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Control of the Cell Cycle During Meiotic Maturation of Mouse Oocytes

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Control of the Cell Cycle During Meiotic Maturation of Mouse Oocytes Control of the cell cycle during meiotic maturation of mouse oocytes Ibtissem Nabti Department of Cell and Developmental Biology University College London London, UK A thesis submitted for the degree of Doctor of Philosophy April 2010 Declaration The work presented in this thesis was conducted under the supervision of Professor John Carroll in the department of cell and developmental biology, University College London, and Professor Keith Jones in the department of reproductive physiology, University of Newcastle upon Tyne. I, Ibtissem Nabti confirm that the data presented in this thesis is my own and from original studies. Also, this dissertation has not previously been submitted, wholly or in part, to any other university for any degree or diploma. Ibtissem Nabti 1 Abstract The overall aim of the work presented in this thesis is to better understand the molecular mechanisms underlying the progression of the meiotic division in mammalian oocytes. The first goal of this project was to investigate the relative contribution of securin and cyclin B1 to the control of the protease separase during the indeterminate period of metaphase II arrest. I found here that although there are conditions in which either securin or MPF can prevent chromosome disjunction, such as during meiosis I, securin is the predominant inhibitor of separase during metaphase II arrest. Thus, CDK1 pharmacological inhibition as well as antibody inhibition of CDK1/cyclin B1 binding to separase, both failed to induce sister chromatid disjunction. Instead, securin morpholino knockdown induced sister chromatid separation, which could be rescued by injection of securin cRNA. I also examined the effect of CDK1 and MAPK activities on securin stability. Inhibition of both kinases, but not either one alone, using roscovitine and UO126, induced the premature destruction of securin during prometaphase of meiosis I. However, this effect is not dependent on securin phosphorylation, given that mutagenesis of CDK1 and MAPK phosphorylation sites on securin did not affect its stability. Finally, I found that CDK1 and MAPK inhibition also causes the premature degradation of other APC/C substrates, such as cyclin B1 and geminin. Interestingly, only the D-box 2 containing substrates and not Ken-box substrates were subject to such effect. However, this prometaphase I instability of the D-box substrates was apparently not mediated by APC/CCdc20 or APC/CCdh1, given that Cdc20 was also targeted for destruction and morpholino-knockdown of both Cdc20 and Cdh1 failed to rescue securin stability. Also, the inhibition of SCF-TrCP using the dominant negative mutant of its F-box protein (- TrCP) could not restore securin levels, suggesting it is not SCF-TrCP-dependent. Future work will explore whether this effect could be attributed to activation of some meiosis- specific APC/C co-activators or the core APC/C, which was recently reported of being able to directly interact with the D-box containing substrates independently of Cdc20 and Cdh1 (Passmore et al., 2003; Yamano et al., 2004). 3 Publications containing work from this thesis Nabti,I., Reis,A., Levasseur,M., Stemmann,O., and Jones,K.T. (2008). Securin and not CDK1/cyclin B1 regulates sister chromatid disjunction during meiosis II in mouse eggs. Dev. Biol. 321, 379-386. Reis,A., Madgwick,S., Chang,H.Y., Nabti,I., Levasseur,M., and Jones,K.T. (2007). Prometaphase APC(cdh1) activity prevents non-disjunction in mammalian oocytes. Nature Cell Biology 9, 1192-1198. 4 Table of contents List of figures...............................................................................................................8 List of tables.................................................................................................................10 Abbreviations ..............................................................................................................11 Chapter 1: General introduction...............................................................................14 1.1. Overview of meiosis ..............................................................................................15 1.2. The mammalian ovum ...........................................................................................16 1.2.1. The structure of the mammalian ovum .........................................................17 1.2.2. Oogenesis: from primordial germ cells to eggs ............................................17 1.2.3. Folliculogenesis: follicular growth ...............................................................19 1.2.4. Ovulation ......................................................................................................20 1.2.5. Fertilization and egg activation.....................................................................21 1.2.5.1. Fertilization..........................................................................................21 1.2.5.2. Egg activation .....................................................................................23 1.3. Regulation of the meiotic cell cycle in oocytes ....................................................27 1.3.1. Maturation-Promoting Factor (MPF) ...........................................................27 1.3.2. MAPK signalling cascade during meiotic maturation..................................31 1.3.3. The Anaphase Promoting Complex/Cyclosome (APC/C)............................35 1.3.3.1. Regulation of the APC/C during the mitotic cell cycle........................40 1.3.3.2. Regulation of the APC/C during the meiotic cell cycle .......................45 1.4. The molecular mechanisms governing cell cycle transitions in meiosis ...............49 1.4.1. Prophase I arrest in oocytes ..........................................................................49 1.4.2. Resumption of meiosis I ...............................................................................52 1.4.3. Metaphase II arrest in oocytes .....................................................................56 1.4.4. Resumption of meiosis II .............................................................................59 1.5. Chromosome attachment and segregation .............................................................62 1.5.1. Chromosome attachment: the cohesin complex .........................................63 1.5.2. Chromosome segregation: dissolving cohesion...........................................64 1.5.3. Separase .......................................................................................................66 1.5.3.1. Structure and function of separase ......................................................66 1.5.3.2. Separase regulation .............................................................................68 5 Chapter 2: Materials & Methods ..............................................................................72 2.1. Materials ...............................................................................................................72 2.2. Methods .................................................................................................................72 2.2.1. Mouse oocyte collection and culture ............................................................72 2.2.1.1. Collection of germinal vesicle oocytes ...............................................72 2.2.1.2. Collection of metaphase-II arrested eggs ...........................................73 2.2.2. Microinjection and imaging .........................................................................76 2.2.3. Western blotting ...........................................................................................77 2.2.4. Chromosome spreads ...................................................................................79 2.2.5. H1 kinase assay ............................................................................................80 2.2.6. Protein knockdown using morpholino antisense oligos ...............................82 2.2.7. Preparation of RNA for microinjection .......................................................83 2.2.7.1. Bacteria transformation and plasmid DNA purification .....................83 2.2.7.2. Synthesis of RNA..................................................................................84 2.2.7.3. Agarose gel electrophoresis.................................................................85 2.2.8. Site-directed mutagenesis .............................................................................85 2.2.9. Statistical analysis.........................................................................................88 Chapter 3: Securin and not MPF regulates sister chromatid disjunction during meiosis II in mouse eggs ............................................................................................90 3.1. Introduction............................................................................................................90 3.2. Results ...................................................................................................................92 3.2.1. Exogenous cyclin B1 and securin show equal stability during Met II arrest..............................................................................................................92 3.2.2. Endogenous cyclin B1 and securin expression varies after PB1 extrusion ..93 3.2.3. CDK1 inhibition does not induce sister chromatid separation .....................96 3.2.4. Inhibition of separase-CDK1 binding fails to induce sister chromatid separation......................................................................................................99
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