Fig. 2. Prenatal androgen exposure causes transgenerational anxiety-like behavior in female offspring.

A Schematic illustration of experimental design. Prior to mating with male mice fed control diet, F₀ mothers were fed control diet or high fat-high sucrose (HFHS) diet for 6 weeks. During embryonic day (E) 16.5−E18.5 pregnant mice were injected subcutaneously with 250 µg of dihydrotestosterone (DHT), a non-aromatizable androgen, dissolved in 50 µL of sesame oil or 50 µL of sesame oil alone (vehicle) resulting in four lineages; CD+Vehicle (control); CD+DHT (androgenized); HFHS+Vehicle (obese); and HFHS+DHT (obese- androgenized). F₁ female and male offspring were mated with unrelated males and females fed CD, respectively, to generate F₂ and thereafter to generate F₃ offspring. Anxiety-like behavior was tested in female and male offspring according to the graph. Male germline refers to F₂ and F₃ male offspring from F₁ males. F₂ and F₃ male siblings are brothers to F₂ and F₃ female offspring. B–D Time spent in the open arms in the elevated plus maze (EPM); time spent in the closed arm of the EPM; time spent in the center of the open filed (OF); time spent in the periphery of the OF. F1: two-way ANOVA, Tukey’s post hoc analysis; F₂ and F₃: one-way ANOVA, Dunnett’s post hoc analysis. All data are presented as mean ± s.e.m. DHT dihydrotestosterone, CD control diet, HFHS high-fat high-sugar, Mat maternal, GMat grand-maternal, GGMat great-grand maternal. Numbers of mice are stated in the bars of each group.