Author Summary In humans, chromosome segregation errors during meiosis are the leading cause of birth defects and miscarriages. Moreover, as women age, these errors increase dramati- cally. For accurate segregation during the first meiotic division, homologous chromosomes must remain physi- cally associated until anaphase I. Normally, attachments along the arms of sister chromatids keep the recombinant homologues together. Human oocytes complete meiotic recombination during fetal development and arrest until ovulation. Therefore, accurate segregation of homologous chromosomes during the first meiotic division requires that recombinant chromosomes remain associated for decades. One hypothesis to explain why segregation errors increase as women age is that the connections between sister chromatids deteriorate over time and allow recom- binant homologues to dissociate prematurely. Here, we address this hypothesis using Drosophila as a model system. We find that when Drosophila oocytes undergo experimentally induced aging, recombinant homologues missegregate during meiosis I. Furthermore, the meiotic stage at which Drosophila oocytes are most vulnerable to age-induced errors is analogous to the stage at which human oocytes remain arrested for decades. Together, our data argue that aging does cause premature loss of the connections between meiotic chromosomes and that this is a major determinant of segregation errors in both Drosophila and human oocytes. a brief arrest at metaphase I before ovulation. However, when egg laying is suppressed, the majority of Drosophila oocytes within each ovariole are halted in developmental progression and ‘‘age’’ within the abdomen of the female (Figure 2B). Such experimen- tally induced aging of Drosophila oocytes can be used to mimic the normal aging process that human oocytes undergo within the ovary during a female’s lifespan [9]. We have used this aging Figure 1. Proposed mechanism for age-dependent loss of chiasma maintenance in human oocytes. Different colors are used to represent the homologous chromosomes, each composed of identical sister chromatids. Meiotic cohesion is represented by solid black circles linking the sister chromatids. Centromeres are depicted as white bi-lobed structures. Sister chromatid cohesion distal to the site of a crossover is necessary to hold recombinant homologues together and this linkage ensures their accurate segregation during meiosis I. During meiotic prophase I, cohesion-dependent association of recombinant homologues is manifest as chiasmata that occur at crossover sites. Because meiotic recombination in human oocytes occurs during fetal development and is followed by a prolonged dictyate arrest, chiasma maintenance requires that sister cohesion remain intact for decades. One explanation for why meiotic segregation errors are more prevalent in older women is that cohesion between sister chromatids deteriorates with age and renders recombinant chromosomes susceptible to missegregation. doi:10.1371/journal.pgen.1000263.g001 Oocyte Age-Dependent NDJ regimen to test the hypothesis that meiotic cohesion deteriorates as the oocyte ages and increases the frequency at which recombinant homologues missegregate. Although maternal age does not dictate the age of the oocyte in fruit flies as it does in humans, for simplicity we will refer to increased NDJ in experimentally aged Drosophila oocytes as ‘‘age-dependent NDJ.’’ Results The cohesin complex consists of four subunits and is required for sister-chromatid cohesion during mitosis and meiosis [6,10]. Although wild-type Drosophila oocytes subjected to our standard four-day aging regimen do not exhibit increased levels of NDJ [9], we reasoned that this time period may be insufficient for cohesion to deteriorate enough to detect a loss in chiasma maintenance and that reduction of functional cohesin might render oocytes more vulnerable to aging effects. Unlike other eukaryotes, meiosis- specific cohesin subunits have not been uncovered in the Drosophila genome [11]. Therefore, to weaken but not eliminate meiotic cohesion, we reduced the dosage of the cohesin subunit SMC1 by utilizing flies heterozygous for a deletion that removes the smc1 gene [12]. Immunoblot analysis confirmed that the level of SMC1 protein is reduced approximately two-fold in ovaries from smc1+/2 females (Figure 3A). Reduction of SMC1 Protein Does Not Eliminate Crossovers In order to assay loss of chiasma maintenance with age, we needed to verify that chiasma formation was not severely disrupted in smc1+/2 oocytes. Cohesion between sister chromatids is required for normal levels of crossovers during meiosis [13–17]. Therefore, we measured the frequency and distribution of exchange in smc1+/2 heterozygous females (Figure 3B). Although reduction of SMC1 protein has a semi-dominant effect on the number and distribution of meiotic crossovers during female meiosis, high levels of homologous exchange were still observed (76% of the wild-type control, Figure 3B). The tetrad exchange rank for Drosophila oocyte bivalents can be estimated from analysis of recombinant and non-recombinant meiotic products [18]. Normally, the X chromo- some is achiasmate (non-recombinant) in 6–12% of Drosophila oocytes [19]. Our recombination data indicates that although crossovers were reduced in smc1+/2 oocytes, the majority of tetrads (74%) had at least one exchange event (Figure 3B). In addition, viability and fertility of smc1+/2 females is normal and they do not exhibit meiotic segregation defects in the absence of an aging regimen (Table S1). Therefore, smc1+/2 oocytes provide an excellent sensitized system that can be used to assay whether cohesion and chiasma maintenance decline with age. Reduced Cohesin Leads to Age-Dependent NDJ To examine the effect of SMC1 dosage on age-dependent NDJ, we subjected smc1+/2 females to the aging regimen and assayed for chromosome missegregation (see Figure 2B, 2C). However, meiotic NDJ did not increase when smc1+/2 oocytes were aged (Table S2). Because Drosophila females harbor a robust mechanism that directs the segregation of achiasmate chromo- somes [20], it is possible that this achiasmate mechanism also ensures accurate disjunction of recombinant chromosomes that fail to maintain chiasmata (Figure 4). Therefore, we reasoned that in order to detect missegregation of recombinant chromosomes in Drosophila oocytes that have lost chiasmata, the achiasmate system must be compromised (Figure 4). Hawley and colleagues have shown that P-element disruption of one copy of matrimony (mtrm[KG08051]/+) disrupts the segregation of achiasmate chromo- PLoS Genetics | www.plosgenetics.org 2 November 2008 | Volume 4 | Issue 11 | e1000263