Reversal of female infertility by Chk2 ablation reveals the oocyte DNA damage checkpoint pathway

Abstract

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.

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

(A, B, D, E, G, H) Histology of 3 weeks postpartum ovaries. Follicle-devoid ovaries are denoted by dotted outline. Arrowheads (A, B, H) indicate primordial follicles. (C, F, I) Oocyte quantification in mutants. Bar = 200μm.

Fig. 2. DSBs in Trip13^Gt/Gt Chk2^−/− newborn oocytes are eventually repaired and yield offspring

(A) Co-immunolabeling of neonatal oocytes. (B) Trip13^Gt/Gt Chk2^−/− oocytes progress to dictyate (“D”) with DSBs. P = pachytene. Boxed nuclei magnified (inset). (C) Female reproductive longevity and (E) fecundity.

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

(A–C) Trip13^Gt/Gt oocyte depletion is partially rescued by p53 deficiency. Bar = 200 μm. (D) DNA damage-induced TAp63 phosphorylation in newborn ovaries is CHK2-dependent. Neonatal ovaries (4) received 3Gy IR before protein extraction 2 hrs later. Note: 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 WT (LxRxxS) or mutant (LxRxxA) CHK2 motifs. Shifted CHK2 (arrowhead) is phosphorylated. IR dose=3Gy. (F) Depletion of p63-positive primordial follicles by IR is CHK2-dependent. Ovaries were cultured 7 days after irradiation. Bar=100 μm. MVH marks oocytes. Inset: ovary cortical region containing primordial follicles.

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

(A–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 hrs post-IR (3Gy). Arrowhead: phosphorylated p63 (15, 16). Trip13 mutants are undergoing oocyte elimination (reflected by MVH), hence use of more ovaries. (G–J) p53 and TAp63 are required for complete elimination of DSB repair-defective oocytes. Ovaries are 3 weeks postpartum. Inset (J) shows primordial follicles. Bar=200 μm.