1. Volume 2, Issue 5, Pages 1077-1087 (November 2012)
Cyclin A2 Is Required for Sister Chromatid Segregation, But Not Separase Control, in Mouse Oocyte Meiosis 
Sandra A. Touati, Damien Cladière, Lisa M. Lister, Ioanna Leontiou, Jean-Philippe Chambon, Ahmed Rattani, Franziska Böttger, Olaf Stemmann, Kim Nasmyth, Mary Herbert, Katja Wassmann 
Cell Reports 
Volume 2, Issue 5, Pages (November 2012)
DOI: /j.celrep
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2. Cell Reports 2012 2, 1077-1087DOI: (10.1016/j.celrep.2012.10.002)
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3. Figure 1
Inhibition of Endogenous Cyclin A Delays GVBD and Prevents Metaphase-to-Anaphase Transition in Meiosis II
(A) Endogenous cyclin A can be detected by western blot analysis in GV and metaphase II oocytes. The number of oocytes used per lane is indicated.
(B) Cyclin A western blot in meiosis I.
(C) Time of GVBD after release in oocytes injected with indicated antibodies (m, mouse; r, rabbit), or controls.
(D) Metaphase II chromosome spreads of oocytes injected in GV stage with anti-cyclin A antibodies as indicated. Kinetochores are stained with CREST (green), chromosomes with propidium iodide (red).
(E) Oocytes were injected with anti-cyclin A antibodies in metaphase II and activated. Chromosome spreads as in (D) after anaphase II, 50 min after activation.
(F) Percentage of sister separation observed in (E). The antibody injection results were assessed by variance analysis and found to be highly significant (∗∗∗p < 0.001) when compared as indicated.
(G) Representative quantitations of cyclin B1-GFP fluorescence levels in meiosis II oocytes that were injected with anti-cyclin A antibodies in metaphase II, and activated as indicated.
(H) Selected time frames of representative movies of oocytes expressing histone H2B-RFP that were injected in metaphase II with control or anti-cyclin A antibodies.
See also Figure S1.
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4. Figure 2
Inhibition of Overall Cdk Activity Prevents Sister Chromatid Separation in Meiosis II, whereas Expression of Stable Cyclin A2 Induces Sister Separation in Meiosis I
(A) Selected time frames of a representative movie of oocytes undergoing metaphase-to-anaphase transition of meiosis II in the presence of roscovitine where indicated. Chromosomes are visualized with H2B-RFP. Time after activation is indicated in minutes. Scale bar represents 20 μm.
(B) Representative chromosome spreads after activation, in the presence of roscovitine where indicated. Kinetochores are stained with CREST (green), and chromosomes with propidium iodide (red). CREST dots are still paired in the presence of roscovitine, indicating that sister chromatids have not been separated. Scale bar represents 5 μm.
(C) Representative quantitations of cyclin B1-GFP fluorescence levels in meiosis II oocytes treated with roscovitine and activated where indicated.
(D) Percentage of oocytes injected with the indicated mRNAs that underwent spontaneous GVBD within 90 min after injection in dbcAmp containing medium.
(E) Percentage of oocytes injected with the indicated mRNAs that extruded PBs.
(F) Chromosome spreads 16 hr after GVBD of oocytes expressing the indicated cyclin mutants. Kinetochores are stained with CREST (green), and chromosomes with propidium iodide (red). The arrows mark examples of single sister chromatids. Scale bar represents 5 μm.
(G) Live imaging of oocytes that have been coinjected with histone H2B-RFP to visualize chromosomes, β-tubulin-GFP to visualize spindles, and ΔNCyclin A2 where indicated. Selected time frames (every 40 min) of collapsed z-sections (ten sections, 3 μm steps), from a representative spinning disk confocal movie of a control and a ΔNCyclin A2-expressing oocyte are shown. Time points after GVBD are indicated. Scale bar represents 20 μm.
See also Figure S2.
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5. Figure 3
Stable Cyclin A2 Induced Precocious Sister Chromatid Separation in Meiosis I
(A) Chromosome spreads of oocytes injected with ΔNCyclin A2 mRNAs where indicated. Spreads were performed at the indicated time points after GVBD, at metaphase-to-anaphase transition. At GVBD + 8 hr, an example of an anaphase I is shown. Kinetochores are stained with CREST (green), and chromosomes with propidium iodide (red). Scale bar: 5 μm. For number of oocytes analyzed see (C).
(B) Percentage of ΔNCyclin A2 expressing oocytes that had undergone the metaphase-to-anaphase transition of meiosis I harboring the indicated proportion of separated sister chromatids at 8, 15, or 16 hr after GVBD. Control oocytes extruded PBs with normal timing at 8–9 hr after GVBD (not shown).
(C) Percentage of oocytes at the indicated time points after GVBD that were in metaphase or anaphase, as determined by chromosome spreads. Important note: all ΔNCyclin A2-expressing oocytes contained at least 20% clearly visible separated single sister chromatids.
