Global Chromatin Reprogramming in Fetal Germ Cells remodeling. We also expected that the global chromatin changes will be sex-specific: they will be more pronounced in male than female fetal germ cells, because at fetal stages only the male but not the female germ line undergoes global de novo DNA methylation. Results DNA methylation We visualized DNA methylation by staining germ cells and somatic cells of the developing male and female fetal mouse gonad at different developmental stages using an anti-5-methyl cytosine (5mC) antibody (Figure 1). Germ cells were distinguished by double staining with antibodies against germ cell markers Oct3/ Oct4 or Ddx4. We found that 5mC levels were very low compared to somatic cells in both male and female germ cells between 11.5 and 15.5 dpc. The global 5mC level sharply increased in male germ cells at 17.5 dpc but remained low in female germ cells at all stages. These results were in agreement with previous observations of methylation erasure and establishment using bisulfite sequenc- ing [4,21,22,23,24,36]. Figure 2. Global changes of H3K9ac in fetal germ cells. Gonad preparations were stained with anti-H3K9ac antibody. DAPI staining was used to visualize nuclei (blue). Other details are as in legend to Figure 1. doi:10.1371/journal.pone.0023848.g002 PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e23848