Characterisation of the Spindle Assembly Checkpoint in Mammalian Oocytes Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) Fakultät für Biologie Universität Bielefeld vorgelegt von Edgar-John Vogt Betreuerin: Prof. Dr. Ursula Eichenlaub-Ritter Bielefeld im Oktober 2009 1 Table of Contents 1 ZUSAMMENFASSUNG / SUMMARY ................................................................................ 4 2 INTRODUCTION ................................................................................................................. 12 2.1 OOGENESIS ....................................................................................................................................... 12 2.2 ANEUPLOIDY IN MAMMALIAN OOCYTES ........................................................................................ 14 2.3 CHROMOSOME SEGREGATION IN MEIOSIS ..................................................................................... 16 2.3.1 CHROMOSOME COHESION ............................................................................................................... 17 2.3.2 REGULATION OF COHESIN DEGRADATION ...................................................................................... 18 2.4 SPINDLE ASSEMBLY IN MEIOSIS ...................................................................................................... 20 2.5 KINETOCHORE – THE INTERFACE BETWEEN SPINDLE MICROTUBULES AND CHROMOSOMES ................................................................................................................................ 21 2.5.1 FUNCTIONAL ROLE OF KINETOCHORE FIBERS IS TO POWER CHROMOSOME MOVEMENT ................ 23 2.5.2 FUNCTIONAL ROLES OF POLEWARD MICROTUBULE FLUX .............................................................. 23 2.6 MOTOR PROTEINS INVOLVED IN CHROMOSOME SEGREGATION .................................................. 24 2.6.1 THE REGULATION OF MCAK BY AURORA KINASE B ..................................................................... 26 2.7 THE SPINDLE ASSEMBLY CHECKPOINT (SAC) PROTECTS FROM ANEUPLOIDY ........................... 28 2.7.1 THE SAC SIGNALING PATHWAY ..................................................................................................... 29 2.7.2 THE SAC IN OOCYTES ..................................................................................................................... 34 3 AIM OF THE WORK ........................................................................................................... 36 4 RESULTS ............................................................................................................................... 38 4.1 DISTRIBUTION OF MAD 2 IN MOUSE OOCYTES ................................................................................ 38 4.1.1 MAD 2 LOCALISES TO KINETOCHORES DURING MEIOSIS I AND II.................................................... 38 4.1.2 MAD 2 LOCALISES TO KINETOCHORES FOLLOWING DISTURBANCES IN SPINDLE FORMATION ........ 40 4.2 MAD 2 IS AN ESSENTIAL COMPONENT OF THE SAC ........................................................................ 42 4.2.1 KNOCKDOWN OF MAD 2 BY SI RNA DOES NOT INTERFERE WITH MEIOTIC PROGRESSION TO METAPHASE II, BUT WITH SPINDLE FORMATION AND CHROMOSOME CONGRESSION ..................... 42 4.2.2 MAD 2 IS REQUIRED FOR INHIBITION OF ANAPHASE I PROGRESSION FOLLOWING SPINDLE DEPOLYMERISATION ....................................................................................................................... 44 4.2.3 MAD 2 PREVENTS ANEUPLOIDY FOLLOWING SPINDLE DEPOLYMERISATION .................................. 46 4.3 DISTRIBUTION OF AURORA KINASE B IN MOUSE OOCYTES ........................................................... 50 4.3.1 AURORA KINASE B LOCALISES TO CHROMOSOMES , CENTROMERES AND THE MID -SPINDLE ......... 50 4.4 AURORA KINASE B IN REGULATION OF MATURATION , SPINDLE FORMATION , CHROMATIN CONSTITUTION AND CHIASMA RESOLUTION IN OOCYTES ............................................................. 53 4.4.1 CHEMICAL INHIBITION OF AURORA KINASE BY ZM447439 CAUSES A BLOCK IN CYTOKINESIS AND PROLONGED SPINDLE ASSEMBLY CHECKPOINT (SAC) ........................................................... 53 4.4.2 CHEMICAL INHIBITION OF AURORA KINASE LEADS TO SPINDLE ABERRATIONS AND CHROMOSOME CONGRESSION FAILURE .......................................................................................... 56 4.4.3 EPIGENETIC MODIFICATIONS OF HISTONES FOLLOWING AURORA KINASE INHIBITION .................. 58 4.5 DISTRIBUTION OF MCAK IN MOUSE OOCYTES .............................................................................. 60 4.5.1 MCAK IS RECRUITED TO CHROMOSOME ARMS AFTER GVBD AND LOCALISES TO CENTROMERE DOMAINS FROM PROMETAPHASE I TO METAPHASE II .............................................. 