Reversal of Female Infertility by Chk2 Ablation Reveals the Oocyte DNA Damage Checkpoint Pathway

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Science  31 Jan 2014:
Vol. 343, Issue 6170, pp. 533-536
DOI: 10.1126/science.1247671

Eggs Well Done

Germ cells can endure extensive DNA damage during their development. Programmed meiotic double-strand breaks (DSBs) are essential for proper segregation of chromosomes to oocytes and sperm. However, incomplete DSB repair by recombination activates a checkpoint that triggers cell death. Exogenous DNA damage is also lethal to oocytes via a highly sensitive checkpoint. Bolcun-Filas et al. (p. 533) show that the CHK2 kinase is a key component of both checkpoints in mouse oocytes. Deletion of Chk2 restored fertility to females that would otherwise be sterile because of a meiotic recombination mutation or radiation exposure.


Genetic errors in meiosis can lead to birth defects and spontaneous abortions. Checkpoint mechanisms of hitherto unknown nature eliminate oocytes with unrepaired DNA damage, causing recombination-defective mutant mice to be sterile. Here, we report that checkpoint kinase 2 (Chk2 or Chek2), is essential for culling mouse oocytes bearing unrepaired meiotic or induced DNA double-strand breaks (DSBs). Female infertility caused by a meiotic recombination mutation or irradiation was reversed by mutation of Chk2. Both meiotically programmed and induced DSBs trigger CHK2-dependent activation of TRP53 (p53) and TRP63 (p63), effecting oocyte elimination. These data establish CHK2 as essential for DNA damage surveillance in female meiosis and indicate that the oocyte DSB damage response primarily involves a pathway hierarchy in which ataxia telangiectasia and Rad3-related (ATR) signals to CHK2, which then activates p53 and p63.

Fertility, offspring health, and species success depend on production of gametes with intact genomes. Particularly crucial is the proper synapsis and segregation of homologous chromosomes at the first meiotic division, processes requiring homologous recombination (HR), a high-fidelity DNA double-strand break (DSB) repair process. Meiocytes initiate HR by producing proteins (namely SPO11) that create DSBs. In mice, ~10% of the >200 induced DSBs are repaired as crossovers (COs), and the rest by non-crossover (NCO) recombination (1).

Aberrant homolog synapsis or DSB repair trigger checkpoints that eliminate defective meiocytes (24). Either defect causes apoptotic elimination of mouse spermatocytes at mid-pachynema of meiotic prophase I (5, 6). In contrast, loss of oocytes defective for both DSB repair and synapsis occurs earlier (within a few days postpartum) than those defective for synapsis alone (~2 months postpartum), suggesting that mammalian oocytes have distinct DNA damage and synapsis checkpoints (2, 7) (fig. S1). Mutations preventing DSB formation (Spo11 and Mei1) are epistatic to those affecting DSB repair (2). The DNA damage checkpoint acts around the time oocytes enter meiotic arrest (dictyate, or resting stage) and presumably persists, because resting primordial follicles are highly sensitive to ionizing radiation (IR) (8).

We focused on checkpoint kinase 2 (CHK2) as a candidate component of the meiotic DNA damage checkpoint. It is a downstream effector of the ataxia telangiectasia mutated (ATM) kinase that responds primarily to DSBs and can also be activated by the ataxia telangiectasia and Rad3-related (ATR) protein kinase that responds primarily to single-stranded DNA (ssDNA) (9, 10). Unlike Atm and Atr, Chk2 is dispensable for fertility and viability. To determine whether Chk2 is required for the meiotic DNA damage checkpoint, we bred mice doubly deficient for Chk2 and Dmc1, a RecA homolog required for interhomolog repair of meiotic DSBs (11). Dmc1 deficiency also prevents synapsis, which is HR-dependent. Whereas 3-week-postnatal wild-type or Chk2−/− ovaries contain primordial through antral follicles (Fig. 1, A and B, and fig. S2), Dmc1−/− ovaries are devoid of follicles (Fig. 1D). Deletion of Chk2 enabled survival of developing oocytes in DMC1-deficient 3-week-old ovaries (Fig. 1, E and F). However, primordial follicles were absent, leading to a nearly complete oocyte depletion by 2 months postpartum (figs. S2 and S3). This pattern of oocyte loss resembles that of Spo11 or Spo11−/− Dmc1−/− mice (fig. S1) (2), suggesting that Chk2 ablation compromises the DSB repair but not synapsis checkpoint.

