nondisjunction in the oocytes (see Figure 2C). To generate 24- hour broods, 7 female flies were mated with 3–5 X^Y, v f B males (per vial). The parents were transferred to new vials every 24-hours and three broods of progeny were analyzed for NDJ. Generation of 8-Hour Sub-Broods of Progeny For simplicity, we have used the term ‘‘sub-broods’’ to differentiate 8-hour broods of progeny from the 24-hour broods of progeny. To generate sub-broods of progeny at the end of the aging regimen, 21 experimental or control females were crossed to 10 X^Y, v f B males (per vial). The parents were transferred to new vials every 8-hours for a total of 48 hours and six sub-broods of progeny were analyzed for NDJ. Nondisjunction Assay Because Drosophila can tolerate certain sex chromosome aneuploidies, segregation errors during meiosis can be monitored in the viable progeny by using differentially marked sex chromo- somes (see Figure 2C). To compensate for the fact that only half of the exceptional progeny survive (see Figure 2C), total NDJ was adjusted according to the following formula: [2*Exceptional Progeny/(2*Exceptional Progeny+Normal Progeny)]*100. Recombinational History of Diplo-X Females To assess whether recombinant bivalents missegregated in oocytes, the Diplo-X female progeny obtained from NDJ tests were genotyped. Each Diplo-X female was crossed to two yw males and the genotype of the X chromosomes of the Diplo-X female was inferred from the sc, cv, v, f, car markers in her male progeny (see Figure 6). Because some fraction of Diplo-X females either died before they could be genotyped or were sterile/sub-fertile, the number of Diplo-X females genotyped was lower than the number of Diplo-X females recovered from the NDJ test. Light Microscopic Analysis of Ovaries and Ovarioles After female flies were subjected to the four-day aging regimen, ovaries from experimental and control females were hand- dissected in modified Robb’s buffer [36] at 8 hour intervals and fixed in 4% formaldehyde, PBS (130 mM NaCl, 7 mM Na2HPO4 and 3 mM NaH2PO4) for 10 min. The ovaries were rinsed in PBS containing 0.01% Tween-20. Images were captured using a SMZ1500 stereo-microscope (Nikon) equipped with a Pixelink camera (Diagnostic Instruments). To quantify the relative number of ooctyes at specific developmental stages, a single fixed ovary from three females was teased to generate individual ovarioles. Oocyte stages were tabulated and averaged for aged and non-aged ovaries at each time point. Oocytes were staged based on the standard morphological criteria described by Mahowald and Kambysellis [43]. Briefly, oocyte stages were distinguished based on the size of the egg chamber, the relative size of the oocyte and the presence of yolk within the oocyte. Yolk deposition begins during stage 8 and provides a convenient marker. Mature (stage 13–14) oocytes were distinguished by the presence of chorionic appendages. Immunoblot Analysis of SMC1 Levels in Ovary Extracts Newly eclosed wild-type and smc1 heterozygous females were fattened with yeast for two days. 30 sets of ovaries from each genotype were dissected in modified Robb’s buffer [36] and frozen in liquid N2. Each frozen tissue sample was homogenized in 240 mL of buffer containing 8 M Urea, 2% SDS, 100 mM Tris HCl pH 6.8 and 5% Ficoll containing 4 mM AEBSF (protease inhibitor, Sigma). The extract was cleared by centrifugation at Oocyte Age-Dependent NDJ 13,000 rpm for 10 min at room temperature and the supernatant was aliquoted and frozen in liquid N2. The protein concentration for each extract was determined using a Bradford assay (Biorad). For each genotype, 10 and 20 mg of total protein was separated by SDS-PAGE on a 7.5% gel and transferred to PVDF membrane. The blot was cut horizontally and anti-SMC1 guinea pig serum [11] was used at 1:1000 dilution for the top half of the blot and monoclonal DM1A (Sigma) was used at 1:15,000 to detect the a- tubulin loading control on the bottom half of the blot. Data Analysis and Statistical Tests To compare the relative frequency of missegregation occurring in aged and non-aged oocytes, an odds ratio (OR) was calculated. The odds ratio provides a method for determining whether the frequency of a certain event (missegregation) is equal for two different treatments (aged and non-aged). In our analysis, it is calculated as: NAAee==ANnAn where Ae = # of exceptional progeny from aged oocytes, An = # of normal progeny from aged oocytes, NAe = # of exceptional progeny from non-aged oocytes, and NAn = # of normal progeny from non-aged oocytes [44]. If the treatments result in equal frequencies of the event, then the odds ratio will be 1. In our analysis, an OR .1 implies that the frequency of missegregation is higher when oocytes undergo aging than when they do not undergo aging. Standard errors (SE) are calculated on the logarithm of the odds ratio according to: SElogOR~ pﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃAﬃﬃﬃnﬃﬃﬃÞﬃ. In Figures 5 and 8, ð1=AeÞzð1=AnÞzð1=NAeÞzð1=N error bars represent 61 SElogOR retransformed back into non- logarithmic units. For the NDJ tests, P values were calculated using a 262 x2 Contingency Test (two-tailed). For these calculations, raw data values (not adjusted values) for a specific brood or sub-brood were compared for aged versus non-aged treatments (# of exceptions and # of normal progeny for aged and non-aged). A 262 x2 Contingency Test also was used to analyze the recombinational history of missegregating chromosomes recovered in Diplo-X progeny. For all tests, a P value of ,0.05 was considered statistically significant (rejection of the hypothesis that the two groups are the same). Supporting Information Figure S1 Excess of mature oocytes in the aged ovaries is cleared by 16 hours after the aging regimen. This figure shows representative images of whole ovaries and single ovarioles from smc1+/2mtrm+/2 control and experimental females dissected at 8- hour intervals after the aging regimen. Mature oocytes (stage13/ 14) at the posterior end of the ovariole can be distinguished (in part) by their chorionic appendages (yellow arrows). The scale bars are 100 microns. At time-point 0 after the aging regimen, mature oocytes are far more abundant in the aged ovary than in the non- aged ovary. It is not uncommon to observe two mature oocytes per ovariole in aged ovaries at this time-point. When both experi- mental and control females are allowed to lay eggs for 8 hours after the regimen, the difference between the number of mature oocytes in the aged ovary and non-aged ovary is reduced. By 16 hours, the excess mature oocytes have been cleared from the aged ovaries, and the distribution of stages found in experimental and control females is very similar (see Figure 7). Found at: doi:10.1371/journal.pgen.1000263.s001 (9.77 MB TIF) Figure S2 Non-recombinant chromosomes exhibit increased missegregation when oocytes undergo aging. The number of non- recombinant (NR) Diplos recovered in sub-broods 1, 3, 4 and 6 is shown for aged oocytes and non-aged oocytes (also see data in PLoS Genetics | www.plosgenetics.org 10 November 2008 | Volume 4 | Issue 11 | e1000263