Perinatal testosterone treatment affects CpG methylation at numerous loci. (a) The dose of testosterone given to female mice at PN0 is masculinizing. Representative pictures of the levator ani (LA) are shown for PN4 XX (left panel) and XX + T (right panel). The muscle size is reduced in the absence of testosterone. The arrows indicate the outline of the LA, which has a much larger surface area in XX + T than in XX (269,150 ± 11,908 μm2 in XX + T vs. 53,721 ± 9,414 μm2 in XX; t = 13.6, p <0.0001). (b) Number of genes where methylation is altered in the XX vs. XX + T comparison in PN4 and PN60 striatum and PN4 and PN60 BNST/POA. (c) Percentage of genes that exhibit testosterone-dependent hypo- or hyper-methylation at each age in each tissue. (d, e) The Venn diagrams show the overlap (green) between genes with sexually dimorphic methylation patterns (blue) and those that are affected by testosterone (yellow) in the (d) striatum and (e) BNST/POA. The numbers of genes belonging to each category are shown within the relevant circles. (f, g) The number of genes that are affected by testosterone in both the striatum and the BNST/POA at (f) PN4 and (g) PN60. The overlap between testosterone-affected genes from the striatum (blue) and BNST/POA (yellow) is shown in green. The numbers of genes belonging to each category are shown within the relevant circles. The bar below the Venn diagram shows the percentage of autosomal vs. X-linked genes in the overlap region.