Fig. 1 Evidence of a specific DNA damage checkpoint in mouse oocytes.

(A, B, D, E, G, H) Histology of 3-week postpartum ovaries. Follicle-devoid ovaries are denoted by dotted outline. Arrowheads in (A), (B), and (H) indicate primordial follicles. Scale bars indicate 200 μm; wt, wild type. (C, F, I) Oocyte quantification in mutants. Box plots show 25th and 75th percentiles (box), median (the line in the center of the box), and minimum and maximum values (whiskers). P values are as follows: ns, not significant; ***, 0.0004; ****, < 0.0001.

To test this, we exploited an allele of Trip13 (Trip13Gt) that causes male and female meiotic failure. Trip13Gt/Gt chromosomes undergo synapsis and CO formation but fail to complete NCO DSB repair (12), causing elimination of the entire primordial follicle pool and nearly all developing oocytes by 3 weeks postpartum (Fig. 1G), coinciding with the oocyte DNA damage checkpoint (fig. S1) (12, 13). Chk2−/− Trip13Gt/Gt ovaries had a large oocyte pool at 3 weeks postpartum (Fig. 1, H and I, and fig. S2), and they retained high numbers of all follicle types after 2 months (fig. S3), indicating that the rescue of surviving oocytes from checkpoint elimination was permanent or nearly so (see below). The rescue was not attributable to activation of an alternative DSB repair pathway during pachynema, a consideration because the Chk2 yeast ortholog MEK1 influences pathway choice (14); all dictyate Chk2−/− Trip13Gt/Gt oocytes (n = 54), like Trip13Gt/Gt oocytes, exhibited abundant histone γH2AX staining, indicative of persistent unrepaired DSBs (versus 7% of Chk2−/− dictyate oocytes; n = 45) (Fig. 2, A and B).

Fig. 2 DSBs in Trip13Gt/Gt Chk2−/− newborn oocytes are eventually repaired and yield offspring.

(A) Co-immunolabeling of neonatal oocytes. (B) Trip13Gt/Gt Chk2−/− oocytes progress to dictyate (“D”) with DSBs. P, pachytene. SYCP3 labels SC axial element. (Right) Boxed nuclei magnified. Female reproductive (C) longevity and (D) fecundity. P values are as follows: ***, 0.0002; ****, <0.0001.

Despite bearing DSBs into late meiotic prophase I, the rescued oocytes proved to be functional. All tested Chk2−/− Trip13Gt/Gt females produced multiple litters (Fig. 2C). Litter sizes were smaller than those of controls (Fig. 2D), attributable to fewer ovulated oocytes and implanted embryos (fig. S4). Chk2−/− Trip13Gt/Gt females sustained fertility for many months, yielding four to seven litters each (Fig. 2C) and over 160 pups collectively. Progeny showed no visible abnormalities up to 1 year of age (n = 28). The results suggested that all or most DSBs persisting into late meiosis were eventually repaired. Indeed, there was no evidence of persistent DNA damage (as indicated by γH2AX) in 2-month-old primordial, growing, or germinal vesicle stage preovulatory Chk2−/− Trip13Gt/Gt oocytes (fig. S5). Thus, repair of DSBs occurred after birth by unknown mechanisms.

Canonically, CHK2 signals to p53 in mitotic cells. In Drosophila melanogaster, CHK2-dependent p53 activation occurs in response to SPO11-induced breaks (3). We therefore tested whether p53 deficiency could rescue Trip13Gt/Gt oocytes. Three-week-old p53−/− Trip13Gt/Gt ovaries had significantly more oocytes than Trip13Gt/Gt single mutants (Fig. 3, B and C, and fig. S2); however, they contained far fewer primordial follicles than Chk2−/− Trip13Gt/Gt ovaries at 3 weeks postpartum, and almost no oocytes remained after 2 months (fig. S3). Therefore, CHK2-mediated elimination of Trip13Gt/Gt oocytes does not occur exclusively via signaling to p53, indicating the existence of another downstream effector(s) that acts perinatally in primordial follicles.

Fig. 3 Genetic and molecular analysis of the oocyte DNA damage checkpoint.