(D) Percentage of oocytes expressing low or high levels of ΔNCyclin A2-GFP with the indicated proportion of separated sister chromatids, as determined by chromosome spreads at GVBD + 16 hr.
See also Figure S3 and Movies S1, and S2, and S3.
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6. Figure 4
Only Stable Cyclin A2 Induces Sister Separation in a Separase-Dependent Manner in Meiosis I
(A) Chromosome spreads 16 hr after GVBD of oocytes expressing ΔNCyclin A2 and ΔDBSecurin as indicated. Kinetochores are stained with CREST (green), and chromosomes with propidium iodide (red). The graph below shows the percentage of oocytes with separated bivalents (either individual chromosomes, or sister chromatids), or bivalents that are not separated.
(B) Chromosome spreads as in (A) 16 hr after GVBD of oocytes expressing ΔDBCyclin B1, and GFP-wt separase or GFP-PM-separase as indicated. The graph below shows the percentage of oocytes with bivalents, or dyads. See also Figure S1 and Movies S1 and S3.
(C) Chromosome spreads of oocytes with the indicated genotype injected with the designated mRNAs, 14–16 hr after GVBD. The number of oocytes analyzed is indicated. Dashed lines show where images of one chromosome spread were assembled to minimize picture size.
Scale bars represent 5 μm. See also Figure S4 and Movie S4.
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7. Figure 5
Cyclin A Is Localized to Centromeres at All Stages of Meiosis Except Anaphase I
(A) Chromosome spreads were performed at the indicated times after GVBD and stained with CREST (green) and anti-cyclin A (red) antibodies. Chromosomes were stained with Hoechst (blue). Note: the CREST signal colocalizes with cyclin A2 at all stages except anaphase I.
(B) Spreads as in (A) at GVBD + 4 hr. Antibody control with epitope-blocked cyclin A antibody.
(C) Localization of ΔNCyclin A2-GFP at centromeres in anaphase I, shown by live imaging, at the indicated time points after GVBD. Chromosomes are visualized with H2B-RFP. Overlays of 15 z-sections (0.7 μm steps) of the individual channels and a merge are shown. (D) One individual z-section of each channel and their merge as in (C). The arrow indicates a bivalent chromosome with cyclin A staining just before anaphase I onset. A total of 16 control oocytes and 24 ΔNCyclin A2-GFP-expressing oocytes were repeatedly analyzed from GVBD + 7.5 hr to GVBD + 9 hr and again 16 hr after GVBD.
Scale bars represent 5 μm. See also Figure S5.
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8. Figure S1
Cyclin A2 Is Present in Oocytes and Inhibiting its Function by Antibody Injections Prevents Sister Chromatid Segregation in Meiosis I, Related to Figure 1
(A) Scheme of meiotic divisions and chromosome segregation in mouse oocytes. (Note: all mouse chromosomes are telocentric.) GV, germinal vesicle stage; GVBD, germinal vesicle breakdown. Cohesin is depicted in the form of black bars, a single Chiasma in the form of an X.
(B) Whole cyclin A western blots shown in Figures 1A and 1B. A control western blot from unsynchronized cell extracts that was performed at the same time and under the same conditions is equally shown. The arrows indicate the cyclin A signal.
(C) Metaphase II antibody and control injections for mouse and rabbit cyclin A antibodies with epitope-blocked and trypsinated antibodies, cyclin E1 antibody, Flag antibodies, and unspecific IgGs. Concentrations were at around 0.5 mg/ ml. All injections were statistically tested using variance analysis and differences of the control injections and cyclin A antibody injections were found to be significant (∗∗p < 0.01).
(D) Chromosome spreads after activation of control, cyclin A, or cyclin E antibody injected oocytes.
(E) Both cyclin A antibodies (mouse ab38 and rabbit H432) immunoprecipitate endogenous cyclin A from unsynchronized mouse cell extracts. For Western blot detection rabbit ab2097 antibody was used.
(F) Kinase assays with immunoprecipitated cyclin A as in (E). Cyclin A immunoprecipitated with antibody (ab38) does not allow histone H1 phosphorylation, whereas cyclin A immunoprecipitated with antibody H432 does phosphorylate histone H1 in vitro.
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9. Figure S2
Stable Cyclin A2 Prevents Exit from Meiosis I, but Unlike Wild-type Cyclin A2, Does Not Prevent Metaphase-to Anaphase Transition, Related to Figure 2
(A) PB extrusion rate of oocytes injected with wild-type cyclin A2 16 hr after GVBD.
(B) Oocytes injected with wild-type cyclin A2 that did not extrude a PB were found arrested with homologous chromosomes aligned at the metaphase plate.
(C) RFP channel of a representative movie in Figure 2G. Shown are time points of 30 min just before and after anaphase onset, as a collapsed image of 10 z-sections (3 μm steps). Scale bar represents 20 μm.
(D) Examples of fixed control or ΔNCyclin A2 expressing oocytes in metaphase I. Chromosomes were stained with Propidium iodide. (Shown is a collapsed image of 15 z-sections of 1 μm steps). Scale bar represents 5 μm.
