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Oocytes can efficiently repair DNA double-strand breaks to restore genetic integrity and protect offspring health - PubMed

  • ️Wed Jan 01 2020

Oocytes can efficiently repair DNA double-strand breaks to restore genetic integrity and protect offspring health

Jessica M Stringer et al. Proc Natl Acad Sci U S A. 2020.

Abstract

Female fertility and offspring health are critically dependent on an adequate supply of high-quality oocytes, the majority of which are maintained in the ovaries in a unique state of meiotic prophase arrest. While mechanisms of DNA repair during meiotic recombination are well characterized, the same is not true for prophase-arrested oocytes. Here we show that prophase-arrested oocytes rapidly respond to γ-irradiation-induced DNA double-strand breaks by activating Ataxia Telangiectasia Mutated, phosphorylating histone H2AX, and localizing RAD51 to the sites of DNA damage. Despite mobilizing the DNA repair response, even very low levels of DNA damage result in the apoptosis of prophase-arrested oocytes. However, we show that, when apoptosis is inhibited, severe DNA damage is corrected via homologous recombination repair. The repair is sufficient to support fertility and maintain health and genetic fidelity in offspring. Thus, despite the preferential induction of apoptosis following exogenously induced genotoxic stress, prophase-arrested oocytes are highly capable of functionally efficient DNA repair. These data implicate DNA repair as a key quality control mechanism in the female germ line and a critical determinant of fertility and genetic integrity.

Keywords: DNA damage; DNA repair; apoptosis; fertility; oocytes.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.

The DNA repair response is activated in prophase-arrested oocytes following DNA damage. WT mice (PN10) were exposed to whole-body γ-irradiation at 0.2, 4.5, or 7 Gy. Prophase-arrested oocytes in primordial and growing follicles were analyzed in untreated controls 0.5, 3, and 6 h later for nuclear localization of pATM (A), γH2AX (B and C), RAD51 (D), and DNA-PKcs (E). Representative immunofluorescence images are shown for controls and at 0.5 h post γ-irradiation. DNA was counterstained with DAPI (blue), and oocytes were labeled with c-Kit or MVH (green) and colocalized with markers of the DNA damage response and repair (red; arrowheads indicate oocytes with positive staining, and arrows indicate negative staining) (Scale bars, 20 µm.) (F) Ovaries were also collected 24 h after γ-irradiation, and oocytes were enumerated (n = 4 to 5 mice/group). All data are expressed as mean ± SEM and analyzed by one-way ANOVA followed by Tukey’s post hoc test or Kruskal–Wallis for nonparametric data. Different letters are significantly different; P < 0.05. Localization of each marker was evaluated in oocytes from ∼50 to 200 primordial follicles and ∼25 to 100 growing follicles per treatment and time point; n = 3–8 animals/group.

Fig. 2.
Fig. 2.

Prophase-arrested oocytes can repair DNA when apoptosis is inhibited. (A) The number of primordial follicles in ovaries collected from TAp63−/− mice 24 h after γ-irradiation at 0.1, 0.45, and 7 Gy or from untreated control mice (n = 3 to 4 mice/group). The percentage of oocytes with γH2AX (B), RAD51 (C), and DNA-PKcs (D) foci and the average number of foci/oocyte in primordial follicles from control mice and TAp63−/− mice 3 h, 24 h, and 5 d after γ-irradiation at 0.1 Gy and 0.45 Gy. Localization of each marker was evaluated in ∼100 to 500 primordial follicle oocytes per treatment and time point; n = 3 to 8 animals/group. (E) γ-Irradiated (0.45 Gy) TAp63−/− mice were treated with vehicle, RAD51 inhibitor (Rad51i; 50 mg/kg, B02), or DNA-PK inhibitor (DNAPKcsi; 10 mg/kg, NU7441), and follicles were enumerated 24 h later (n = 4/group). Primordial follicles in vehicle and DNA-PKcs inhibitor-treated ovaries were of normal histological appearance (indicated by dashed-line white circles in PAS images), but apoptotic oocytes were observed in ovaries treated with the RAD51 inhibitor (indicated by white arrowheads in PAS images). The percentage of primordial follicle oocytes with γH2AX foci was calculated for each treatment group. All data are expressed as mean ± SEM and analyzed by one-way ANOVA followed by Tukey’s post hoc test or Kruskal–Wallis for nonparametric data. Different letters are significantly different; P < 0.05. Absence of error bars in #H2AX foci/oocyte day 5 graphs (B–D) indicates too few oocytes with foci for statistical analysis.

Fig. 3.
Fig. 3.

DNA repair is sufficient to restore functional fertility. (A) Number of ovulated oocytes harvested from γ-irradiated wild-type and TAp63−/− mice following exogenous hormonal stimulation. Representative images of oocytes obtained from TAp63−/− females stained with f-actin (red) to mark the oolema, αβ-tubulin (green) to label the spindle, and DAPI to label the DNA on the metaphase plate. (B) Number of zygotes obtained from γ-irradiated WT and TAp63−/− mice at E0.5 after mating with untreated males. Representative images of zygotes obtained from TAp63−/− females stained with f-actin (red) to mark the zygote boundary and with DAPI to label the DNA in pronuclei (circled by dotted white lines) to confirm fertilization. (Scale bar, 20 μm.) (C) Number of blastocysts obtained from γ-irradiated WT and TAp63−/− mice at E3.5 after mating with untreated males. The ratio of inner cell mass to trophectoderm (TE) is shown. Representative images of blastocysts obtained from TAp63−/− females stained with f-actin (red) to mark the cell boundaries and with DAPI to label the DNA and CDX2 (aqua) to label TE. (Scale bar, 20 μm.) Fertility of mice (D) from continuous mating trial. All data are expressed as mean ± SEM and analyzed by one-way ANOVA followed by Tukey’s post hoc test or Kruskal–Wallis for nonparametric data or t tests for pairwise comparisons. Different letters are significantly different; *P < 0.05, **P < 0.005, ****P < 0.0001.

Fig. 4.
Fig. 4.

Offspring from γ-irradiated TAp63−/− dams are healthy. (A) TAp63−/− mice were untreated or γ-irradiated at PN10. Adults were then mated with untreated WT males, and offspring health analyzed. (B) Weight of female and male offspring from control, 0.1 Gy, and 0.45 Gy TAp63−/− dams at 5, 19, and 65 d after birth. Dots represent weights for individual animals. Female/male n = 62/55, 62/74, 64/65 for control, 0.1 Gy, and 0.45 Gy. (C) Percentage of offspring from control, 0.1 Gy, and 0.45 Gy TAp63−/− dams that survive to 65 d (adulthood). (D) Percentage of fat, bone mineral density, and bone mineral composition were determined by DEXA in male and female offspring from control and 0.45-Gy TAp63−/− dams at 65 d. Female/male n = 19/40, 51/110 for control and 0.45 Gy. Data for B–D are expressed as mean ± SEM and analyzed by one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.005, ****P < 0.0001. No significant differences were observed in B and C. (E) The genomes of two offspring from unirradiated TAp63−/− control dams (n = 2) and 18 offspring from 0.45-Gy–treated TAp63−/− dams (n = 5) were sequenced. Box and whisker plots of mutations in the 1.4 Gbp assayed per animal are shown. TAp63+/− offspring from irradiated dams have a significantly lower mutation rate than controls (1.2 × 10−9 ± 0.44 × 10−9 vs. 5.2 × 10−9 ± 2.7 x10−9 P = 4.2 × 10−6 Poisson generalized linear model mutations ∼ genotype).

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