Figure 1.
Neocarzinostatin, an ionizing radiation mimetic, fragments DNA and blocks meiosis in oocytes.
(A) First polar body (PB1) extrusion rates following maturation of oocytes treated with NCS. (B) Chromatin in oocytes from (A); (i) depicting a typical metaphase II egg that was not treated with NCS, (ii, iii) meiotic arrest caused by NCS with varying levels of fragmentation. White dotted line represents egg and polar body outlines. (C) Percentage of oocytes treated with or without NCS that display fragmented DNA. (D) Nuclear staining of γH2AX in GV oocytes with or without NCS treatment; asterisks is at center of nucleolus. (E) Percentage of γH2AX positive oocytes treated with or without NCS. (A, C, E) Pooled data from 2 replicates. In parentheses, total number of oocytes examined. (A) Different letters denote significant difference, p<0.05 (Fisher's exact test). (B, D) Scale bar, 10 µm.
Figure 2.
Interstrand crosslinking has no effect on completion of meiosis in oocytes.
(A) First polar body (PB1) extrusion rates in oocyes treated with MMC after milrinone washout. (B, C) Meiotic spindles from mature metaphase II stage eggs obtained in (A) were imaged (B) and the sister chromatids on the spindle classified as aligned or mis-aligned (C). Arrow, indicates a non-aligned chromosome. Scale bar, 10 μm. (D, E) Rates of second polar body extrusion (D) and pronucleus formation (E) in parthenogenetically activated eggs following addition of MMC to GV stage oocytes that were then in vitro matured. (A, C, D, E) Pooled data from between 4 to 5 replicates; in parentheses, number of eggs examined; n.s., p>0.05 (Fisher's exact test).
Figure 3.
Comparable impaired embryo development following ICL induction at either GV or 1-embryo stage.
(A) Schematic showing the experimental design. MMC was either added after milrinone washout or following parthenogenetic activation with Sr2+. Blastocyst rates were assessed at 120 hours. (B) Blastocyst rates from those oocytes treated as in (A) with the MMC concentration stated. In parentheses, number of eggs examined from 3 pooled replicates; different letters denote significantly difference (p<0.05; Fisher's exact test).
Figure 4.
Interstrand crosslinking increases FANCD2 nuclear foci in HeLa cells.
(A) FANCD2 immunostaining of synchronised HeLa cells in S-, G2- and M-phase. (B) FANCD2 nuclear foci during S-phase with or without MMC. (C) Nuclear FANCD2 foci counted per cell (mean ± standard deviation) from images in (B). In parentheses, total number of cells examined from 2 pooled replicates, *p<0.0001 (t-test). (D) Nuclear foci were calculated using a software-based approach. Confocal images were converted from 32-bit RGB to an 8-bit gray scale. ImageJ software was used to interrogate images, identify and count all nuclear foci of greater than 1.7x the mean nuclear background. In the 3 cells depicted the foci are numbered. (A, B) Scale bar, 10 μm.
Figure 5.
FANCD2 association with spindle microtubules and poles but not nuclear foci in oocytes.
(A) FANCD2 immunostaining of the nuclei in GV oocytes with or without previous MMC addition at the concentrations stated. No FANCD2 foci were observed. (B, C) FANCD2 immunostaining of either meiotic spindles in meiosis I (B) or at metaphase II arrest (C). FANCD2 was present on spindle microtubules and poles, and its localisation was unaffected by MMC addition. Images are representative of at least 10 oocytes from at least two replicates. Scale bar, 10 μm.
Figure 6.
Induction of γH2AX foci in GV oocytes following MMC addition.
(A) γH2AX immunostaining shown in GV oocytes treated with or without 20 µg/ml MMC. Scale bar, 10 μm. (B) γH2AX foci (mean ± standard deviation) counted per oocyte from images in (A). In parentheses, total number of oocytes examined, pooled from 2 independent experiments. *p<0.0001 (t-test).
Figure 7.
FANCD2 nuclear foci increase with ICLs in 1-cell embryos.
(A) FANCD2 nuclear foci in 1-cell embryos treated with or without 3 µg/ml MMC. Scale bar, 10 μm. (B) Nuclear FANCD2 foci counted per embryo (mean ± standard deviation). MMC had been added at either GV stage oocytes, or to 1-cell embryos as indicated. Both groups had been in vitro matured and activated as described in Fig 3A. In parentheses, total number of embryos examined, pooled from 2 independent replicates. Different letters denote significantly difference (p<0.05; ANOVA, Tukey's post-hoc test).