Loosing one allele of mad2 affects the fidelity of chromosome segregation in meiosis I and fertility.

<p>(A) Chromosome spreads were performed in metaphase I (6 hours after GVBD) and metaphase II (2 hours after PBE). Chromosomes are stained with propidium iodide (red), and kinetochores with CREST serum <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001165#pone.0001165-Furuta1" target="_blank">[30]</a>. Shown are one euploid and one aneuploid metaphase II spread of <i>mad2+/−</i> oocytes. (B) Quantification of aneuploid oocytes in metaphase I and II harboring more or less than 20 chromosomes. n indicates the number of interpretable metaphase spreads obtained. 8 <i>mad2+/−</i> mice were analysed in four independent experiments. (C) 21% of female mad2+/− mice are sterile. (D) <i>mad2−/−</i> embryos are not viable and therefore the litter size is expected to be 25% lower in the heterozygote crosses. The average litter size from <i>mad2+/−</i> crosses is reduced to 43% compared to wild type crosses, and to 57,6% compared to the expected litter size.</p

Cyclin B and Securin are degraded before PBE in <i>mad2+/−</i> oocytes.

<p>(A) Time lapse video microscopy to establish the time of anaphase onset and PBE. Anaphase onset can sometimes be observed in the timepoint before PBE. (B) Quantitation of GFP-Cyclin B in <i>mad2+/+</i> and <i>mad2+/−</i> oocytes relative to anaphase onset and PBE. Live video analysis shows that GFP-Cyclin B (quantifications of the fluorescence signal are shown as a graph, arbitrary units set to maximal levels of 100) reaches its lowest levels before PBE. Timepoints were taken every 20 minutes, chromosomes were labelled with Hoechst. A representative oocyte is shown and the number of successfully analyzed oocytes is indicated. No differences between anaphase onset, PBE and GFP-Cyclin B degradation were detected between control and <i>mad2+/−</i> oocytes. (C) Quantifications of Securin-YFP fluorescence signal intensities in <i>mad2+/+</i> and <i>mad2+/−</i> oocytes relative to anaphase onset and PBE, as described in (B). No differences between anaphase onset, PBE and Securin-YFP degradation were detected. (D) Kinase assays to assess MPF activity during meiosis I in <i>mad2+/+</i> and <i>mad2+/−</i> oocytes. Histone H1 (H1) was used as a substrate. Control (ctrl): GVBD+8 h (-PB) without substrate addition. (E) Endogenous Securin levels in <i>mad2+/+</i> and <i>mad2+/−</i> oocytes 3 hours after GVBD. 20 oocytes each were used.</p

Anaphase onset is accelerated in <i>mad2+/−</i> mice.

<p>(A) Entry into the first meiotic division in mouse oocytes is induced by removing dbcAMP from the culture medium. Synchronized oocytes undergo GVBD around 1,5 hours after release, and extrude a PB around 8–9 hours after GVBD. MPF (M-phase promoting factor) activity increases from GVBD until metaphase I, drops when the first PB is extruded, and increases again as oocytes progress into meiosis II. (B) Time lapse video microscopy of oocytes with the indicated phenotype undergoing the first meiotic division. Chromosomes were labelled with Hoechst. Anaphase onset and PBE were observed. Only movies with at least 80% of oocytes extruding a PB at times comparable to control oocytes in the incubator without exposure to Hoechst excitation light were used. (C) Cumulative times of PBE and (D) distribution of PBE (same data set) in <i>mad2+/+</i> (n = 53) and <i>mad2+/−</i> (n = 70) oocytes. The peak time average of PBE in <i>mad2+/−</i> oocytes is significantly earlier (33 min) than in <i>mad2+/+</i> oocytes (497 min and 530 min respectively, p<0,01 with both the T and the U test, p value of the T test (2 tail, type2) = 0,00232, p value of the U test (2 tail) = 0,00572). The results of three independent experiments are shown.</p

SAC control is impaired in <i>mad2+/−</i> oocytes.

