Figure 6
P4–PGRMC1 interaction decreased intra-oocyte cAMP levels.
For this, 16.5 dpc ovaries were isolated and cultured with vehicle, 1 μM P4 alone, 5 μM AG-205 alone, or both AG-205 and P4. After 1 h or 2 days of culture, cAMP levels in the ovaries were measured by performing radioimmunoassay (RIA) (A). Different letters denote statistical significance at P < 0.05 (ANOVA and post hoc test, 3 independent replicates, in each replicate every treatment contained 30 ovaries). After 2 days of culture, Adcy2 mRNA levels in ovaries in all the groups were analyzed by performing qRT-PCR (B). *Denotes statistical significance at P < 0.05 between vehicle and treated ovaries (ANOVA and post hoc test, 3 independent replicates, in each replicate every treatment contained 10 ovaries). Next, the ovaries at 19.5 dpc were subjected to immunofluorescence analysis by staining with anti-ADCY2 antibody (green) and anti-PGRMC1 antibody (red) (C). Results of immunofluorescence analysis showed that ADCY2 and PGRMC1 were predominately colocalized in the cytoplasm of oocytes. Arrows indicate labeled oocytes; scale bar = 25 μm. Then, oocytes and somatic cells were purified from fetal ovaries, and their expression of oocyte marker Ddx4 and somatic cell marker Foxl2 was measured by qRT-PCR (D). ***Denotes statistical significance at P < 0.001 (t-test, 3 independent replicates, in each replicate every treatment contained 10 ovaries). 16.5 dpc ovaries were cultured with vehicle, 1 μM P4 alone, 5 μM AG-205 alone, or both AG-205 and P4 for 2 days, and then oocytes and somatic cells were purified and performed RIA (E). Different letters denote statistical significance at P < 0.05 (ANOVA and post hoc test, 3 independent replicates, in each replicate every treatment contained 20-30 ovaries).
