UNIVERSITY OF SOUTHAMPTON FACULTY OF NATURAL AND ENVIRONMENTAL SCIENCES Biological Sciences Understanding the Meiotic Competence of Oocytes Derived from Oogonial Stem Cells by Larissa Zárate-García Thesis for the degree of Doctor of Philosophy June 2017 UNIVERSITY OF SOUTHAMPTON ABSTRACT FACULTY OF BIOLOGICAL AND ENVIRONMENTAL SCIENCES School of Biological Sciences Thesis for the degree of Doctor of Philosophy UNDERSTANDING THE MEIOTIC COMPETENCE OF OOCYTES DERIVED FROM OOGONIAL STEM CELLS By Larissa Zárate-García This thesis investigates whether Oogonial Stem Cells (OSCs) exist and can be isolated from the adult mouse ovary. It also examines their ability to differentiate into oocyte-like cells in vitro. Transmembrane domains in the DEAD-box polypeptide 4 (DDX4) – the germline marker used in this antibody-based isolation – are examined using in silico protein modelling. The specificity of this antibody to DDX4 is tested in male and female germline cells. It is shown that the DDX4 antibody may have some specificity for DDX4, but the existence of a surface-bound DDX4 is unlikely. OSC-like cells can be isolated from the ovary using the DDX4 antibody by Fluorescence-Activated Cell Sorting (FACS). However, gene expression analysis and protein immunofluorescence show that these cells do not initially express DDX4 or possess germline identity. Despite this, they acquire some pre-meiotic and oocyte-specific markers in culture, including DDX4. Critically, the cells never express meiosis-specific markers even in the presence of meiotic enhancers BMP4 and retinoic acid. It is unlikely that these ovarian cells are being sorted by means of a cell surface DDX4 expression, because another antibody to a larger DDX4 epitope fails to detect DDX4 in isolated cells. These findings highlight that freshly isolated OSCs are not germ stem cells, and are not being isolated by their DDX4 expression, but instead may reflect an artefact of the antibody used or procedure adopted. Altogether this work offers support to the established dogma that the adult ovary is populated at birth by a fixed number of oocytes, and that adult de novo production is a rare or insignificant event. Table of Contents List of Figures .................................................................................................................... vii List of Tables ...................................................................................................................... xi Declaration of Authorship .............................................................................................. xiii Acknowledgements ............................................................................................................ xv Definitions and Abbreviations ....................................................................................... xvii 1. Chapter 1: Introduction ............................................................................................ 1 1.1 General Considerations of the Female Mammalian Germline .............................. 1 1.2 Determination of Primordial Germ Cells in the Mouse Embryo ........................... 3 1.2.1 Role of Bone Morphogenetic Factors .............................................................. 3 1.2.2 Founding Primordial Germ Cells ..................................................................... 7 1.2.3 Further Modifications and Migration Towards the Genital Ridges ................. 8 1.3 Determination of Female Germ Stem Cells ......................................................... 10 1.3.1 Formation of Intercellular Bridges in Germ Cells ......................................... 10 1.3.2 Activation of Secondary Germline-specific Markers ..................................... 11 1.4 Meiosis and Folliculogenesis ............................................................................... 12 1.4.1 Role of Retinoic Acid .................................................................................... 12 1.4.2 Onset of Meiosis ............................................................................................. 15 1.4.3 Prophase I, Chromosome Recombination and First Meiotic Arrest .............. 16 1.4.4 Formation of Germinal Vesicle Oocytes and Perinatal Apoptosis of Female Germ Stem Cells ............................................................................................ 17 1.4.5 Primordial Follicle Assembly and Establishment of Follicle Reserve ........... 18 1.4.6 Primary to Pre-antral Follicle Growth ............................................................ 19 1.4.7 Graafian Follicles, Germinal Vesicle Breakdown and Second Meiotic Arrest 20 1.4.8 Cumulus Expansion, Ovulation and Resumption of Metaphase II ................ 21 1.5 Stem Cells in Animals ......................................................................................... 22 1.5.1 Classification and History of Stem Cells ....................................................... 23 1.5.2 In Vivo Pluripotency/Differentiation Balance in Stem Cells ......................... 24 1.5.3 Methods to Derive Embryonic Stem Cell Cultures ....................................... 26 1.5.4 In Vivo Maintenance of Pluripotency, Proliferation and Self-renewal .......... 28 1.5.5 In Vitro Differentiation of Embryonic and Induced Pluripotent Stem Cells .. 30 1.5.6 Oocyte-like cells from Embryonic and Induced Pluripotent Stem Cells ....... 30 1.6 Germ Stem Cells in the Adult Mammal Ovary ................................................... 31 i 1.6.1 Early Studies on De Novo Oogenesis ............................................................ 31 1.6.2 Early Mathematical Models of Follicular Dynamics ..................................... 33 1.6.3 Modern Mathematical Models of Follicular Dynamics ................................ 35 1.7 The Bone Marrow as a Source of Germ Stem Cells ........................................... 37 1.8 Oocytes Obtained from Adult Oogonial Stem Cells ........................................... 40 1.8.1 Ddx4-based Method for OSCs Isolation ........................................................ 40 1.8.2 Ifitm3- and Pou5f1-based Methods for OSCs Isolation ................................. 43 1.8.3 Studies Not Supporting the Existence of OSCs ............................................ 44 1.9 Thesis Aims ......................................................................................................... 45 1.9.1 Do OSCs exist? ............................................................................................. 45 1.9.2 Can they provide us with oocytes? ................................................................ 46 1.9.3 Are these oocytes meiotically competent cells? ............................................ 46 2. Chapter 2: General Materials and Methods ........................................................ 49 2.1 Mice ..................................................................................................................... 49 2.1.1 Ethics ............................................................................................................. 49 2.1.2 Mice ............................................................................................................... 49 2.1.3 Hormonal Priming ......................................................................................... 49 2.1.4 Tissue Collection ........................................................................................... 50 2.2 Oocyte Handling and Collection ......................................................................... 50 2.2.1 Manufacture of Mouth Pipetting System ...................................................... 50 2.2.2 GV Oocyte Handling and Collection ............................................................. 51 2.2.3 MII Oocyte Handling and Collection ............................................................ 51 2.2.4 Enzymatic Digestion of Organs ..................................................................... 51 2.3 Gene Expression Analysis ................................................................................... 52 2.3.1 Total RNA Extraction from Oocytes ............................................................. 52 2.3.2 Total RNA Extraction from Tissue and Cell Suspensions ............................ 52 2.3.3 Complementary DNA Synthesis .................................................................... 53 2.3.4 Real-Time Polymerase Chain Reaction ......................................................... 53 2.3.5 Agarose Gel Electrophoresis .......................................................................... 55 2.4 Western Blot ........................................................................................................ 55 2.4.1 Protein Extraction and Measurement ............................................................. 55 2.4.2 SDS-PAGE Gel Running ............................................................................... 55 2.4.3 Transfer of Proteins to Nitrocellulose Membranes ........................................ 56 2.5 Cell Culture ......................................................................................................... 56 ii 2.5.1 Control Culture Media (CM) .......................................................................... 56 2.5.2 OSC Culture Media (OSCM) ......................................................................... 56 2.5.3 MEF Growth Culture Medium ....................................................................... 57 2.5.4 Coating of Culture Dish Surfaces