Aging Negatively Impacts DNA Repair and Bivalent Formation in the C. elegans Germ Line

Abstract

Defects in crossover (CO) formation during meiosis are a leading cause of birth defects, embryonic lethality, and infertility. In a wide range of species, maternal aging increases aneuploidy and decreases oocyte quality. In C. elegans which produce oocytes throughout the first half of adulthood, aging both decreases oocytes quality and increases meiotic errors. Phenotypes of mutations in genes encoding double-strand break (DSB)-associated proteins get more severe with maternal age suggesting that early meiosis reflects a particularly sensitive node during reproductive aging in the worm. We observed that aging has a direct effect on the integrity of C. elegans meiotic CO formation, as observed by an increase of univalent chromosomes and fusions at diakinesis, with a considerable increase starting at 4 days. We also characterize the possible causes for the age-related changes in CO formation by analyzing both steady-state levels and kinetics of the ssDNA binding proteins RPA-1 and RAD-51. Profound reductions in numbers of both RPA-1 and RAD-51 foci suggests that both DSB formation and early meiotic repair are compromised in aging worms. Using laser microirradiation and γ-irradiation to induce exogenous damage, we show specifically that recruitment of these homologous recombination proteins is altered. Repair defects can be seen in two-and-one-half day-old adults making the loss of germline repair capacity among the earliest aging phenotypes in the worm.

Keywords: C. elegans; DSB repair; RAD51; RPA; aging; germline; meiosis.

FIGURE 1

Chiasmata formation is compromised in aging germ lines. Crossover formation is assessed by the number of DAPI-staining bodies at diakinesis. (A) Quantification of DAPI-bodies in N2 diakinesis nuclei from the indicated ages. (B–D) Representative DAPI-stained images of young (B) and aged (C,D) N2 worms showing bivalents (day 1), “separated bivalents” (arrowheads) and fusion (arrow). (E) Percent of late pachytene-stage nuclei containing the indicated number of COSA-1 foci in gfp::cosa-1 worms. Numbers on top of bars indicate the total number of nuclei analyzed per age. Statistical significance was determined using Chi-square test. ^****p < 0.0001.

FIGURE 2

Fewer RAD-51 foci in meiotic nuclei from aged mothers. (A) DAPI-stained C. elegans germ line showing the regions in which RAD-51 foci were quantified. Each zone corresponds to a particular meiotic stage: Transition zone (TZ) corresponds to leptotene and zygotene, Zones 1 and 2 to early pachytene, Zones 4 and 5 to middle pachytene and Zones 5 and 6 to late pachytene. (B) The percentage of nuclei in each zone containing the indicated number of RAD-51 foci. Four gonads per age in N2 worms were quantified. (C) Quantification of RAD-51 foci in the rad-54(ok615) mutant background. Shown is the percentage of nuclei in late pachytene nuclei containing the indicated number of RAD-51 foci. Numbers on top of bars indicate the total number of nuclei analyzed per age. Statistical significance was determined using Chi-square test. ^****p < 0.0001.

FIGURE 3

Dynamics of RPA-1 and RAD-51 are altered in aging, meiotic germ cells. spo-11 mutant worms at day 1 and day 4 of adulthood were exposed to 10Gy of γ-irradiation. (A) Average number of GFP::RPA-1 or B) or RAD-51 in mid-pachytene nuclei at indicated timepoints post-exposure. A total of 10 worms per age/timepoint were analyzed in gfp::rpa-1; spo-11 (A) or spo-11 (B) worms. (C) Percent of late pachytene nuclei containing the indicated number of COSA-1 foci in gfp::cosa-1, spo-11 worms at 8 h post-exposure. Statistical significance was determined using Chi-square test. ns p > 0.1, ^∗p < 0.05, ^****p < 0.0001.

FIGURE 4

Recruitment of RPA-1 to complex DNA damage decreases with maternal age upon DSB formation. Live imaging of mid-pachytene nuclei post-exposure to microirradiation. (A) Left: Representative images from each age of worms examined showing GFP::RPA-1 fluorescence in 20 min post- microirradiation. Images are extracted from the 45-min live-imaging sequence. Scale bars are 3μm. Right: Key for number of recruitment regions represented by each color in panels (B,C). (B) Percent of mid-pachytene (MP) nuclei for each age of worm (days post-L4) with the indicated number of GFP::RPA-1 recruitment regions. (C) Percent of nuclei in 1-day- and 2.5-day-old adults with the indicated number of GFP::RPA-1 recruitment regions (PMT, pre-meiotic tip; TZ, transition zone; MP, mid-pachytene; LP, late pachytene). Statistical significance was determined using the Fisher’s Exact test and p-values are indicated as follows: ns ≥ 0.1234, 0.0332–0.1233 = ^∗, ≤ 0.0001 = ^****. Nuclei n-values as indicated for each condition.

FIGURE 5

RPA-1 dynamics complex DNA damage attenuates with age. (A–C) Percent of mid-pachytene nuclei with RPA-1 recruitment regions out of total number of nuclei 12 min (A), 25 min (B) and 4 h (C) after microirradiation, in worms at 1, 2, 3, and 4 days post-L4. (D–F) Percent RPA-1 recruitment regions appearing as individual foci or as clustered foci per nucleus at 12 min (D), 25 min (E) and 4 h (F) after microirradiation, in worms at 1, 2, and 3 days post-L4. (G–I) Percent of nuclei with the indicated number of RPA-1 recruitment regions per nucleus 12 min (A), 25 min (B) and 4 h (C) after microirradiation, in worms at 1, 2, and 3 days post L4. Statistical significance was determined using the Fisher’s Exact test and p-values are indicated as follows: ns ≥ 0.1234, 0.0332–0.1233 = ^∗0.0021–0.0331 = ^∗∗0.0002–0.0020 = ^∗∗∗, ≤ 0.0001 = ^****. N values as indicated for each condition.