(E and F) One representative quantitation of YFP-securin (E) and cyclin B1-GFP (F) in a ΔNCyclin A2 expressing oocyte as indicated. PB extrusion in the control oocyte and the number of oocytes analyzed in (E) and (F) is indicated. a.u., arbitrary units.
(G) Examples of chromosome spreads of control and ΔNCyclin A2 KD expressing oocytes, stained as in Figure 2F. The number of oocytes analyzed is indicated. Scale bar represents 5 μm.
(H) Still pictures of collapsed z-sections (2 μm steps, 12 sections) of a Spinning Disk Confocal movie of ΔNCyclin A2, H2B-RFP, and β-tubulin-GFP injected oocytes treated with Roscovitine. Time after GVBD is indicated. Scale bar represents 20 μm.
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10. Figure S3
Precocious Sister Separation Is Not due to Lower Expression Levels of Stable Cyclin A2 Compared to Wild-type Cyclin A or Stable Cyclin B1, Related to Figure 3
(A) Levels of endogenous cyclin A compared to the exogenous expression of wild-type and stable cyclin A2. Antibody controls are included. All acquisitions were done with exactly the same settings and at the same time, images were not treated. Scale bar represents 20 μm. Quantitations of cyclin A staining as described in Experimental Procedures.
(B) Quantitation of GFP fluorescence signal at the start of the experiment in GV and 6 hr after GVBD in prometaphase I. Oocytes were co-injected with H2B-RFP, and either ΔNCyclin A2-GFP or ΔDBCyclin B1-GFP. Fluorescence intensities were compared using the two-sample t test.
(C) Phenotypes observed by chromosome spreads in oocytes 16 hr after GVBD that have been injected with the indicated mRNAs at 0.1 μg/μl, or diluted 1:10.
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11. Figure S4 Only Cyclin B1 Can Inhibit Separase, Related to Figure 4
(A) Oocytes were injected with Histone H2B-RFP alone, or with ΔDBCyclin B1 with or without GFP-PM-separase as indicated (GFP was used to control for separase expression, an overlay of the GFP and RFP channel is shown). The movie was started at GVBD + 6h, shown are 40 min time points on a Nikon TE2000E microscope with PrecisExite High Power LED Fluorescence (overlay of 8 z-sections of 2 μm steps).
(B) ZZ-Tev4-tagged separase (WT or PM), stabilized (ΔDB) cyclin B1 fused with full-length eYFP and/or ΔDBCyclin A2 fused with a C-terminal eYFP fragment were co-expressed in Hek293T cells as indicated. Separase was purified from prometaphase arrested cells by immunoaffinity chromatography and Tev-protease elution. Samples, in which Tev-protease was added prior to immunoaffinity purification served as negative controls (lanes 8, 11, and 13). Both cyclins were simultaneously detected on the same membrane with an anti-eG/YFP antibody. Note that despite higher accumulation of cyclin A2 over -B1 upon co-expression (lanes 1-3), only the latter associates with separase specifically (lanes 8–10).
(C) Metaphase II oocytes of the indicated genotype injected with control mRNA or ΔNCyclin A2. Spreads were performed 2 hr after injections, without activation.
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12. Figure S5
Monopolar Attachment of Kinetochores and Localization of Sgo2 and PP2A Are Not Affected in Oocytes Expressing ΔNCyclin A2, but Cyclin A May Induce Sister Separation through a PP2A-Dependent Mechanism, Related to Figure 5
(A) Oocytes expressing ΔNCyclin A2 where indicated were analyzed by chromosome spreads in metaphase I. Kinetochores are stained with CREST (green), chromosomes with Propidium iodide (red). A magnification of one bivalent each is shown below. Scale bar represents 5 μm.
(B) Whole mount immunofluorescence of metaphase I oocytes expressing ΔNCyclin A2 where indicated. Cold stable microtubules were stained with anti-β-tubulin antibody (green), kinetochores with CREST (red), and chromosomes with Hoechst (blue).
(C) Oocytes expressing ΔNCyclin A2 or ΔDBCyclin B1 where indicated, analyzed by chromosome spreads at GVBD + 9h30. Oocytes were maintained in metaphase I with MG132 where indicated. Spreads were stained with Hoechst (DNA, blue), and either CREST (green), and anti-Sgo2 (red); or CREST (red), and anti-PP2A (catalytic subunit, green) antibodies. The triple merge is shown above, the merge of Sgo2 or PP2A with DNA is shown below. Scale bar represents 5 μm.
(D) Control or cyclin A antibody injected oocytes were treated with okadaic acid for 5 hr where indicated. The percentage of oocytes with separated sister chromatids was shown to be highly significant for each condition compared as indicated by variance analysis (∗∗∗p < 0.001).
(E) Cyclin A staining on chromosome spreads at the indicated meiotic stages, without CREST staining to show that there is no cross-reactivity of secondary antibodies.
(F) Additional cyclin A anaphase I stainings (see Figure 5A). Cyclin A (green), chromosomes (Propidium iodide, red). Scale bar represents 5 μm.
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