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

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

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

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

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

²²⁵Ac-E4G10 treatment results in a relatively normal remaining tumor vasculature.

<p>A, Greater coverage of tumor blood vessels (CD31 positive) by pericytes (α-SMA-positive cells) in ²²⁵Ac-E4G10 treated tumor relative to dual control. B, Transmission electron micrographs of blood vessels in dual control and ²²⁵Ac-E4G10 treated tumor. The dual control tumor contains extravasated RBC-filled vascular spaces that are not lined with endothelial cells, whereas blood vessels in ²²⁵Ac-E4G10 treated tumor display a continuous endothelial lining (arrow) resting on a basement membrane (BM) that is shared with the surrounding pericyte. Scale bar, 50 µm</p

²²⁵Ac-E4G10 therapy inhibits the growth of LnCap prostate tumors.

<p>A, Flow cytometric analysis depicting the lack of E4G10 binding to LnCap cells; J591, mouse-anti prostate specific membrane antigen is the positive control. Mouse and rat isotype controls were also evaluated. B, Photographs of in situ (left) and excised tumor (right) in a representative dual control and ²²⁵Ac-E4G10 treated animal. C, Tumor volume in various treatment groups at described time-points. D, Serum prostate specific antigen (PSA) levels in the three treatment groups at 22 days post-implantation with 5 million LnCap cells. E, Kaplan Meier curve showing enhancement of survival with ²²⁵Ac-E4G10 treatment. Data in C, D are mean ± S.E.M. <i>Scale bar</i>, 1 cm.</p

A combination of ²²⁵Ac-E4G10 with paclitaxel enhances the anti-tumor response.

<p>A, Tumor volume in the four treatment groups over time. Data are mean ± S.E.M. B, Kaplan Meier survival curve of treated animals showing significant enhancement of animal survival when ²²⁵Ac-E4G10 therapy is followed by a course of paclitaxel. C, Absence of histopathologic damage in normal organs, assessed 10 days after cessation of ²²⁵Ac-E4G10 treatment.</p

Effect of ²²⁵Ac-E4G10 therapy on tumor histology, vascularity and apoptosis.

<p>A, Light microscopy depicting numerous RBC-filled vascular spaces (arrows) in dual control tumor and fewer, but relatively normal-looking vessels (arrowheads) in the ²²⁵Ac-E4G10 treated tumor. B, Top: Immunohistochemical staining of tumor-sections for vWF, an endothelial cell marker (top). TUNEL staining of tumor sections to detect apoptosis (bottom). Quantification of vWF staining (C) and apoptosis (D) in 4 randomly selected fields. Data are mean ± S.E.M.</p