Impairment of BRCA1-related DNA double-strand break repair leads to ovarian aging in mice and humans

Abstract

The underlying mechanism behind age-induced wastage of the human ovarian follicle reserve is unknown. We identify impaired ATM (ataxia-telangiectasia mutated)-mediated DNA double-strand break (DSB) repair as a cause of aging in mouse and human oocytes. We show that DSBs accumulate in primordial follicles with age. In parallel, expression of key DNA DSB repair genes BRCA1, MRE11, Rad51, and ATM, but not BRCA2, declines in single mouse and human oocytes. In Brca1-deficient mice, reproductive capacity was impaired, primordial follicle counts were lower, and DSBs were increased in remaining follicles with age relative to wild-type mice. Furthermore, oocyte-specific knockdown of Brca1, MRE11, Rad51, and ATM expression increased DSBs and reduced survival, whereas Brca1 overexpression enhanced both parameters. Likewise, ovarian reserve was impaired in young women with germline BRCA1 mutations compared to controls as determined by serum concentrations of anti-Müllerian hormone. These data implicate DNA DSB repair efficiency as an important determinant of oocyte aging in women.

Fig. 1. Aging-related DSBs in GV oocytes and ovarian tissue from young and old mice and humans

(A) Bar graphs show a higher percentage of γH2AX-positive follicles in old (11 to 12 months) compared to young (4 to 5 weeks) mice, N=8/group; *P<0.01; Student’s t test. Adjacent photomicrographs represent γH2AX staining of young and old FVB mice ovarian sections; Inset (upper left corner of old mice ovarian tissue) shows a higher magnification of the right γH2AX(+) primordial follicle surrounded by γH2AX(-) stromal cells in old ovarian tissue. (B) Bar graphs show a higher number of γH2AX-positive foci per oocyte in old compared to young mice, N=13/group; *P<0.05; Student’s t test. Adjacent photomicrographs represent γH2AX (green) staining and counter staining of DAPI of young and old FVB mice oocytes. (C) Bar graph shows higher percentage of γH2AX-positive follicles in age group >20 yrs (N=4/group, age range 21 to 28yrs) compared to age group ≤20yrs (N=3/group, age range 2 to 14yrs; *P<0.01; Student’s t test). Adjacent photomicrographs represent γH2AX staining of human ovarian sections in two different age groups. (D) Bar graphs show higher numbers of γH2AX-positive foci per oocyte in human women age group ≥30 yrs (N=4/group, age range 35 to 42yrs) compared to age group <30yrs (N=5, age range 23 to 28yrs; *P<0.05; Student’s t test). Adjacent photomicrographs represent γH2AX (red) staining and counter staining for 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) in human oocytes. Blue arrow head: γH2AX-negative follicle; black arrow: γH2AX-positive follicle; yellow arrow: γH2AX foci. All bar graphs show the mean ± standard error of the mean (S.E.M.).

Fig. 2. Expression of DNA repair genes and proteins in mouse oocytes

Significant decrease in the expression of DNA repair genes in old mice (11 to 12 months) compared to young mice (4 to 5 weeks) shown by real time PCR. All results are mean ± S.E.M., N=8/group. Bar graphs represent the gene expressions and photomicrographs represent the protein expressions. The bar graphs show significantly lower levels of expression for (A) BRCA1, (C) MRE11, (D) RAD51, and (E) ATM in old mice compared to young mice (**P<0.001; Student’s t test). (B) Shows a nonsignificant change in BRCA2 expression levels in old mice compared to young mice (P=0.2; Student’s t test). The representative photomicrographs show lower amounts of (F) BRCA1 (green), (H) MRE11 (red), (I) RAD51 (red), and (J) ATM (green) protein expression in old mice compared to young mice. Oocytes were counterstained with DAPI (blue). (G) No significant difference was detected in BRCA2 protein expression patterns in young and old mice. White arrow points to the cytoplasm and pink arrow to the nucleus. All bar graphs show the mean ± S.E.M.

