Figure 7. Quantification of oocyte stages present in non-aged and aged ovaries at different time-points following the aging regimen. The number of oocytes at each of the indicated stages was tabulated for one ovary from three different smc1+/2 mtrm+/2 females and the average is presented. Immediately after the aging regimen (time-point 0), the number of mature oocytes (stage 13/14) is ,2.5 fold higher in the aged ovaries than in non-aged ovaries. This contrasts sharply with the decline in stage 10 oocytes at this same time point. The number of stage 8 oocytes is similar in aged and non-aged ovarioles immediately following aging regimen. These results are consistent with a previous report [23] that when egg-laying is inhibited, stages 1–8 halt in development and ‘‘age’’ while stages 9–13 continue to develop and only arrest once they reach stage 14. After egg-laying resumes, the relative abundance of each of the different stages is similar in aged and non-aged ovaries by 16 hours although fewer oocytes are present in the aged ovaries. doi:10.1371/journal.pgen.1000263.g007 Because the achiasmate segregation pathway is not completely disrupted in mtrmKG08051 heterozygotes, we also have been able to assess the effect of aging on the segregation of non-recombinant chromosomes. Interestingly, our data demonstrate that when the dosage of SMC1 is reduced, MI missegregation of non- recombinant chromosomes also increases with age. This result is reminiscent of our previous findings that NDJ of achiasmate chromosomes increases with age when activity of the meiotic Oocyte Age-Dependent NDJ cohesion protein ORD is compromised by a hypomorphic missense mutation [9]. In Drosophila oocytes, accurate segrega- tion of achiasmate chromosomes relies on homologous pairing of centromere proximal heterochromatin [28–30] and both ORD and the cohesin complex are highly enriched at the percentric heterochromatin in oocytes [11,17]. In addition, our recent data indicate that heterochromatin-mediated pairing is moderately disrupted by ord mutations, even in the absence of aging (VVS and SEB, unpublished results). Together, these data suggest that in addition to holding sister chromatids together, cohesion proteins play an important role in maintaining the heterochromatic pairing of achiasmate homologues in Drosophila oocytes. Whether cohesion proteins function directly to ‘‘glue’’ homologues together or play a more indirect role, such as influencing the structure of heterochromatin, remains to be determined. Regardless of the mechanism, loss of heterochromatin-associated cohesion proteins with age could account for the increased missegregation of non- recombinant chromosomes that we observe. Our data support the hypothesis that age-dependent NDJ in women is caused at least in part by progressive loss of sister- chromatid cohesion over time. One unresolved issue regarding meiotic cohesion in human oocytes is whether the original cohesin molecules used to establish cohesion are maintained for decades or continually replaced as oocytes age. Addressing this question will be essential in understanding the specific mechanisms that lead to loss of meiotic cohesion and chiasma maintenance during the aging process. Early experiments in yeast led to the widely accepted model that cohesion between sister chromatids can only be established during DNA replication [31,32]. However, recent evidence in mammalian tissue culture cells indicates that cohesin association with chromatin is much more dynamic than originally predicted [33]. In addition, experiments in yeast have recently demonstrated that cohesion can be re-established on a genome- wide scale during G2/M in a DNA damage-dependent manner [34,35] and support the model that non-canonical mechanisms can establish cohesion after S phase is completed. The power of genetic manipulation combined with the ability to age oocytes makes Drosophila an ideal model system to address whether re-establishment of meiotic cohesion occurs during prophase I. These future studies will be pivotal in understanding how cohesion dynamics during meiosis govern chiasma mainte- nance and ultimately, why and how loss of cohesion occurs as oocytes age. Materials and Methods Drosophila stocks Flies were reared at 25uC on standard cornmeal molasses media. The smc1ex46 allele is a deletion that removes the gene [12] and is denoted as smc12. The mtrm[KG08051] allele results from a P- element disruption of the gene [21] and is denoted as mtrm2. Descriptions of the other genetic markers and chromosomes used in this study can be found at http://www.flybase.org. Although several proteins (Axs, Ald/Mps1, Nod and Mtrm) are required for the accurate segregation of achiasmate chromosomes in Drosophila oocytes, we considered a mtrm allele to be the ideal choice for our studies [21,22]. In contrast to nod, achiasmate segregation is severely affected in the mtrm heterozygote and chromosome loss is not observed [36]. In addition, unlike Axs oocytes, spindle defects and premature onset of anaphase I have not been observed in mtrm heterozygotes [37–39]. We thought it unwise to compromise achiasmate segregation using ald mutations given the role of this protein in the spindle assembly checkpoint; ald oocytes also exhibit low levels of NDJ of the exchange bivalents PLoS Genetics | www.plosgenetics.org 8 November 2008 | Volume 4 | Issue 11 | e1000263