Mechanisms of mRNA post-transcriptional regulation in mammalian oocytes Karl-Frédéric VIEUX Department of Biology McGill University Montreal, QC, Canada July 2018 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy © Karl-Frédéric Vieux 2018 Table of contents Abstract ......................................................................................................................................... iv Résumé ........................................................................................................................................... v Acknowledgements ...................................................................................................................... vi Publications and author contributions .................................................................................... viii List of figures ................................................................................................................................ ix List of tables ................................................................................................................................. xi List of abbreviations ................................................................................................................... xii Chapter 1 – INTRODUCTION ................................................................................................... 1 I – OOGENESIS ........................................................................................................................ 2 1) In-utero (pre-natal) stages of oogenesis ...................................................................... 2 A. The making of germ cells ....................................................................................... 3 A.1. Repression of intrinsic somatic signals and activation of pluripotency .................. 3 A.2. Epigenetic reprogramming ...................................................................................... 4 B. PGC migration, proliferation and gonad colonization ........................................ 7 B.1. Migration and colonization ..................................................................................... 7 B.2. Proliferation and survival ........................................................................................ 8 C. Making oogonial: sex determination and meiosis ................................................... 11 C.1. Gonad differentiation ............................................................................................ 11 C.2. Initiation of meiosis ............................................................................................... 12 2) Post-natal stages of oogenesis ..................................................................................... 15 A. Oocyte Growth ...................................................................................................... 16 A.1. Primordial follicle recruitment, folliculogenesis and oocyte growth .................... 16 A.2. Granulosa cells support oocyte growth ................................................................. 20 A.3. Meiotic and developmental competence ............................................................... 22 B. Oocyte maturation ................................................................................................ 26 B.1. Maturation: GVBD and chromosome segregation ................................................ 26 B.2. Meiotic arrest......................................................................................................... 27 B.3. Molecular triggers of meiosis ................................................................................ 28 i B.4. CDK1/Cyclin B1 orchestrate maturation .............................................................. 29 C. Postnatal neo-oogenesis……………………….……………………………………….……….... 31 II – THE REGULATION OF GENE EXPRESSION IN THE OOCYTE ........................ 34 1) Gene expression ........................................................................................................... 34 A. Transcription ......................................................................................................... 34 B. Translation............................................................................................................. 37 2) Post-transcriptional mechanisms of translational regulation ................................. 40 A. The role of the 5'-methyl cap and the poly(A) tail in translational regulation 40 A.1. RNA stability ........................................................................................................ 40 A.2. Translational rate................................................................................................... 42 A.3. Deadenylation ....................................................................................................... 45 B. Cis-elements and the recruitment of trans-factors.................................................. 46 B.1. The untranslated regions, the open-reading frame ................................................ 46 B.2. Sequence-mediated regulation .............................................................................. 47 C. RNA localization and microenvironment (RNPs) .............................................. 53 C.1. Characteristics ....................................................................................................... 53 C.2. Formation .............................................................................................................. 54 3) RNA processing and management in the oocyte ...................................................... 57 A. The challenges for gene expression in the oocyte ............................................... 57 A.1. Transcription during Oogenesis ............................................................................ 57 A.2. NSN to SN ............................................................................................................ 57 A.3. Histone modification profile in oocytes ................................................................ 58 B. The need for translational regulation ................................................................. 59 B.1. Meiotic and developmental competence: a complex ordeal ................................. 59 B.2. Spatio-temporal regulation .................................................................................... 61 C. Mechanisms of RNA processing in mammalian oocytes ................................... 62 C.1. Polyadenylation and translational activity ............................................................ 62 C.2. Decapping, RNAi and degradation ....................................................................... 63 C.3. Oocyte RNPs: germ granules, P-bodies and stress granules ................................. 64 D. Rationale and hypothesis ...................................................................................... 66 Chapter 2 – MANUSCRIPT I .................................................................................................... 67 Preface ...................................................................................................................................... 68 ii Significance statement............................................................................................................. 69 Abstract .................................................................................................................................... 71 Introduction ............................................................................................................................. 72 Results ...................................................................................................................................... 75 Discussion ................................................................................................................................. 81 Materials and Methods ........................................................................................................... 84 Acknowledgements .................................................................................................................. 89 Figures ...................................................................................................................................... 90 Supplemental data ................................................................................................................. 103 Chapter 3 – MANUSCRIPT II ................................................................................................ 106 Preface .................................................................................................................................... 107 Significance statement........................................................................................................... 108 Abstract .................................................................................................................................. 110 Introduction ........................................................................................................................... 111 Materials and Methods ......................................................................................................... 114 Results .................................................................................................................................... 119 Discussion