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. 2010 Dec 15;123(Pt 24):4292-300.
doi: 10.1242/jcs.067447.

The chromosome passenger complex is required for fidelity of chromosome transmission and cytokinesis in meiosis of mouse oocytes

Bedra Sharif  1 , Jie Na, Karin Lykke-Hartmann, Stephen H McLaughlin, Ernest Laue, David M Glover, Magdalena Zernicka-Goetz
Affiliations

The chromosome passenger complex is required for fidelity of chromosome transmission and cytokinesis in meiosis of mouse oocytes

Bedra Sharif et al. J Cell Sci. .

Abstract

The existence of two forms of the chromosome passenger complex (CPC) in the mammalian oocyte has meant that its role in female meiosis has remained unclear. Here we use loss- and gain-of function approaches to assess the meiotic functions of one of the shared components of these complexes, INCENP, and of the variable kinase subunits, Aurora B or Aurora C. We show that either the depletion of INCENP or the combined inhibition of Aurora kinases B and C activates the anaphase-promoting complex or cyclosome (APC/C) before chromosomes have properly congressed in meiosis I and also prevents cytokinesis and hence extrusion of the first polar body. Overexpression of Aurora C also advances APC/C activation and results in cytokinesis failure in a high proportion of oocytes, indicative of a dominant effect on CPC function. Together, this points to roles for the meiotic CPC in functions similar to the mitotic roles of the complex: correcting chromosome attachment to microtubules, facilitating the spindle-assembly checkpoint (SAC) function and enabling cytokinesis. Surprisingly, overexpression of Aurora B leads to a failure of APC/C activation, stabilization of securin and consequently a failure of chiasmate chromosomes to resolve - a dominant phenotype that is completely suppressed by depletion of INCENP. Taken together with the differential distribution of Aurora proteins B and C on chiasmate chromosomes, this points to differential functions of the two forms of CPC in regulating the separation of homologous chromosomes in meiosis I.

