Oocyte Age-Dependent NDJ Figure 3. Chiasma formation is not severely disrupted when SMC1 is reduced. (A) Whole ovary extracts from wild-type and smc1+/2 females were analyzed by immunoblot analysis using SMC1 antiserum [11]. a-tubulin was used as a loading control. Note that the relative intensity of SMC1 signal is lower in the smc1+/2 extract than in wild type. A visual comparison suggests that SMC1 is reduced by approximately two-fold (SMC1 signal intensity is similar in the lanes containing 20 mg of smc1+/2 extract and 10 mg of wild-type extract). (B) Meiotic recombination in wild-type and smc1+/2 females was measured in four intervals along the X chromosome: sc-cv, cv-v, v-f, f-car. Crossovers were reduced in the distal intervals (sc-cv, cv-v) in smc1+/2 oocytes. The tetrad exchange rank was computed from the recombination data and the percentage of bivalents for each rank is shown. Note that E0 tetrads increase in the mutant females at the expense of E1, E2, E3 tetrads. doi:10.1371/journal.pgen.1000263.g003 NDJ events (70/72) occurred during meiosis I (Table S5). Approximately one-third of all Diplo-X females arose from nondisjunction of exchange tetrads (R) in both aged and non- aged oocytes (Figure 5B). Notably, recombinant bivalents missegregated 1.8 times more frequently in the aged oocytes than in non-aged oocytes (compare R/N for aged and non-aged, Figure 5B). In addition, the number of recombinant bivalents that missegregate is under-represented in our assay because it is possible for a Diplo-X female to inherit two non-recombinant chromatids from a recombinant tetrad (see Figure 6). These data argue that upon reduction of SMC1, recombinant bivalents become more vulnerable to missegregation when oocytes age. Although the achiasmate pathway is compromised in mtrm+/2 mutants, our data indicate that it is not completely disengaged. An estimated 26% of the tetrads are achiasmate in smc1+/2 oocytes (Figure 3B) but we only observe 4–9% NDJ in non-aged oocytes when the achiasmate pathway is compromised (smc1+/2 mtrm+/2, Table S3). Moreover, segregation of an X chromosome (In(1)dl-49) harboring an inversion that severely suppresses crossovers is not completely random in mtrm+/2 oocytes (Table S1). The data in Figure 5B indicate that when the level of SMC1 protein is reduced, aging further increases the frequency at which achiasmate chromosomes missegregate in mtrm+/2 oocytes (compare NR/N for aged and non-aged). These data implicate the cohesin complex in accurate disjunction of achiasmate as well as chiasmate bivalents. However, the low number of MII NDJ events that we observe (as determined by the centromere proximal car marker) argues that the increased missegregation of non-recombinant chromosomes is not due to loss of centromeric cohesion between sister chromatids. Oocytes Are Most Susceptible to Age-Related Segregation Defects After Pachytene but Before Metaphase I Our analysis of age-dependent NDJ in smc1+/2 mtrm+/2 oocytes indicates that broods 1 and 2 are most susceptible to age effects. Each Drosophila ovariole consists of multiple oocytes at progressive stages of development. At the posterior end of the ovariole (stages 13/14), the mature oocytes undergo nuclear envelope breakdown and spindle assembly. Stage 14 oocytes remain arrested at metaphase I until passage through the oviduct triggers resumption of the meiotic divisions. After an aging regimen, the most mature oocytes are laid first and therefore will contribute to the progeny in brood 1 (see Figure 2A). In mature oocytes, the chromosomes have already made stable connections with the meiotic spindle microtubules and premature loss of chiasmata during the aging regimen should not affect their segregation. However, if the mature oocytes develop gross defects in the spindle machinery as they age, recombinant chromosomes could missegregate by a mechanism unrelated to chiasma maintenance. Conversely, oocytes that undergo aging at a stage prior to meiotic spindle assembly would depend on chiasma maintenance for proper segregation. Therefore, it was important to determine what fraction of broods 1 and 2 correspond to metaphase I arrested oocytes. To compare the relative distribution of oocytes at different developmental stages in ovaries from smc1+/2 mtrm+/2 control females and those subjected to the aging regimen, we examined ovaries at 8-hour intervals after completion of the four-day regimen. One striking difference was the increased number of mature oocytes (stages 13/14) following aging (Figure 7 and Figure S1). Previous reports by other investigators also have documented that mature oocytes accumulate when egg laying is suppressed [23,24]. Quantitative analysis indicated that our aging regimen resulted in a ,2.5-fold increase in mature oocytes compared to the ovarioles of control females (Figure 7). However, after 16 hours of egg laying, this excess of mature oocytes was no longer observed. These results argue that in our NDJ tests, at least some fraction of progeny in brood 1 (0–24 hours) arise from oocytes that have not yet assembled meiotic spindles. Evaluation of the distribution of different developmental stages in fixed ovaries also indicated that following the aging regimen, the increase in mature oocytes occurred primarily at the expense of stage 9–12 oocytes (Figure 7) while oocytes at earlier stages (stages PLoS Genetics | www.plosgenetics.org 4 November 2008 | Volume 4 | Issue 11 | e1000263