Institute of Reproductive Medicine University of Veterinary Medicine Hannover __________________________________________________________ Analysis of the protein kinase p90rsk during in vitro and in vivo maturation of porcine oocytes and its dependence on the mitogen-activated protein kinase (MAPK) THESIS submitted in partial fulfilment of the requirements for the degree DOCTOR OF PHILOSOPHY -P H.D.- in the field of Reproductive Medicine at the University of Veterinary Medicine Hannover by Carolin Schuon, née Bösebeck, Meppen, Germany Hannover, Germany 2005 Supervisor: Prof. Dr. B. Meinecke Institute of Reproductive Medicine University of Veterinary Medicine Hannover, Foundation, Hannover, Germany Advisory committee: Prof. Dr. B. Meinecke Prof. Dr. G. Breves Institute for Physiology University of Veterinary Medicine Hannover, Foundation, Hannover, Germany Prof. Dr. K.-D. Hinsch BfA-Reha-Klinik Borkum Riff Borkum, Germany External evaluation: Prof. Dr. S. Kölle Institute for Veterinary Anatomy, Histology and Embryology, Justus-Liebig-University, Gießen Oral examination: November 10 th 2005 This thesis was founded by the H. Wilhelm Schaumann Stiftung Es gibt nichts Schöneres als das Mysteriöse. Aus ihm entspringt alle wahre Kunst und Wissenschaft. Albert Einstein This study has been published in part: Conferences (Oral presentation): Bösebeck, C., S. Ebeling u. B. Meinecke Phosphorylation and MAPkinase-accociated activation of p90rsk during in vitro maturation of porcine oocytes. Vet. Med. Austria/ Wien. Tierärztl. Mschr. 91, Suppl. 2, S. 12-13, 2004 Conferences (Poster): Bösebeck, C., S. Ebeling u. B. Meinecke P90rsk and MAPKinase interdependence: Phosphorylation and activation patterns during in vitro maturation of porcine oocytes 38. Jahrestagung der Physiologie und Pathologie der Fortpflanzung, 30. Veterinär- Humanmedizinische Gemeinschaftstagung, 10.-11.02.Zürich Schweizer Archiv für Tierheilkunde 147 , 51, 2005 INDEX A INTRODUCTION ..................................................................9 B LITERATURE REVIEW......................................................12 1 OOCYTE DEVELOPMENT ................................................................. 12 1.1 Oogenesis............................................................................................... 12 1.2 Intercommunication between the oocyte and its surrounding somatic cells ................................................................................................................ 14 2 OOCYTE MATURATION ................................................................... 16 2.1 Meiotic competence................................................................................ 16 2.2 Nuclear maturation.................................................................................. 17 2.2.1 Morphological changes ........................................................................... 17 2.3 Cytoplasmic maturation........................................................................... 19 2.3.1 Morphological changes ........................................................................... 20 2.4 Biochemical changes .............................................................................. 21 2.4.1 Transcription and translation................................................................... 21 2.4.2 Protein synthesis and phosphorylation.................................................... 22 3 CELL CYCLE ASPECTS OF OOCYTE MATURATION ........................... 23 3.1 Maturation-Promoting-Factor (MPF) ....................................................... 24 3.1.1 MPF activation and inactivation .............................................................. 25 3.1.2 Substrates and function of the MPF........................................................ 31 3.2 Mitogen-activated protein kinase (MAPK) ............................................... 32 3.2.1 MAP-Kinase activation and inactivation .................................................. 33 3.2.2 Substrates and function of the MAP-Kinase............................................ 37 3.3 P90rsk..................................................................................................... 41 3.3.1 P90rsk activation and inactivation........................................................... 42 3.3.2 Function and substrates of the p90rsk .................................................... 45 3.4 Interactions between MPF, MAP-Kinase, and p90rsk............................. 47 C MATERIALS AND METHODS ...........................................50 1 CELL CULTURE TECHNIQUES .......................................................... 50 1.1 General handling and precautions .......................................................... 50 1.2 Media ...................................................................................................... 50 1.2.1 Supplements........................................................................................... 51 1.3 Preparation and selection of cumulus-oocyte-complexes ....................... 52 1.4 Cultivation of cumulus-oocyte-complexes ............................................... 53 1.4.1 Regular cultivation conditions ................................................................. 53 1.4.2 Cultivation conditions with addition of an inhibitor................................... 53 1.5 Cytogenetic assessment of oocytes........................................................ 54 1.5.1 Fixation and staining of oocytes.............................................................. 54 1.5.2 Assessment of the nuclear status of oocytes .......................................... 54 2 PROTEIN CHEMICAL TECHNIQUES .................................................. 55 2.1 Preparation and storage of oocytes and cumulus cells........................... 55 2.2 One-dimensional electrophoresis............................................................ 56 2.3 Immunoblot (Western Blot) ..................................................................... 57 INDEX 2.4 Chemiluminescence................................................................................ 57 2.5 P90rsk kinase assay ............................................................................... 59 2.5.1 Kinase assay kit ...................................................................................... 59 2.5.2 ‘In-Gel’ renatured kinase assay............................................................... 61 2.6 Autoradiography...................................................................................... 62 2.7 Quantitative measurement of radioactivity .............................................. 62 3 ANIMAL EXPERIMENT ..................................................................... 62 3.1 Animals ................................................................................................... 62 3.2 Experimental design................................................................................ 63 3.3 Surgical techniques................................................................................. 64 3.4 Preparation of oocytes and cumulus cells............................................... 65 3.5 Analysis of in vivo matured oocytes and cumulus cells........................... 65 D RESULTS ...........................................................................66 1 CONTROL OF MATURATION ............................................................ 66 2 P90RSK IN OOCYTES AND CUMULUS CELLS .................................... 67 3 MAPK IN OOCYTES ......................................................................... 68 4 PHOSPHORYLATION PATTERN OF P90RSK AND MAPK DURING IN VITRO MATURATION ....................................................................... 69 5 INHIBITION EXPERIMENTS .............................................................. 71 6 KINASE ASSAY ............................................................................... 74 6.1 Kinase assay kit...................................................................................... 74 6.2 ‘In-Gel’ renatured kinase assay............................................................... 76 7 ANIMAL EXPERIMENT ..................................................................... 76 E DISCUSSION .....................................................................80 1 KINETICS OF NUCLEAR MATURATION .............................................. 80 1.1 Kinetics of nuclear maturation during in vitro maturation......................... 80 1.2 Kinetics of nuclear maturation during IVM with an addition of U0126 ..... 81 1.3 Kinetics of nuclear maturation during in vivo maturation......................... 81 2 P90RSK AND MAPK IN PORCINE OOCYTES AND CUMULUS CELLS .... 82 2.1 P90rsk in porcine oocytes and cumulus cells.......................................... 82 2.2 MAPK in porcine oocytes and cumulus cells........................................... 82 3 PHOSPHORYLATION PATTERN OF SK AND MAPK ............................. 83 3.1 Phosphorylation pattern of p90rsk during in vitro maturation .................. 83 3.2 Phosphorylation pattern of MAPK during in vitro maturation................... 83 3.3 Phosphorylation pattern of p90rsk during IVM with an addition of U0126 ................................................................................................................ 84 3.4 Phosphorylation pattern of p90rsk during in vivo maturation................... 85 4. P90RSK ACTIVITY DURING IVM WITH AND WITHOUT AN ADDITION OF U0126 ............................................................................................