Age-dependent alteration in COUP-TFII expression in fetal rat Leydig cells in vehicle-exposed control rats and after in utero exposure to dibutyl phthalate (DBP; 500 mg/kg/day).

(A–B) Triple immunofluorescence for SMA (blue), 3β-HSD (red) and COUP-TFII (green) on fetal testis sections from vehicle (control) and DBP-exposed animals. Arrows in A indicate examples of individual Leydig cells positive for COUP-TFII whereas asterisks indicate DBP-induced aggregates of Leydig cells which are predominantly COUP-TFII-immunopositive. SC = seminiferous cords. Scale bar A = 20 µm, B = 200 µm. (C) Quantification of the percentage of COUP-TFII positive fetal Leydig cells in vehicle (control) and DBP-exposed animals using tiled high resolution images as shown in panel B. Values are means ± SEM for 5–8 animals at each age (minimum of 3 litters per group). ***p<0.001, in comparison with respective control; other comparisons are indicated by capped lines.