<p>(A) Schematic outline of the experimental procedure. Nocodazole was used at a final concentration of 200 nM. (B) Chromosome spreads in metaphase II. Shown are one euploid <i>mad2+/+</i> oocyte and one aneuploid <i>mad2+/−</i> oocyte harboring 21 chromosomes. Chromosomes are stained with propidium iodide (red), and kinetochores with CREST serum <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001165#pone.0001165-Furuta1" target="_blank">[30]</a>. (C) Percentage of aneuploid oocytes, as determined by metaphase II spreads and kinetochore staining. 4 independent experiments using 4 mice of each genotype were performed. (D, E) Time lapse video microscopy of oocytes expressing Securin-YFP as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001165#pone-0001165-g003" target="_blank">Figure 3</a>. The graphs show the fluorescence measurements of Securin-YFP at the indicated times after GVBD, and the times of anaphase onset and PBE. Timepoints were taken every 20 minutes, chromosomes were labelled with Hoechst. A representative oocyte is shown and the number of successfully analyzed oocytes is indicated. (D) Oocytes treated with nocodazole. (E) Oocytes released from a 2 hour nocodazole arrest as in (A–C).</p

Overexpression of Mad2 leads to chromosome missegregations in meiosis I.

<p>(A) Schematic outline of the experimental procedure. Oocytes arrested in GV stage are injected with mRNA encoding GFP-tagged Mad2, and released to undergo meiotic maturation. Oocytes that extrude a PB are examined for GFP-Mad2 expression by fluorecence microscopy, and those expressing detectable levels of Mad2 are used for chromosome spreads. The experiment was repeated 4 times. (B) Percentage of oocytes bypassing the metaphase I arrest with moderate GFP-Mad2 expression. (C) Percentage of aneuploidies in metaphase II (gain or loss of one or more univalent chromosomes) of oocytes injected with injection buffer (control), or GFP-Mad2 mRNA (same as in (B)). (D) Model to explain the observed phenotypes after changing Mad2 levels. See text for details.</p

Epistasis analysis between OSD1, TAM, and TDM.

<p>Meiotic spreads of (A to C) <i>osd1-3/tam-2</i> double mutant, (D to F) <i>osd1-3/tdm-3</i> double mutant, (G to L) tam-2/tdm-3 double mutant and (M to O) <i>osd1-3/tam-2/tdm-3</i> triple mutant. Scale bar = 10 µM.</p

OSD1 and UVI4 are synthetically essential for female gametogenesis and somatic growth.

<p>(A and B) Cleared female gametophyte. Nuclei and nucleoli have been artificially highlighted in blue and red, respectively. (A) Wild type at the 8 nuclei stage. A nucleolus is visible in the center of each nucleus (arrows) (B) A female gametophyte in the <i>uvi4+/− osd1-2</i>+/− plant at a comparable stage, showing a single giant nucleus with a massive nucleolus. (C to D) Double staining of female gametophytes with DAPI and propidium iodide. The DNA is stained in blue and the nucleoli appear in red. (C) Wild type at the 8 nuclei stage (arrows) (D) One female gametophyte in <i>uvi4+/− osd1-2</i>+/− containing a single large nucleus with a great amount of DNA, and a large nucleolus. Scale bar = 10 µM. (E) A 5 weeks old <i>osd1/uvi4</i> double mutant. Scale bar = 5 mm.</p

Structural comparison of OSD1 and Mes1 proteins.

<p>OSD1 and Mes1 share co-aligned putative APC/C interacting domains.</p

TAMΔD provokes the entry into a third meiotic division.

<p>(A to F) Meiotic spreads of wild type plants transformed by TAMΔD. (A) Pachytene. (B) Metaphase I. (C) Metaphase II. (D) late anaphase II (E) Aberrant third division (F) Resulting telophase III with seven nuclei. (G) Meiotic product stained by toluidine blue. Scale bar = 10 µM. (H) Alexander staining of an anther, showing the complete absence of pollen grains (Compare to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002865#pgen.1002865.s005" target="_blank">Figure S5A</a>). Scale bar = 100 µM.</p

Complementation test of <i>osd1-3</i> by wild-type and mutated versions of OSD1.

<p>Male meiotic products stained by toluidine blue: (A) a dyad of spores from the <i>osd1-3</i> mutant. (B) A tetrad of spores from <i>osd1</i> complemented by the wild type <i>OSD1</i> gene. Note that one of the spores is out of focus because they are organized in a tetrahedron. (C to I) Male meiotic products from <i>osd1-3</i> transformed by versions of the <i>OSD1</i> gene with a GxEN-box mutation (OSD1ΔGxEN), a D-box mutation (OSD1ΔD), a MR-tail mutation (OSD1ΔMR) or combination of these mutations. Some versions induced complementation, with a majority of tetrads (E, H), while others did not restore tetrad formation (C, D, F, G, I). Scale bar = 10 µM.</p