Oocyte Age-Dependent NDJ Figure 2. Scheme for aging Drosophila oocytes and measuring nondisjunction. (A) Each Drosophila ovary is composed of several ovarioles that contain a linear array of oocytes at progressive stages of development. Meiosis is initiated at the anterior tip of the ovariole, in the germarium (left). After exit from the germarium, the oocytes move posteriorly through the ovariole (stages 2 through 14). Not all oocyte stages are present at any given time in a single ovariole. This schematic is adapted from Robinson et al. [45]. (B) For the aging regimen, virgin females of the same genotype are divided into two groups. Control females (left) are incubated with males and lay eggs continuously. Experimental females (right) are deprived of males and egg-laying is suppressed. In these females, developmental progression of oogenesis is halted and oocytes ‘‘age’’ within the female. After four days of this aging regimen, control and experimental females are placed in vials with males and the flies are transferred at 24-hour intervals to generate ‘‘24 hour broods’’ of progeny for NDJ analysis. Each brood corresponds to oocytes that were halted at specific stages of development during the aging regimen (see panel A). Meiotic NDJ is measured in all three broods of both aged and non-aged oocytes (see below). (C) To measure NDJ, we made use of an X chromosome dominant marker, Bar (B2), that affects eye shape. Bar+ experimental females are crossed to Bar2 males that contain an attached X^Y chromosome. The sperm generated by these males will contain either X^Y with the Bar2 marker or no sex chromosome (designated ‘‘0’’). If meiotic segregation is normal in the female, oocytes will be produced that contain one X chromosome. However, segregation errors during female meiosis will result in exceptional gametes that contain either two X chromosomes (Diplo) or no X chromosomes (Nullo). The adult progeny resulting from normal and exceptional gametes can be distinguished based on their sex and eye shape (due to Bar2). In this test, all of the normal progeny survive, but only half of the exceptional progeny are viable. Therefore, the number of exceptional progeny is doubled and the total number of progeny is adjusted when calculating the %NDJ. doi:10.1371/journal.pgen.1000263.g002 somes in Drosophila oocytes [21]. However, meiotic cohesion, synaptonemal complex assembly and crossover frequency appear to be unaffected in mtrm+/2 oocytes [21,22]. Therefore, to monitor whether exchange bivalents lose chiasmata with age, we compromised the achiasmate pathway by reducing the dosage of mtrm in smc1+/2 females. We observed increased segregation errors in smc1+/2 oocytes following the four-day aging regimen when the achiasmate pathway was also compromised (mtrm+/2) (Figure 5A). Significant levels of age-dependent nondisjunction were observed for broods 1 and 2, which represent oocytes that are developmentally most mature during the aging regimen (see Figure 2A). We have performed multiple experiments to compare NDJ in smc1+/2 mtrm+/2 aged and non-aged oocytes and have also assayed missegregation of different X chromosomes (data not shown). Although the absolute level of NDJ may vary between experi- ments, we have repeatedly observed higher levels of NDJ in smc1+/2 mtrm+/2 aged oocytes in the first two 24 hour broods (see Tables S3A and S3B for examples of raw data from two independent experiments). In contrast, mtrm+/2 oocytes with normal levels of SMC1 do not exhibit age-dependent NDJ in the first brood (Table S4); we did detect a significant increase in brood 2, but the absolute level of NDJ was relatively low (Table S4). The finding that increased meiotic chromosome missegrega- tion in aged Drosophila oocytes occurs when the dosage of a cohesin subunit is reduced is consistent with deterioration of cohesion and loss of chiasma maintenance as an underlying cause for age-dependent NDJ. Recombinant Chromosomes Missegregate in Older smc1+/2 mtrm+/2 Oocytes To ask whether deterioration of chiasmata contributed to the increase in age-dependent nondisjuntion in smc1+/2 mtrm+/2 oocytes, we assayed the recombinational history of the misse- gregating chromosomes. For this analysis, we collected ‘‘Diplo-X’’ female progeny that received two maternally contributed X chromosomes due to meiotic missegregation (see Figure 2C) and performed an additional cross to genotype the two X chromosomes (Figure 6). Using this strategy, we determined whether missegre- gating chromosomes had undergone one or more crossovers (Table S5). In addition, a centromere-proximal marker (car) allowed us to assess whether NDJ events involved homologous chromosomes (MI NDJ) or sister chromatids (MII NDJ). The genotype of the Diplo-X females suggested that the majority of PLoS Genetics | www.plosgenetics.org 3 November 2008 | Volume 4 | Issue 11 | e1000263