Figure 4. Downregulation of PGRMC1 by RNAi reversed P4-induced inhibition of primordial follicle formation in vitro.

For this, 16.5 dpc ovaries were transfected with scrambled siRNA (Scra-siRNA) or siRNA against Pgrmc1 (Pgrmc1-siRNA) for 4 days, and transfection efficiency was determined by performing qRT-PCR (A); ***denotes statistical significance at P < 0.001 between vehicle and treated ovaries (t-test, 3 independent replicates, in each replicate every treatment contained 10 ovaries). Next, Scra-siRNA- or Pgrmc1-siRNA-transfected ovaries were treated with vehicle or P4 at the same time, and Pgrmc1 expression was measured by performing qRT-PCR after 4 days of culture (B). Different letters denote statistical significance at P < 0.005 (ANOVA and post hoc test, 3 independent replicates, in each replicate every treatment contained 10 ovaries). After culturing for 6 days, the ovaries were fixed and embedded in paraffin, and the paraffinized sections were stained with anti-DDX4 antibody (green) and PI (red) for morphology analysis (C–F). Then the numbers of oocytes and follicles were counted after staining the sections with hematoxylin (G). Arrowheads indicate oocytes in cysts or naked oocytes, and arrows indicate primordial follicles; scale bar = 25 μm. Different letters denote statistical significance at P < 0.05 (ANOVA and post hoc test, n = 3–5, 3 independent replicates).