Oocyte Age-Dependent NDJ Figure 4. Scheme to assay loss of chiasmata with age in Drosophila oocytes. In the absence of crossovers, accurate segregation of bivalents in Drosophila oocytes is governed by the achiasmate pathway which relies on pericentric heterochromatin. In the presence of a functional achiasmate system, bivalents that lose their chiasmata will still segregate properly. Therefore, the achiasmate pathway must be compromised to determine whether chiasmata are lost as Drosophila oocytes age. Top panel: A functional achiasmate segregation system (depicted by green stars between homologues) will ensure accurate segregation of recombinant bivalents that have lost chiasmata during the aging process. Bottom panel: When the achiasmate pathway is compromised (depicted by the absence of green stars), recombinant bivalents that have lost chiasmata should missegregate. doi:10.1371/journal.pgen.1000263.g004 1–8) remained abundant. Significantly, we found that 16 hours after the aging regimen ceased and egg laying commenced, the distribution of late stages in the aged group resembled that in control ovaries, although the total number of oocytes between stages 8 and 14 was slightly less in the aged ovaries. Consistent with our findings, King [23] reported that stages 9–12 are significantly under-represented in ovaries of 4 day old virgins. Together, these data indicate that oocytes at stages1–8 halt in development and age during the four-day aging regimen, while oocytes at later stages (stages 9–13) continue to mature and do not ‘‘age’’ until they arrest at metaphase I (stage 14). Given the altered distribution of oocytes at different developmen- tal stages following the aging regimen, we carried out NDJ tests to determine which stages during oocyte development are the most susceptible to age-dependent segregation errors. We subjected smc1+/2 mtrm+/2 females to our standard four-day aging regimen and set up crosses to assay meiotic NDJ (see Figure 2). However, this time, we transferred the parents to new vials every 8 hours (instead of every 24 hours) to generate 8-hour ‘‘sub-broods’’ of progeny. This allowed us to measure age-related segregation errors in ‘‘sub-broods’’ of progeny that represented snapshots of oocytes at different meiotic stages (pachytene, diplotene-like and metaphase I). Following the aging regimen, age-dependent NDJ was observed in sub-broods 3 and 4, which arose from oocytes that were fertilized 16–32 hours after the aging regimen was completed (Figure 8A, Table S6). Our cytological analysis (Figure 7 and Figure S1) and the published time-frame for oogenesis progression [19,25], indicate that these sub-broods correspond to oocytes at stages 7 and 8 which have already disassembled their synaptone- mal complex before initiation of the aging regimen [26,27]. In contrast, a significant increase in meiotic segregation errors was not observed during the first 16 hours in aged smc1+/2 mtrm+/2 oocytes (Figure 8A, Table S6). These results argue that metaphase I arrested smc1+/2 mtrm+/2 oocytes (stage 14) are not vulnerable to age-related defects. Similarly, oocytes that undergo aging during pachytene (sub-broods 5 and 6) also appear to be refractory to age effects. Therefore, only oocytes that age during a very specific window of oogenesis (post-pachytene/pre-metaphase I) exhibit age dependent NDJ. To determine what fraction of missegregating chromosomes were recombinant in the aged versus non-aged oocytes, we genotyped the Diplo-X progeny for a subset of sub-broods obtained from the NDJ analysis shown in Figure 8A. In addition to sub-broods 3 and 4 which exhibited age-dependent nondis- PLoS Genetics | www.plosgenetics.org 5 November 2008 | Volume 4 | Issue 11 | e1000263