(A to C) Trip13Gt/Gt oocyte depletion is partially rescued by p53 deficiency. Scale bars, 200 μm. (D) DNA damage–induced TAp63 phosphorylation in newborn ovaries is CHK2-dependent. Neonatal ovaries (four) received 3 Gy IR before protein extraction 2 hours later. Increased p63 in Chk2−/− is likely due to increased oocytes in this genotype. (E) p63 contains a CHK2 phosphorylation site. HeLa cells bearing FLAG-tagged TAp63 with wild-type (WT) (LxRxxS) or mutant (LxRxxA; A, Ala) CHK2 motifs. Shifted CHK2 (arrowhead) is phosphorylated. IR dose = 3 Gy. (F) Depletion of p63-positive primordial follicles by IR is CHK2-dependent. Ovaries were cultured 7 days after irradiation. Scale bars, 100 μm. MVH marks oocytes. (Insets) Ovary cortical regions containing primordial follicles.

One candidate is p63, a p53 paralog. A predominant isoform called TAp63 (TA, transactivation) appears perinatally in late pachytene and diplotene oocytes, approximately coinciding with DNA damage checkpoint activation. Because TAp63 was implicated in the elimination of dictyate oocytes subjected postnatally to DSB-causing IR (15, 16) and it contains a CHK2 consensus substrate motif LxRxxS (L, Leu; R, Arg; S, Ser; x, any amino acid) (17), we speculated that CHK2 might activate TAp63 in response to DSBs. Indeed, whereas IR induces phosphorylation in wild-type ovaries (15, 16), TAp63 remained unphosphorylated in CHK2-deficient ovaries (Fig. 3D). Moreover, mutating serine to alanine in the CHK2 phosphorylation motif in p63 also prevented IR-induced TAp63 phosphorylation in cultured cells (Fig. 3E). We next tested whether CHK2 is required for the elimination of DSB-bearing dictyate oocytes, presumably via TAp63 activation. Whereas the entire primordial follicle pool was eradicated 1 week after IR treatment of wild-type ovaries, CHK2 deficiency prevented oocyte elimination despite the presence of p63 protein (Fig. 3F). Furthermore, irradiated Chk2−/− females remained fertile, with an average litter size (6.3 ± 1.8, n = 7) similar to that of unirradiated controls (6 ± 2.3, n = 3). If this rescue of fertility was due entirely to abolition of TAp63 activation, then deletion of TAp63 should also restore fertility to irradiated females. Previous studies (15, 16) found that p63−/− and TAp63−/− oocytes survived 5 days after 0.45 to 5 gray (Gy) of IR, but longer-term survival was not evaluated. We found that 0.45 Gy IR completely eradicated primordial oocytes after 7 days in females homozygous for a viable, TA domain–specific deletion allele of p63 (TAp63) (18, 19), identical to wild type (Fig. 4, A and B).

Fig. 4 CHK2 signals to both p63 and p53 in oocytes.

(A to E) Depletion of primordial follicles by IR requires p53 and TAp63. Week-old ovaries were irradiated, cultured 7 days, then immunostained. p63 and MVH are oocyte-specific. (F) Dynamic signaling to p53 and p63 in response to meiotic and induced DSBs. Shown are Western blots of neonatal ovarian protein. The irradiated sample was collected 2 hours post-IR (3 Gy). Arrowhead, phosphorylated p63 (15, 16). Trip13 mutants are undergoing oocyte elimination (reflected by MVH), hence use of more ovaries. (G to J) p53 and TAp63 are required for complete elimination of DSB repair–defective oocytes. Ovaries are 3 weeks postpartum. (J Inset) Primordial follicles. Scale bars, 200 μm.

These results suggested IR-induced DSBs (and perhaps meiotic DSBs) stimulate CHK2 signaling to a protein(s) in addition to TAp63. Suspecting p53, we found that, whereas irradiated p53−/− ovaries were essentially devoid of oocytes (Fig. 4C) (15, 16), p53−/− TAp63−/− oocytes (including those in primordial follicles) were rescued (Fig. 4D) to a degree similar to Chk2 mutants (Fig. 3F). Irradiated p53+/− TAp63−/− (Fig. 4E) but not p53−/− TAp63+/− oocytes were partially rescued, indicating that CHK2 signals to both p53 and p63 and that they act in a partially redundant fashion to eliminate DSB-bearing resting oocytes. The marked effects of p53 haploinsufficiency, and the possible inconsistencies with earlier reports showing that deletion of p63 alone could rescue primordial follicles from IR over the short term, indicate that checkpoint responses may be sensitive to quantitative variation.

