Figure 5. Both chiasmate and achiasmate homologues exhibit age-dependent NDJ in smc1+/2 mtrm+/2 oocytes. (A) An odds ratio is used to represent the relative level of NDJ in aged versus non-aged oocytes for 24-hour broods. An odds ratio .1 indicates that NDJ is more likely in the aged oocytes than in the non-aged oocytes. The error bars correspond to 61 standard error. Two independent experiments are shown. In both experiments, NDJ in aged oocytes is significantly higher than in non-aged oocytes for broods 1 and 2 (**P = 0.0010, *P = 0.0062; NNP = 0.0017, NP = 0.0439) but age-dependent NDJ is not observed for Brood 3 (P = 0.4751, P = 0.1416 for Experiments I and II respectively). Although the absolute levels of NDJ vary, the same pattern is observed for both experiments. For each experiment, at least 1000 progeny were scored for each brood. The raw data for the two independent experiments are shown in Tables S3A and S3B. (B) Diplo-X females from brood 1 of Experiment II were genotyped (see Figure 6). R denotes Diplo-X females containing at least one recombinant X chromosome. NR denotes Diplo-X females in which both X chromo- somes were non-recombinant (as judged by the markers scored). N is the number of total progeny scored in the NDJ assay. The frequency at which R and NR chromosomes missegregate also is shown (R/N and NR/ N). Missegregation of both recombinant and non-recombinant chro- mosomes is more prevalent in aged oocytes. doi:10.1371/journal.pgen.1000263.g005 junction, we also analyzed the recombinational history of missegregating-X chromosomes in sub-broods 1 and 6, which represent opposite ends of the developmental spectrum examined in this study (Tables S7, S8). Again, we found that both recombinant and non-recombinant chromosomes missegregated in aged and non- aged oocytes (Tables S7, S8). In addition, a striking trend emerged for the aged oocytes when we calculated the frequency at which recombinant (R) chromosomes missegregated in each sub-brood (R/ N; Figure 8B). NDJ of recombinant chromosomes in sub-brood 3 aged oocytes was significantly higher than that for aged oocytes in sub-brood 1 (P = 0.0029) or sub-brood 6 (P = 0.0179). In addition, this trend holds true when the NDJ of recombinant chromosomes in sub-brood 4 aged oocytes is compared to that for sub-broods 1 or 6 aged oocytes, although the difference is not statistically significant. These data contrast strongly with those for non-aged oocytes, in which recombinant chromosomes missegregate at similar frequen- cies in the four sub-broods analyzed. The data shown in Figure 8 provide compelling evidence that only oocytes at a specific meiotic stage are vulnerable to age- Oocyte Age-Dependent NDJ dependent segregation errors even when all oocytes within the ovary are subjected to the aging regimen. The increased nondisjunction observed in specific sub-broods arises in part because segregation of recombinant bivalents becomes more error- prone (Figure 8B). When one takes into account the well- documented time-line for oocyte development while evaluating the sub-brood NDJ results, a striking pattern emerges (Figure 8C). When pachytene oocytes undergo the aging regimen, recombinant chromosomes do not exhibit an age-dependent increase in nondisjunction (sub-broods 5 and 6). In contrast, sub-broods 3 and 4, which display the greatest age-related defects, are composed primarily of oocytes at stages 7 and 8 of oocyte development. Both electron microscopy and immunofluorescence analyses have verified that disassembly of the synaptonemal complex in Drosophila oocytes initiates prior to these stages [26,27]. Although Drosophila chromosomes do not exhibit typical diplotene/ diakinesis morphology in prophase I oocytes, stages 7 and 8 of oogenesis correspond to these classical meiotic stages [19]. Transit through each of these stages takes approximately 5–8 hours during normal oogenesis, but when their developmental progression is halted by our aging regimen, meiotic chromosomes remain suspended in this state up to 19 times longer than usual. Of special note is the finding that recombinant chromosomes are most susceptible to segregation errors if Drosophila oocytes undergo aging during diplotene (Figure 8) because this is the meiotic stage at which human oocytes remain arrested for decades. Our sub-brood analysis also suggests that non-recombinant chromosomes in smc1+/2 mtrm+/2 oocytes missegregate more frequently when aging is induced in oocytes prior to metaphase I (Figure S2), although the difference between sub-brood 1 and the other sub-broods is not statistically significant (0.10,P,0.27). However, non-recombinant chromosomes in sub-brood 3 are significantly more vulnerable to missegregation after aging (P = 0.029). These results support the model that the connections that keep achiasmate bivalents together depend at least in part on meiotic cohesion proteins [9]. Discussion Together, our data support the hypothesis that weakening of meiotic cohesion with age is a significant determinant of age- dependent nondisjunction in both fly and human oocytes. One major advantage of these studies is that we have lowered but not eliminated the level of the cohesin subunit SMC1. Although smc1+/2 oocytes are sensitized to the effects of aging, meiotic cohesion is initially intact. Therefore our regimen allows us to observe the effect of aging upon the normal cohesin complex that functions during Drosophila meiosis. In this respect our analysis differs markedly from previous studies using knock-out mice in which the meiosis-specific subunit SMC1-b was eliminated [8]. Here we demonstrate that deterioration of normal meiotic cohesion during the aging process causes loss of chiasma maintenance. Loss of arm cohesion in aged oocytes accounts for the increased frequency with which recombinant chromosomes missegregate during meiosis I. In contrast, low levels of MII NDJ indicate that centromeric cohesion remains intact, at least for the duration of our aging regimen. Recombinant chromosomes in Drosophila oocytes that undergo aging after they have disassem- bled their synaptonemal complex (diplotene-like) are most vulnerable to age effects and these oocytes bear a striking resemblance to the stage at which human oocytes remain arrested for decades. Interestingly, Drosophila oocytes that are subjected to aging during pachytene do not exhibit significant age-dependent nondisjunction. Using epifluorescence microscopy to compare PLoS Genetics | www.plosgenetics.org 6 November 2008 | Volume 4 | Issue 11 | e1000263