DNA double strand break levels increase with age in <i>fog-2</i> feminized germlines.

A) Schemes used to isolate young (1 day post-L4), aged (4 days post L4), and old (6 days post L4) fog-2(q71) worms for experiments. Days count ~18–24 hour periods after hermaphrodites were isolated as L4 larvae and are separated by alternating grey shaded boxes. B) Representative whole germline images of young, aged, and old fog-2 germlines. The RAD-51 zone is indicated with a green dashed line. All germlines are oriented with the distal mitotic tip on the left and the end of pachytene on the right. Scale bars represent 20μm. Grey numbered boxes indicate the positions of the images presented in panel C. C) Representative images of the peak levels of RAD-51 foci observed in meiotic nuclei of young, aged, and old fog-2 germlines. Scale bars represent 5μm. Each panel is numbered to indicate the position in the germlines displayed in panel B that each inset was taken from. D) RAD-51 foci per nucleus in fog-2(q71) oocytes. Line plots represents the mean RAD-51 foci per nucleus along the length of the germline in a sliding window encompassing 0.1 units of normalized germline distance with a step size of 0.01 germline distance units. Mean RAD-51 foci were calculated from nuclei analyzed in n = 9 total germlines for young, aged, and old fog-2 and from n = 7 total germlines for aged sperm depleted fog-2. For all groups, the analyzed germlines were derived from ≥3 experimental replicates. Shaded areas around each line represent ± SEM. Average nuclei quantified in each bin ± standard deviation: Young fog-2 173±21.3, Aged fog-2 143±32.0, Old fog-2 148.5±29.6, Aged Sperm Depleted fog-2 119±40.5. Germline distance was normalized to the premeiotic tip (0) and end of pachytene (1) based on DAPI morphology (see Methods). Arrowheads indicate the “peak RAD-51” windows, defined as the windows along the length of the germline of each age group with the highest RAD-51 foci per nucleus. P values were calculated by Mann-Whitney U test comparisons of RAD-51 counts within these peak windows with Holm-Bonferroni correction for multiple comparisons (n.s. = p>0.05, *** = pS1 Data.