Figure 1. Morphological and molecular characterization of cultured ovarian somatic cells. (A–D) Phase contrast (PH) observations after 24 hrs of culture. (A) White and black arrowheads indicate scattered epithelioid and fibroblastoid cells, respectively. (B) Fibroblastoid cells at higher density. (C) A little colony of small epithelioid cells (red arrowheads) surrounded by fibroblastoid cells. (D) A large colony of polygonal epithelioid cells. Scale bar = 100 μm. (E) RT-PCR analyses for granulosa (AMH and FOXL2) and theca (LHCGR, Gli1 and CYP17A1) cells markers (GAPDH was used as housekeeping gene). (F) WB analysis for LHCGR and FOXL2 after increasing culture times. (GAPDH was used as housekeeping protein). (G, H) Densitometric quantification of proteins relative expression is reported. Data are expressed as mean ± SEM of three analyses. (I-M) Representative field of IF for FOXL2, αSMA and pan-CK after 24 hrs of culture, scale bar = 100 μm. (I, J) FOXL2 identifies cells with different morphologies: (I) scattered epithelioid and fibroblastoid cells (white and yellow arrowheads, respectively); blue arrowheads pointed fibroblastoid FOXL2 negative cells and (J) a large colony of polygonal epithelioid cells (white circle). (K, L) Double IF for αSMA and FOXL2. Cells positive for αSMA showed a fibroblastoid morphology. Note that IF for αSMA and FOXL2 never overlappe3d (red arrowheads indicate αSMA positive and FOXL2 negative cells, white arrowheads FOXL2 positive and αSMA negative cells; blue arrowheads indicate a double negative cell). (M) Little colony of pan-CK positive small epithelioid cells. (N) Quantification of the FOXL2, αSMA and pan-CK positive and triple-negative cells. (O, P) Follicles isolated from 16dpp mouse ovaries cultured for 24 hrs. (O, P) Follicular cells, spread out to form an epithelioid cell monolayer, showed FOXL2 (red) positivity.