Fig. 4. E-cad is responsible for maintaining the primordial follicle pool by regulating NOBOX expression in oocytes.

a Immunofluorescence staining showed that the expression of NOBOX (green) was specifically localized to the nuclei of all germ cells and that NOBOX expression is increasing with follicle formation and activation (arrows). Oocytes were stained with DDX4 (red). The nuclei were dyed with Hoechst counter-stain (blue). b NOBOX expression profile in neonatal ovaries. The expression of NOBOX in the ovary increased over time from 1 dpp to 7 dpp. c NOBOX and E-cad were colocalized to the oocytes of ovaries at 3 dpp. NOBOX (green) and E-cad (red) expression levels were elevated in the oocytes of primordial and activated follicles. d, e Knockdown of E-cad suppressed NOBOX expression as shown by western blot assay d and immunofluorescence staining e. The nuclei were dyed with a Hoechst counter-stain (blue). f, g Histological and oocyte counting analyses showed that overexpression of Nobox in E-cad-KD transfected ovaries partially rescued the loss of oocytes compared with the ovaries transfected with E-cad-KD alone (refer to Table S5). The experiments were repeated at least three times, and representative images are shown. The data are presented as the means ± S.D. and considered statistically significant at P < 0.05. Scale bars: a, f 50 μm; c, e 20 μm