Fig. 6. Downregulation of Bmp15 expression and suppression of Smad pathway activation in ovaries of Zp3-Foxo3a Tg mice. Ovaries from 6-, 8- and 15- to 17-day-old Tg or WT mice were collected and used for RNA and protein extraction. (A-C) Levels of Bmp15 mRNA from Tg and WT ovaries at PD6, PD8 and PD15-17, as measured by quantitative real-time PCR. (D) Levels of Gdf9 mRNA from Tg and WT ovaries at PD15-17, as measured by quantitative real-time PCR. The relative levels of Bmp15 or Gdf9 mRNA were normalized against levels of ß-actin mRNA. The mRNA levels in WT mice were defined as 1.0. mRNA levels in Tg ovaries were calculated relative to those of WT mice and are shown as mean±s.e.m. (E-I) Western blot analysis for levels of activated Smad1/5/8 (E, p-Smad1/5/8), total Smad1 (F), ß-actin (G), activated Smad2 (H, p-Smad2), and total Smad2 (I) in WT and Tg ovaries at PD8 and PD15-17. All experiments were repeated at least three times. For each experiment with PD6 and PD8 mice, n=10-15; for experiments with PD15-17 mice, n=5-8. Representative results are shown.