship is unlike any other, and you keep me sane by keeping insane. Never change. To my wonderfully crazy and loving family- Mum, Dad, Tom, Leanne, thank you for simply everything. Everything I have achieved has been achieved with you by my side, and I simply cannot express how utterly grateful I am to you all. Finally, to Simon- the overwhelming love, support and laughter you provide makes life a walk (dance) in the park. iii Abstract Successful development of mammalian oocytes, eggs and embryos relies on the production of ATP by their mitochondria, through metabolism of pyruvate and fatty acids. Imaging of lipid droplets in mammalian eggs has proven difficult due to the invasive, unspecific and unquantitative nature of fluorescent lipophilic stains. Here, we show that coherent anti-Stokes Raman scattering (CARS) microscopy can be used to image lipid droplets in live mouse oocytes and pre-implantation embryos, in a label-free, chemically-specific manner. CARS enables visualisation of lipid droplet distributions, and quantitation of droplet size, number and spatial distribution, notably whilst maintaining their developmental viability. CARS also allows examination of the type of lipids comprising lipid droplets, through means of hyperspectral imaging. It is shown that the chemical composition of these droplets differ in oocytes matured in media supplemented with saturated and unsaturated fatty acids. Correlation of CARS measurements with simultaneous two-photon fluorescence (TPF) microscopy of conventionally used lipid dyes demonstrates only partial correlation, and shows their lack of specificity and unpredictable staining patterns. Dynamic monitoring of lipid metabolism in eggs allows determination of the extent of fatty acid oxidation occurring in mouse oocytes and embryos. Investigation into how inhibition of fatty acid metabolism affects the mitochondrial redox state, membrane potential and ATP level allows further understanding of why fatty acid metabolism is significant for egg and pre-implantation embryo development. Starvation and fatty acid-feeding of oocytes shows that the lipid droplet distribution reflects their level of lipid metabolism, while the detrimental effects of palmitic acid are shown to involve its action at the endoplasmic reticulum SERCA pumps. iv Contents ABSTRACT .............................................................................................................................................. IV CONTENTS .............................................................................................................................................. V LIST OF FIGURES AND TABLES ............................................................................................................. VIII ABBREVIATIONS ................................................................................................................................... XII CHAPTER 1. INTRODUCTION .................................................................................................................. 1 1.1. OOCYTE, EGG AND EMBRYO DEVELOPMENT ................................................................................... 1 1.1.1. Follicular Development and Oogenesis ................................................................................ 1 1.1.2. Oocyte Maturation and Ovulation ....................................................................................... 3 1.1.3. Fertilisation .......................................................................................................................... 5 1.1.4. Embryonic Development ...................................................................................................... 7 1.2. INFERTILITY AND DEVELOPMENTAL QUALITY ........................................................................................... 9 1.2.1. Causes of Infertility ............................................................................................................ 10 1.2.2. Assisted Reproductive Techniques ..................................................................................... 12 1.2.3. Assessment of oocyte, egg and embryo quality ................................................................ 14 1.3. METABOLISM IN EGGS AND EMBRYOS................................................................................................. 17 1.3.1. Mitochondria ..................................................................................................................... 17 1.3.2. Metabolic Substrates ......................................................................................................... 19 1.3.3. The TCA Cycle and Oxidative Phosphorylation .................................................................. 24 1.4. LIPID DROPLET BIOLOGY .................................................................................................................. 30 1.4.1. Lipid Droplet Formation ..................................................................................................... 30 1.4.2. Lipid Droplet Growth and Fusion ....................................................................................... 31 1.4.3. Lipid Droplet Degradation ................................................................................................. 34 1.5. CHARACTERISATION OF LIPID DROPLETS IN MAMMALIAN EGGS ................................................................ 35 1.5.1. Conventional Methods of Investigating LDs and Lipid Content ......................................... 35 1.5.2. LD content of Different Mammalian Species ..................................................................... 37 1.6. MULTIPHOTON MICROSCOPY ........................................................................................................... 40 1.6.1. Raman Spectro-Microscopy ............................................................................................... 41 1.6.2. Coherent anti-Stokes Raman Scattering (CARS) Microscopy ............................................. 44 1.6.3. Two-photon Fluorescence (TPF) Microscopy ..................................................................... 45 1.6.4. Raman and CARS Microscopy of Lipids in Biological Tissues ............................................. 46 1.6.5. Raman and CARS Microscopy of oocytes eggs and embryos ............................................ 47 1.7. SUMMARY AND AIMS ..................................................................................................................... 49 CHAPTER 2. MATERIALS AND METHODS.............................................................................................. 51 2.1. ANIMALS ...................................................................................................................................... 51 2.2. GAMETE COLLECTION AND MANIPULATION .......................................................................................... 51 2.2.1. Female gamete collection .................................................................................................. 51 2.2.2. Male gamete collection ..................................................................................................... 52 2.3. CULTURE CONDITIONS ..................................................................................................................... 52 2.3.1. In vitro Maturation ............................................................................................................ 52 2.3.2. In vitro Fertilisation ............................................................................................................ 52 2.3.3. Embryonic maturation media
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