DNA damage response during mouse oocyte maturation

Abstract

Because low levels of DNA double strand breaks (DSBs) appear not to activate the ATM-mediated prophase I checkpoint in full-grown oocytes, there may exist mechanisms to protect chromosome integrity during meiotic maturation. Using live imaging we demonstrate that low levels of DSBs induced by the radiomimetic drug Neocarzinostatin (NCS) increase the incidence of chromosome fragments and lagging chromosomes but do not lead to APC/C activation and anaphase onset delay. The number of DSBs, represented by γH2AX foci, significantly decreases between prophase I and metaphase II in both control and NCS-treated oocytes. Transient treatment with NCS increases >2-fold the number of DSBs in prophase I oocytes, but less than 30% of these oocytes enter anaphase with segregation errors. MRE11, but not ATM, is essential to detect DSBs in prophase I and is involved in H2AX phosphorylation during metaphase I. Inhibiting MRE11 by mirin during meiotic maturation results in anaphase bridges and also increases the number of γH2AX foci in metaphase II. Compromised DNA integrity in mirin-treated oocytes indicates a role for MRE11 in chromosome integrity during meiotic maturation.

Keywords: DNA damage; MRE11; double strand DNA breaks; meiotic maturation; mouse oocytes.

Figure 1.

DSBs induced by Neocarzinostatin in GV-stage oocytes result in chromosome segregation problems in anaphase I. (A) Immunofluorescence of control and NCS treated GV-stage oocytes labeled with pS139 H2AX (γH2AX) and MDC1 antibodies.  Maximum z-projection of confocal section across nucleus is shown. (B) Number of γH2AX foci in control and NCS treated GV-stage oocytes (n=137, 22, 118, 29, 21). (C) Selected time frames of live H2B-EGFP control and NCS treated oocytes imaged by confocal microscopy. Arrowheads show chromosome segregation errors in anaphase and arrow shows diffusing chromosome fragment in MII. Maximum z-projection of H2B-EGFP and single bright filed section is shown. Time in hh:mm. (D) Percentage of segregation errors in anaphase I and (E) timing of anaphase onset in control and NCS treated oocytes (for C and D n=98, 17, 20, 56, 47, 113). (F) Percentage of 1^st polar body resorption after anaphase onset in control, 10 and 100 ng/ml NCS treated oocytes (n=98, 55, 76). Data in D), E) and F) are from analysis of time lapse movies of control and NCS treated H2B-EGFP oocytes as shown in C).

Figure 2.

Chromosomes are fragmented in NCS-treated oocytes. (A) Time-lapse imaging of NCS-treated oocytes. Oocytes expressing 2mEGFP-CENP-C (green) and H2B-mCherry (red) were treated with NCS at 100 ng/ml. Green arrowheads indicate the homologous kinetochores of a bivalent, which is magnified on the right. Red arrowheads indicate lagging chromosomes. Time after NEBD (h:mm). Scale bar, 10 μm. (B) Chromosome hyperstretching in NCS-treated oocytes. The distance between homologous kinetochores was measured in 3D in oocytes 6 h after NEBD (n=252, 409 bivalents).  ***p < 0.0001. (C) NCS-treated oocytes exhibit lagging chromosomes without kinetochores. The number of lagging chromosomes at anaphase was counted (n=11, 17 oocytes). The lagging chromosomes were categorized. Error bars, s.d.

Figure 3.

H2AX phosphorylation changes during meiotic maturation. (A) Immunofluorescence of control and NCS treated GV-stage, prometaphase I (PMI), metaphase I (MI) and metaphase II (MII) oocytes labeled with γH2AX and MDC1 antibodies. Maximum z-projection of confocal section across chromatin region is shown. In the case of MII oocytes, maximum z-projection for DNA and γH2AX but single confocal sections for MDC1 are used. Insets show maximum z-projection of metaphase II plate area. (B) Quantification of γH2AX during meiotic maturation in control and NCS treated oocytes (n = 26, 28, 26, 27, 43, 8, 28, 23). (C) Quantification of γH2AX foci number in GV-stage and MII oocytes treated with NCS in GV stage before meiotic maturation. Acute NCS treatment of metaphase II eggs (control damage MII) is also shown (n = 137, 118, 98, 84, 24). (D) Percentage of MII eggs with at least one γH2AX focus (n = 106, 55, 21). (E) Immunofluorescence of MII eggs treated acutely with NCS and labeled with γH2AX and MDC1 antibodies. Maximum z-projection of confocal sections is shown.

Figure 4.

MRE11 is essential for H2AX phosphorylation in meiosis I and its inhibition during meiotic maturation causes the increase in DSBs in metaphase II. (A) Imunofluorescence of GV-stage oocytes stained with MRE11 antibody. Maximum z-projection is shown. (B) Expression of MRE11 (81 kDa) in control morpholino (ctrl) and Mre11 morpholino (k_d, knock down) injected GV-stage and MII oocytes detected by western blot (n=200 oocytes/lane). M1 and M2 are protein markers of indicated MW. Control NIH3T3 cells were used as a positive control (12 µg/lane). We have seen similar expression of MRE11 in noninjected GV and MII oocytes (data not shown). (C) Immunofluorescence of control, mirin and NCS treated GV-stage, metaphase I and metaphase II oocytes labeled with γH2AX and MDC1 antibodies. Maximum z-projection of confocal section across chromatin region is shown. GV-stage oocytes were treated with Mirin, NCS or both for 1 h in milrinone supplemented medium before fixation. Metaphase I and II oocytes matured in the presence of Mirin for 7 or 16 h before fixation. (D) Quantification of γH2AX foci number in GV-stage and MII oocytes treated with mirin in GV stage before meiotic maturation (n=137, 33, 200, 109). E) Quantification of γH2AX foci number in GV-stage oocytes non-treated (control) or treated with mirin, NCS or both for 1 h in milrinone-supplemented medium before fixation (n=153, 48, 118, 24). (F) Quantification of γH2AX during meiotic maturation in control and mirin-treated oocytes (n=26, 8, 15, 21, 14, 32). G) Percentage of oocytes with at least one γH2AX focus matured into the metaphase II in control or mirin-supplemented medium (n=31, 58).

Figure 5.

MRE11 controls chromosome segregation and integrity.(A) Time-lapse imaging of control and mirin treated H2B-EGFP oocytes.  Maximum intensity z-projection of H2B-EGFP and single section of bright-field images in selected time frames are shown. (B) Imaging of chromosome dynamics and integrity in live oocytes expressing H2B-mCHERRY and CENPC-EGFP and maturing in control or Mirin supplemented medium. Maximum z-projection of confocal section of selected time intervals is shown. Arrowheads show DNA anaphase bridges.