Fig. 3. Expression of DNA repair genes and proteins in human oocytes

Scatter plots represent the relative expression of DNA repair genes assessed by qRT-PCR in human oocytes from 24 patients aged 24 to 41 years, some of which show significant age-related declines. Relative expression is defined as. (A) BRCA1 (r=0.60, P<0.001; linear regression analysis and Student’s t test); (C) MRE11 (r=0.447, P<0.05; linear regression analysis and Student’s t test); (D) RAD51 (r=0.5, P<0.01; linear regression analysis and Student’s t test); (E) ATM (r=0.4, P <0.01; linear regression analysis and Student’s t test) show a decrease in the expression in old compared to young human oocytes. (B) BRCA2 does not show any significant change in the expression levels as the age progresses (r=0.1, P=0.75; linear regression analysis and Student’s t test). Photomicrographs represent the protein expression in human oocytes grouped as old (≥35 yrs), young (≤27 yrs) and all results are mean ± s.e.m. (N=4/group). Representative photomicrographs (F) BRCA1 (grey), (H) MRE11 (red), (I) RAD51 (red), and (J) ATM (green) show decreased protein expression in old (36 to 41 yrs) compared to young (24 to 35 yrs) human oocytes. (G) No decline in expression of BRCA2 protein (red) was observed in old compared to young human oocytes. White arrow points to cytoplasm and pink arrow to the nucleus. Oocytes were counterstained with DAPI (blue).

Fig. 4. BRCA-mutant mouse ovarian function

(A, B) Relative BRCA gene expression levels in BRCA-mutant mice. We observed significant BRCA-deficiency in heterozygous BRCA1^+/Δ11 and BRCA2^+/Δ27 mutant mice and the absence of BRCA gene expression in BRCA2^Δ27/Δ27 homozygous mice (HM) compared with wild-type mice (WT) (*** P<0.0001 by Student’s t test, **P<0.001 by ANOVA, * P<0.05 by ANOVA). BRCA1 HT mice also showed significantly lower MII oocyte yield per female (C) (* P<0.01; Student’s t test), (E) reduced litter size (* P<0.05; Student’s t test), reduced primordial follicles per ovary (G, L for 5-day ovary, P for 4-month ovary; *P <0.05; Student’s t test), and a higher percentage of γH2AX-positive follicles (I, R, only in 4-month-old mice, * P<0.01; Student’s t test) verses BRCA1 wild-type mice (K, O, and Q). No difference was observed for the same comparisons for BRCA2 HT or HM mice with WT (D, F, H, and J, analyzed by ANOVA). Panels M and N are enlarged views of K and L, respectively. Black arrows in panels K, L, M, N, O, and P point to primordial follicles; black arrows in Q and R point to γH2AX (+) primordial follicles, and blue arrowheads in Q and R point to γH2AX (-) primordial follicles. All bar graphs show the mean ± S.E.M.

Fig. 5. Impact of siRNA silencing of DNA repair genes on genomic integrity and survival of mouse oocytes in response to genotoxic stress

In response to H₂0₂ treatment (250 μM) (A) mean number of γH2AX foci increased (P<0.05; Student’s t test) and (B) AC3 levels increased (P<0.05; Student’s t test), wherease (C) survival decreased in the oocytes in which expression of DNA repair genes had been silenced compared to controls (scrambled siRNA) (Scram) (P<0.05 Fisher’s exact test). Photomicrographs are representative of the γH2AX foci (green) and AC3 (red) levels in the siRNA-silenced oocytes: (D) scrambled, (E) BRCA1, (F) MRE11, (G) RAD51, (H) ATM. All bar graphs show the mean ± S.E.M. Arrowheads shows nuclear region. Oocytes are counterstained with DAPI (blue). non inj ctrl, noninjected control oocytes.

Fig. 6. Diminished ovarian reserve in BRCA1-deficient individuals

Women with significant BRCA1 (P<0.0001; analyzed by ANOVA) but not BRCA2 (P=0.127; analyzed by ANOVA) mutations had lower mean serum AMH concentrations compared to those with no BRCA mutations. All bar graphs show the mean ± S.E.M.