Because Chk2 but not p53 deficiency reversed Trip13Gt/Gt female infertility, an outcome similar to the results with postnatal ovary irradiation, we hypothesized that the same DNA damage checkpoint was operative in both pachytene/diplotene and dictyate oocytes. To test this, we first examined patterns of p53 and TAp63 activation in different genotypes of ovaries, with or without IR exposure. As expected for wild type, TAp63 phosphorylation and p53 stabilization and/or expression occurred only after exposure to IR (Fig. 4F). Importantly, we observed p53 protein in unirradiated Trip13Gt/Gt neonatal ovaries but not in wild type (Fig. 4F), implying a role for p53 in the elimination of mutant oocytes with unrepaired meiotic DSBs (and consistent with partial rescue of Trip13Gt/Gt p53−/− oocytes; Fig. 3C). Stabilization of p53 in response to unrepaired meiotic DSBs is CHK2-dependent, because we did not detect p53 in Chk2−/− Trip13Gt/Gt ovaries (Fig. 4F). TAp63 was absent from neonatal Trip13Gt/Gt ovaries bearing residual oocytes (Fig. 4F). Normally, TAp63 appears in late meiotic prophase I, when meiotic DSBs have been repaired, and is robustly activated in resting oocytes in response to exogenous DNA damage (15, 16). Nevertheless, the absence of TAp63 in Trip13Gt/Gt oocytes predicts that it is not responsible for their death. Indeed, no oocyte rescue was observed in wean-age TAp63−/− Trip13Gt/Gt ovaries (Fig. 4I). A potential explanation for TAp63 repression in Trip13Gt/Gt oocytes was suggested by our observation (Fig. 4F) that unphosphorylated TAp63 was present in Chk2−/− Trip13Gt/Gt ovaries lacking detectable p53. These results suggest a regulatory relationship between p53 and TAp63 in the meiotic DNA damage response.

The mutual exclusivity of TAp63 and p53 in Trip13Gt/Gt oocytes gives insight into the failure of either single mutant to rescue fertility. We hypothesized that unrepaired DSBs that persist into late pachynema trigger CHK2-dependent p53 activation and oocyte elimination independent of TAp63 but that, in the absence of p53, TAp63 can be expressed and activated by CHK2 to drive oocyte elimination. This predicts that removal of both proteins would abolish the CHK2-dependent checkpoint. Indeed, we found that p53 heterozygosity could rescue TAp63−/− Trip13Gt/Gt oocytes (Fig. 4J). This rescue included primordial follicles (Fig. 4J, inset; nullizygosity for all three genes is embryonically semilethal). These and previous results with single mutants indicate that the DNA damage checkpoint pathway that monitors repair of SPO11-induced DSBs involves CHK2 signaling to both p53 and TAp63 and that this pathway also operates in postnatal resting oocytes (fig. S6).

A remaining question concerns the upstream activator(s) of CHK2. Canonically, ATM phosphorylates CHK2 in response to DSBs, whereas ATR responds to ssDNA by activating CHK1 (20, 21). However, ATR and ATM have other activities in mouse meiosis. ATM negatively regulates SPO11, causing Atm−/− oocytes to sustain extensive DSBs and triggering elimination by the meiotic DNA damage checkpoint (fig. S1) (2, 22). Therefore, CHK2 is likely activated by a different kinase. Indeed, Chk2 deficiency rescued Atm−/− oocyte depletion (fig. S7) to a degree similar to the rescue of DMC1-deficient ovaries. The facts that (i) CHK2 can trigger apoptosis in the absence of ATM in somatic cells (9), (ii) CHK2 can be activated in an ATR-dependent manner (10), and (iii) ATR localizes to sites of meiotic DSBs in mice (23) prompt us to propose that the DNA damage checkpoint pathway in mouse oocytes involves signaling of ATR to CHK2, which in turn signals to p53 and TAp63 (fig. S6). Additionally, spermatocytes may have a distinct DNA damage response pathway; we did not observe histological evidence for rescue of DSB repair-defective but synapsis-proficient spermatocytes by deletion of Chk2 or p53 (fig. S8).

Our results are of biomedical interest with respect to the primordial follicle pool depletion and premature ovarian failure that can occur after cancer radiotherapy or chemotherapy. CHK2 is an attractive target because chemical inhibitors are available, and Chk2 insufficiency is of minor phenotypic consequence in mice (24).

Supplementary Materials

Materials and Methods

Figs. S1 to S8

References (2531)

References and Notes

  1. Acknowledgments: This work was supported by NIH grant GM45415 to J.C.S. and contract CO26442 from the NY State Stem Cell Program. We thank A. Mills for providing p63 mutant mice and M. A. Handel for critical reading of the manuscript.

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