Oocyte Age-Dependent NDJ Figure 8. Diplotene-like oocytes are most vulnerable to age-dependent nondisjunction. (A) For each of the different 8-hour sub-broods indicated, the relative level of NDJ in aged versus non-aged smc1+/2 mtrm+/2 oocytes is represented as an odds ratio. The error bars correspond to 61 standard error. Oocytes laid between 16–32 hours after the aging regimen (sub-broods 3 and 4) exhibit significant age-dependent NDJ (**P = 0.0009 and *P = 0.0035). The data shown represent pooled data from two independent experiments in which at least 1200 progeny were scored for each sub- brood. The combined raw data are shown in Table S6. (B) For aged oocytes, the frequency at which recombinant chromosomes missegregate (R/N) in sub-brood 3 is significantly different from that for both sub-broods 1 and 6 (P = 0.0037, for sub-brood 3 versus 1, P = 0.022 for sub-brood 3 versus 6). In contrast, the frequency of R Diplos is similar in all four sub-broods analyzed from non-aged oocytes (0.73,P,0.92 for pair-wise comparisons). (C) Schematic of an ovariole following the aging regimen reflects the accumulation of metaphase I arrested oocytes (stage 14) that occurs at the expense of stages 9–13 (compare with Figure 2A). Unlike stages 9–13 which continue to progress, oocytes at stages 1–8 halt in development during the aging regimen. However, only oocytes that undergo aging during diplotene (stages 7–8) exhibit significant levels of age-dependent NDJ. In contrast, oocytes that remain arrested at metaphase I are not susceptible to age effects. Schematic is adapted from Robinson et al. [45]. doi:10.1371/journal.pgen.1000263.g008 [40–42]. The specificity of the mutant phenotype in mtrm+/2 oocytes combined with the observation that sister-chromatid cohesion appears to be normal convinced us that this was the best approach to disrupt the achiasmate segregation pathway. Recombination Analysis To assay X-chromosome crossover frequency and distribution in smc1+/2 oocytes, 5–7 females were crossed to 3 yw males per vial. Crossover frequency and distribution was measured by assaying sc, cv, v, f, car markers in the male progeny. The recombination data was used to estimate tetrad exchange ranks [18]. Aging Regimen and Generation of Broods To age Drosophila oocytes, the aging regimen described by Jeffreys et al. was modified [9] such that the glucose agar media was prepared without the addition of fungal inhibitors (methyl paraben and ethyl acetate). The glucose agar media contained 2% agar (Fisher) and 5% dextrose (Fisher) and was prepared with milli-Q grade water. Yeast paste was prepared by dissolving 30 g of active dry yeast (Red Star) in 50 mL milli-Q grade water. A schematic of the aging regimen is shown in Figure 2B. Approximately 200 virgin females of the desired genotype were collected within an 8-hour period and the females were fed yeast overnight in vials with cornmeal molasses media to promote vitellogenesis. Overnight incubation of females with yeast allows yolk deposition and maturation of oocytes so that the ovaries contain a complete complement of oocytes at the different stages. The following day, females were split into two groups and placed in separate plexi-glass laying bottles containing glucose agar plate with a smear of yeast paste. The control group of females was supplied with an equal number of male flies and laid their eggs continuously. Their oocytes were ‘‘non-aged’’. The experimental group of females were deprived of males. Because oviposition was suppressed in these females and the developmental progression of oogenesis was halted, oocytes ‘‘aged’’ within their abdomens. This experimentally induced aging of oocytes mimics the aging of oocytes in human females. Control and experimental flies were held in the laying bottles for four days with fresh yeast paste/ glucose-agar plates supplied each day. Generation of 24-Hour Broods of Progeny At the end of the four-day aging regimen, the experimental females (with aged oocytes) and the control females (with non-aged oocytes) were crossed to X^Y, v f B males to measure meiotic PLoS Genetics | www.plosgenetics.org 9 November 2008 | Volume 4 | Issue 11 | e1000263