60 4.6 MCAK IS INVOLVED IN THE METAPHASE I-ANAPHASE I TRANSITION ......................................... 64 4.6.1 KNOCKDOWN OF MCAK BY SI RNA INDUCES A MEIOTIC ARREST ................................................. 64 4.6.2 DOUBLE KNOCKDOWN OF MCAK AND MAD 2 OVERCOMES THE MEIOTIC ARREST LEADING TO SPINDLE AND CHROMOSOME CONGRESSION DEFECTS AT METAPHASE II ....................................... 65 4.6.3 DELAY IN ANAPHASE I PROGRESSION AND ANAPHASE LAGGING AFTER CHEMICAL INHIBITION OF AURORA KINASE BY ZM447439 FROM PROMETAPHASE I ......................................................... 70 2 Table of Contents 5 DISCUSSION ......................................................................................................................... 74 5.1 MAD 2 IS LOCALISED AT KINETOCHORES FOR PROPER SAC FUNCTION DURING MEIOSIS I........ 74 5.2 LOSS OF MAD 2 FUNCTION DOES NOT PREDISPOSE MAMMALIAN OOCYTES TO ANEUPLOIDY .... 77 5.3 LOSS OF MAD 2 FUNCTION MAKES MAMMALIAN OOCYTES HIGHLY SUSCEPTIBLE TO ANEUPLOIDY WHEN EXPOSED TO SPINDLE POISONS ...................................................................... 81 5.4 AURORA KINASE B PROMOTES BIPOLAR ATTACHMENT ................................................................ 83 5.5 ROLE OF AURORA KINASE B IN CHIASMA RESOLUTION AND DEPOLYMERISATION OF MICROTUBULES IN THE MIDBODY ................................................................................................... 88 5.6 TENSION FACILITATES BI-ORIENTATION TO SILENCE THE SAC ................................................... 89 5.7 MCAK IS NOT PRIMARILY INVOLVED IN THE CORRECTION OF KINETOCHORE - MICROTUBULE ATTACHMENT ERRORS DURING MEIOSIS I ............................................................ 92 5.8 MCAK IS INVOLVED IN SATISFYING THE SAC IN MEIOSIS I ......................................................... 96 5.9 MEIOSIS -SPECIFIC VERSUS OOCYTE -SPECIFIC FUNCTIONS OF MCAK AND ITS POSSIBLE INVOLVEMENT IN SEXUAL DIMORPHISM IN CHROMOSOME SEGREGATION ............................... 100 5.10 REGULATION OF THE SAC DURING MEIOSIS I IN MAMMALIAN OOCYTES : CONSEQUENCES OF ALTERED EXPRESSION WITH RESPECT TO AGE AND ANEUPLOIDY ........................................ 102 6 MATERIALS AND METHODS ........................................................................................ 104 6.1 CHEMICALS , ENZYMES , AND MATERIALS ..................................................................................... 104 6.2 ANIMALS AND CULTURE OF MOUSE OOCYTES .............................................................................. 104 6.3 MICROINJECTION ........................................................................................................................... 104 6.4 INHIBITOR TREATMENT AND CHECKPOINT ACTIVATION ............................................................ 105 6.5 KNOCKDOWN OF EXPRESSION BY SI RNA ..................................................................................... 105 6.6 QUANTITATIVE REAL -TIME RT-PCR ........................................................................................... 106 6.7 IMMUNOFLUORESCENCE ............................................................................................................... 107 6.8 MICOSCOPY AND IMAGE ACQUISITION ......................................................................................... 109 6.9 C-BANDING FOR CHROMOSOMAL ANALYSIS ................................................................................ 110 6.10 STATISTICS ..................................................................................................................................... 110 7 REFERENCES .................................................................................................................... 111 LIST OF ABBREVIATIONS ................................................................................................... 141 ACKNOWLEDGEMENTS ...................................................................................................... 143 3 Zusammenfassung 1 Zusammenfassung / Summary Die Fehlverteilung von Chromosomen, die auf vorzeitiger Trennung von Chromatiden und Non-Disjunction beruhen, erhöht sich dramatisch in Oozyten der Frau mit zunehmendem Alter und eine geringe, wenn auch nicht so auffällige Erhöhung, beobachtet man ebenfalls bei Eizellen einiger Mausstämme. Untersuchungen zur relativen Menge von Boten-RNA (messenger RNA, mRNA) bei diesen Eizellen führte zu der Hypothese, dass das Altern der Eizelle mit einer veränderten Genexpression, z.B. von Zellzyklus-regulierenden Kinasen, Motorproteinen und Kontrollpunkt-Komponenten, assoziiert ist. Desweiteren wurde vorgeschlagen, dass ein Verlust der Kohesinproteine, welche die Homologe