FIGURE 5.
Proliferation and apoptosis in TGF-β1- and SD208-treated ovaries. Ovaries at 18.5 dpc were cultured alone as a control, with 10 ng/ml of TGF-β1, or with 1 μm SD208 for the staining of PCNA, a BrdU incorporation assay, and the TUNEL assay. Incorporation of BrdU into the granulosa cells was decreased in TGF-β1-treated ovaries and increased in SD208-treated ovaries compared with control ovaries (A–C, arrows: BrdU positive granulosa cells) after 7 days of culture. Immunostaining of PCNA expression in ovarian sections from TGF-β1- and SD208-treated ovaries (D–F, arrows, PCNA negative granulosa cells; arrowheads, PCNA positive granulosa cells) after 7 days of culture. TUNEL assays for apoptosis were performed in control, TGF-β1-, and SD208-treated ovaries (G–I, arrows, TUNEL positive oocytes) after 5 days of culture and the number of apoptotic germ cells was analyzed in the largest cross-section of all treated ovaries (K). Western blot was performed using anti-cleaved caspase-3 and anti-GAPDH antibodies after 5 days of culture (J). Data were obtained from at least six ovaries. Scale bars: 40 μm (A–I). Bars with different letters are significantly different (p < 0.05).