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Figures

Fig. 1.
Fig. 1.
Depletion of INCENP leads to misaligned chromosomes, advancement of APC/C activation and prevents cytokinesis. (A,B) Green fluorescence of Securin–GFP and a digital interference contrast (DIC) image of oocytes is presented for a time-series of a control oocyte injected with an irrelevant siRNA (A) or siRNA against INCENP. (B). The scale bar for all time-lapse images represents 50 μm. (A′,B′) Quantitation of total fluorescence of Securin–GFP in three representative oocytes showing degradation profiles for RNAi control (blue) and INCENP-RNAi-treated (red) oocytes. ‘RF’ in this and subsequent figures refers to the relative fluorescence of Securin–GFP (see the Materials and Methods section and the supplementary material for a description of the kinetic parameters). (C,D) Maximal APC/C activity and time taken to reach maximal activity, respectively, for every oocyte in the indicated groups. The minus sign (−) denotes oocytes otherwise untreated except for the injection of mRNA encoding Securin–GFP (green). Oocytes labeled ‘control’ are treated with an irrelevant siRNA. (E) Oocytes stained to reveal DNA (red) and microtubules (green) for controls in meiosis I (MI) and meiosis II (MII; upper row) and INCENP RNAi treated at MI and at 10 hours having failed to extrude the PB (lower row). Preparations of spread chromosomes are shown alongside each stained panel. The scale bar for the immunofluorescence images in A and B represents 10 μm and, for the chromosome spreads, 20 μm. (F) Knockdown of INCENP transcripts assessed by qrtPCR for the indicated concentrations of siRNA.
Fig. 2.
Fig. 2.
Pharmacological inhibition of Aurora kinases B and C phenocopies INCENP depletion. (A,B) Green fluorescence of Securin–GFP and a DIC image of oocytes are presented for a time-series of control oocytes (A) or oocytes treated with AZD1152 (B). The scale bar for all time-lapse images represents 50 μm. Arrows point to the initiation of cytokinesis, followed by subsequent regression of the PB. (A′,B′) Quantitation of total fluorescence of Securin–GFP in three representative oocytes, showing degradation profiles for control oocytes (green) and AZD1152-treated oocytes (blue). (C,D) Maximal APC/C activity and time taken to reach maximal activity, respectively, for every oocyte in the indicated groups. (E) Oocytes stained to reveal DNA (red) and microtubules (green) at 10 hours post GVBD. Preparations of spread chromosomes are shown alongside each stained panel. The scale bar for the immunofluorescence images in E and F represent 5 μm.
Fig. 3.
Fig. 3.
Overexpression of AurC mRNA prevents cytokinesis; overexpression of AurB mRNA prevents APC/C activation and leads to arrest with chiasmate dyads. Green fluorescence of Securin–GFP and a DIC image of oocytes are presented for each series in which images are presented at the indicated time-points. (A) Time-lapse series of Securin in three representative control oocytes. (A′) Quantitation of total fluorescence of Securin–GFP in the three oocytes shown in (A). (B) Time-lapse series of Securin in three representative oocytes into which AurC mRNA had been injected at 0.5 μg/μl. (B′) Quantitation of total fluorescence of Securin–GFP in the three oocytes shown in (B). Note that Securin destruction occurs in all oocytes, although cytokinesis [PB extrusion (PBE)] fails. 58% of oocytes degraded Securin and extruded PBs (black data-points in panels D and E). (C) Time-lapse series of Securin in three representative oocytes into which AurB mRNA had been injected at 0.5 μg/μl. The scale bar referring to all time-lapse images represents 100 μm. (C′) Quantitation of total fluorescence of Securin–GFP in the three oocytes shown in C. Note the slowed destruction of Securin in these oocytes that arrested in MI. 44% of oocytes progressed to MII and showed destruction of Securin (red data-points in panels D and E). (D) Values of maximal APC/C activity and (E) time to maximal activity post GVBD for every oocyte in all groups analysed. Oocytes expressing synthetic AurC mRNA have been divided into two categories, depending upon whether they failed to exclude a polar body (no PBE; purple data-points) or succeeded (PBE; black data-points). Oocytes expressing synthetic AurB mRNA have also been divided into two categories, depending upon whether they arrested in MI (MI; blue data-points) or progressed to MII (MII; red data-points). (F) Oocytes injected with synthetic mRNA encoding HA-tagged Aurora kinases B or C, fixed 10 hours post GVBD and stained to reveal HA–Aurora-B or HA–Aurora-C (green), tubulin (red) and DNA (blue). Oocytes overexpressing HA–Aurora-C have a meiosis II spindle with some misaligned chromosomes. Oocytes overexpressing HA–Aurora-B have a meiosis I spindle with a chiasmate arrangement of dyads. (G) Preparations of chromosome spreads from the indicated number of oocytes that had failed to extrude a PB as a result of expressing exogenous Aurora C (note the 40 univalents), and preparations of chromosome spreads from the indicated number of oocytes that had failed to extrude a PB as a result of expressing exogenous Aurora B (note the 20 chiasmate bivalents). The scale bar for the immunofluorescence images represents 10 μm and, for the chromosome spreads, 20 μm.
Fig. 4.
Fig. 4.
Depletion of INCENP prevents localization of exogenous Aurora B and suppresses the dominant phenotype of its expression. (A) Time-lapse series showing chromatin (histone-H2B–RFP; red) from oocytes injected with synthetic mRNA encoding Aurora-B–GFP (green). Note the localization of Aurora B to paired homologues held in a chiasmate configuration for at least 10 hours. (B) Time-lapse series showing chromatin (histone-H2B–RFP; red) from INCENP-RNAi-treated oocytes injected with synthetic mRNA encoding Aurora-B–GFP (green). Note the failure of Aurora B to localize to chromosomes or the central spindle and the completion of meiosis I, as described in the text, in the absence of cytokinesis. Scale bar: 10 μm.
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