Figure 4. Dnmt inhibition outside of the critical period increases dendritic spine markers in the POA and masculinizes behavior.
(a) Animals were treated with Zeb or estradiol at PN10 and PN11. In adulthood, animals were gonadectomized and implanted with testosterone-releasing capsules and male sexual behavior was assessed 20 days later. Brains were collected and Neurabin II levels were quantified in the POA following the completion of three weeks of behavioral testing. (b) Zeb administration after the close of the critical period for sexual differentiation of the brain masculinized Neurabin II protein levels (a priori t-test between females and females + zeb: t(8) = –2.833, p = 0.0221), although there was no main effect across all treatment groups (ANOVA, F(4,22) = 2.22, p = 0.1002). Cropped representative western bands are shown and full length blots are provided in Supplementary Figure 10. (c) Number of mounts (F(4,27) = 5.23, p = 0.003), the latency to mount (F(4,26) = 2.44, p = 0.0718, a priori t-test between females and females + zeb: t(10) = 2.862, p = 0.0169), and thrust latency (F(4,21) = 6.67, p = 0.0013) were also masculinized by Dnmt inhibition whereas estradiol treatment administered at this same time point did not induce masculinization of brain or behavior. Although there were large differences in the number of thrusts between males and females (F(4,27) = 14.861, p < 0.0001), Zeb did not masculinize number of thrusts in females. n = 7 male, 7 male zeb, 6 female, 4 female + e, 4 female + zeb (b), Mounts: n = 7 male, 8 male zeb, 7 female, 4 female + e, 6 female + zeb. Mount latency: n = 7 male, 8 male zeb, 6 female, 4 female + e, 6 female + zeb. Thrusts: n = 7 male, 8 male zeb, 6 female, 4 female + e, 7 female + zeb. Thrust latency: n = 7 male, 8 male zeb, 4 female, 4 female + e, 3 female + zeb (c). *p < 0.05, **p < 0.01, ***p < 0.001 one-way ANOVA main effect compared to vehicle-treated female. #p<0.05 t-test compared to vehicle-treated female. Data are represented as mean ± s.e.m.