Fig. 3

Asynchronous activity of PI3K in germ cells of embryonic ovaries leads to the formation of first wave follicles. a Detecting the expression of FOXO3 showed an asynchronous activity of PI3K signaling in embryonic germ cells. No FOXO3 expressions were observed in germ cells at 15.0 dpc, whereas part of germ cells in cysts expressed FOXO3 (arrows) in the cytoplasm (c-FOXO3⁺), indicating an activity of PI3K signaling in them at 18.0 dpc. At 3 dpp, all oocytes expressed FOXO3 whereas only the oocytes in the medulla region were with c-FOXO3⁺ (arrow), and the oocytes in cortical region were with nuclear-FOXO3 localization (arrowhead). DDX4, red; FOXO3, green. CR, cortical region; MR, medulla region. b The germ cell counting result showing the ratio of total FOXO3⁺ (t-FOXO3⁺) germ cells significantly increased from 18.0 dpc to 3 dpp, but the c-FOXO3⁺ germ cells kept a stable proportion in the ovaries (n = 5). c Representative in situ karyotyping images of the meiotic phases in embryonic germ cells. SYCP3, red; HOE, blue. B/W, the red fluorescence of SYCP3 was inverted to black/white (B/W) to highlight the meiotic stages in the right panel. d Co-localization of FOXO3 and SYCP3 in 18.0-dpc mouse ovaries, showing the majority of FOXO3⁺ germ cells were at diplotene or dictyate stage (arrows), while most of FOXO3⁻ germ cells were at pachytene stage (arrowheads). SYCP3, red; FOXO3, green. e Counting the ratio of meiotic stages in FOXO3⁺, FOXO3⁻ and total germ cells in 18.0 dpc, confirming that the FOXO3⁺ germ cells present a faster meiotic process (n = 3). All experiments were repeated at least three times, and representative images are shown. Data are presented as the mean ± SD and analyzed by a two-tailed unpaired Student’s t-test, n.s. P ≥ 0.05 and ***P < 0.001. Scale bars, 50 μm (a), 5 μm (c), 10 μm (d)