Figure 1

Comparison of Global DNA methylation patterns in the chromosomes of mouse embryonic fibroblasts and neonatal spermatogonia. A-C) Fluorescent micrographs of metaphase chromosomes from primary mouse embryonic fibroblasts showing highly methylated autosomal centromeric domains (thin arrow) as determined by 5-methyl cytosine (5-mC) staining (red). D-F) Chromosomes in neonatal spermatogonia lack global DNA methylation at pericentric heterochromatin, while 5-mC (red) decorates entire chromatids in the majority of autosomes (thin arrow). Two chromosomes on each metaphase spread consistently exhibit lack of 5-mC staining (bold arrow and arrowhead). These patterns are reproducible after using two standard chromosome fixation protocols using either paraformaldehyde (PFA) or methanol/acetic acid (MeOH/AA) as shown in (G-I). J-L) Fluorescent in situ hybridization (FISH) and subsequent determination of DNA methylation patterns (5-mC; red) confirms that the two hypo-methylated chromosomes observed on each spread correspond to the X chromosome (green; arrow head) and the Y chromosome (red; Inset and bold arrow in K-L). Note the lack of 5-mC staining on the Y chromosome and the low levels of global methylation on the X chromosome compared to autosomes (thin arrows) in germ cell metaphases. DNA was counterstained with HOECHST 33258 (blue). Scale bars = 10 μm.