Age-related molecular variability in Caenorhabditis elegans, T. R. Golden et al. 183 Fig. 4 Incorporation of BrdU into the DNA of aging nematodes. N2 nematodes at 9 days of age (a,b,c) or 11 days of age (d,e,f) were exposed for 24 h to BrdU. BrdU was detected immunohistochemically using a monoclonal antibody to BrdU, and a fluorescent secondary antibody. In younger worms, incorporation of BrdU is apparent in the gonad, near the distal tips (a), and at higher magnification in (b) and (c). As worms age, label is still apparent in the gonad, although fewer nuclei are labeled, and the gonad appears disordered (d), and at higher magnification in (e) and (f) (see arrows). Also, label begins to appear in amorphous regions that are similar in appearance to those detected by DAPI staining [arrowheads in (e) and (f)]. Five of five 11-day-old animals examined displayed nongonad staining, whereas such staining was not seen in 7- or 9-day-old animals. et al. reported necrotic death of hypodermal and intestinal nuclei, but this was occasional, stochastic and mostly end stage. DAPI staining also detected the age-related accumulation of masses of DNA in the body cavity of wild-type nematodes (Fig. 2 and Supplemental movie). These masses appear to contain DNA that is actively replicating, as foci within the masses labeled with BrdU (Fig. 4), and the masses do not accumulate in wild- type worms grown on the DNA synthesis inhibitor FUDR (data © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2007 No claim to original US government works not shown). This leads us to consider these masses to be ‘tumor- like’ growths, which represent uncontrolled DNA replication. These ‘tumors’ likely account for the heterogeneity between middle-aged and old animals with respect to DNA content, and we hypothesize may be detrimental to the health of the animals. In some animals the mass occupies so large a volume of the mid-body region that it potentially impedes the function of the intestine, which could compromise the nutritional status of aging worms. The age-related increase in mean nuclear genome content we observed in wild-type C. elegans via quantitative real-time PCR (Fig. 5a) is likely substantially due to these DNA masses. The increase in nuclear genome copy number was unexpected, as C. elegans have ceased reproduction by 10 days of age (John- son, 1987) and the animal is essentially post mitotic at this stage with the exception of the germ line. The increase in mean nuclear genome copy number is concurrent with the age at which BrdU was incorporated into the masses (Fig. 3), and the maximal mean copy number is achieved at the same age where the masses reach their maximal size (14 days, Fig. 2 and Sup- plemental movie). The source of the massive accumulation of DNA appears to be the hermaphrodite germ line, as males do not develop the masses, and also do not demonstrate an increase in nuclear genome copy number (Fig. 5c). Growth of wild-type worms on plates containing FUDR, which results in sterility, prevents mass accumulation and the increase in nuclear genome copy number (Fig. 5d). Early studies of germ-line development and fertiliza- tion found that in mature hermaphrodites where the sperm were depleted, the nuclear membrane of unfertilized oocytes disappears, and meiosis resumes (Wood, 1988). One round of nuclear division was observed, with subsequent replication of DNA resulting in a highly polyploid oocyte. These unfertilized oocytes are normally expelled. We hypothesize that with age, these unfertilized oocytes are retained and continue to replicate, resulting in the accumulation of massive amounts of nuclear material within the animal. The long-lived daf-2 strains did not accumulate masses to the same extent as N2 (Fig. 3 and Supplementary Fig. S1). Additionally, an age-related increase in mean genomic DNA content is not observed in the long-lived strain daf-2(e1370) (Fig. 5b). A doubling of DNA content is observed between 4 and 6 days of age, which may reflect an increase in embryo content, or the increase in body size that occurs between these ages. After this point, however, mean genomic DNA content does not increase. This is in accord with our theory that unfertilized oocytes are a source of the masses, as daf-2(e1370), which rarely developed masses, also lays almost no unfertilized oocytes (Gems et al., 1998). As an additional test of whether the hermaphrodite germ line was required for the DNA masses and changes in nuclear genome copy number, we examined the strain glp-4, which contains a temperature-sensitive mutation resulting in sterility and the production of only a few germ cells when grown at the restrictive temperature. We found that this strain’s genomic DNA did not amplify at the restrictive temperature, giving an