Oocyte Age-Dependent NDJ Figure 6. Scheme to determine the recombinational history of X chromosomes that missegregate in the NDJ test. A hypothetical example is presented in the two panels to illustrate how we determine the genotype of the two missegregating X chromosomes that give rise to a Diplo-X female. Top panel: In this scenario, a single crossover between cv-v on the X chromosome in the oocyte generates a bivalent with two recombinant and two non-recombinant chromatids. Missegregation during meiosis I results in an egg with two X chromosomes that is fertilized and develops into a ‘‘Diplo-X’’ adult female. In this schematic, the Diplo-X female inherited one recombinant and one non-recombinant X chromosome from the oocyte. Bottom panel: The Diplo-X female is crossed to a normal male and her male progeny are scored for the different markers on the X chromosome. Genotyping is complicated by the fact that crossovers can occur between the two X chromosomes in the oocytes of the Diplo-X females. However, the genotype of the X chromosomes in the Diplo-X female (right, top panel) can be assigned because in any recombination analysis, the parental chromosomes are the most predominant. In this example, the parental chromosomes are shown in red. Note that it is possible for a Diplo-X female to inherit two non-recombinant chromatids from a recombinant tetrad. In addition, a double crossover between two adjacent markers will be invisible. For these reasons, the frequency at which recombinant chromosomes nondisjoin is underestimated in our analyses. doi:10.1371/journal.pgen.1000263.g006 smc1+/2 mtrm+/2 aged and non-aged oocytes, we have not observed any obvious differences in SC morphology, or the timing of its assembly or disassembly (VVS and SEB, unpublished results). Therefore, we speculate that the synaptonemal complex may play a role in protecting sister-chromatid cohesion from age-induced deterioration. The differential response of pachytene and diplo- tene-like Drosophila oocytes to the aging process has profound implications regarding the vulnerability of human oocytes to age- induced loss of meiotic cohesion. Our data argue that the meiotic stage (dictyate) at which human oocytes remain suspended as women age is also the stage during which recombinant chromosomes are at highest risk for loss of chiasmata. Because the achiasmate pathway in Drosophila oocytes will ensure the accurate segregation of recombinant chromosomes that lose chiasmata due to deterioration of arm cohesion during the aging process, we needed to dismantle this system for our analysis. Therefore, we used a P-element insertion in matrimony (mtrmKG08051) as a genetic tool to disrupt the achiasmate pathway. However, we do not think that heterozygosity for mtrm contributes to age-dependent loss of arm cohesion. Recent work has demonstrated that Mtrm protein physically interacts with and inhibits the activity of Polo kinase in Drosophila oocytes from stage 11 until nuclear envelope breakdown (NEB) at stage 13 [22]. When Mtrm protein is reduced (mtrm+/2), NEB can occur prematurely (at stage 12) and the spindle assembles around a karyosome that is less compact than in wild type. Although this may account for the eventual missegregation of achiasmate chromosomes in mtrm+/2 heterozygotes, sister chroma- tid cohesion remains intact in these oocytes as evidenced by the striking chiasmata visible in the individualized bivalents [22]. In addition, premature NEB in mtrm+/2 oocytes still occurs much later than the stages that exhibit sensitivity to aging in our experiments (stages 7–8). Therefore, we conclude that reducing the dosage of Mtrm is not causing the increased missegregation of recombinant chormosomes that we observe in aged oocytes. PLoS Genetics | www.plosgenetics.org 7 November 2008 | Volume 4 | Issue 11 | e1000263