Figure 1. Characterization of granulosa cells in primary culture.

Granulosa cells (GCs) were collected from 3 weeks old C57BL/6 mice ovaries and seeded at a density 1×10⁵ cells/well. 24 h later, GCs were incubated without primary antibodies (IgG) for the control condition (A) or with an anti-AMH antibody (B). The secondary antibody was coupled to FITC and DAPI was used to visualize the nucleus. AMH expression was detected in the cytoplasm as expected. To asssay the transfection efficiency, primary GCs were transfected either with a β-galactosidase vector (C, 1 µg), pMax-GFP vector (D, 1 µg) or GAPDH-cy3 siRNA (E). After 24 h, GC were fixed, stained with X-Gal and counterstained with nuclear fast red (C) or fluorescence was visualized (D). Alternatively, siRNA transfection efficiency was assayed with a GAPDH-cy3 siRNA on isolated GCs using flow cytometry analysis (E). (F), Markers of immature GCs were analysed by RT-PCR to confirm their status.