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Fig. 9. Inhibition of oocyte- and GDF9-stimulated CC and mural GC proliferation using the ALK 4/5/7 kinase inhibitor, SB431542. (A) Cumulus cells (OOX) were treated with GDF9 (250 ng/ml) or DOs (0.8/µl), with or without 4 µM SB431542 and cultured for 6 hours, and Ccnd2 mRNA levels were examined using real-time RT-PCR. Columns are means ± s.e.m. from six replicate experiments and columns with different superscripts are significantly different (P<0.05). (B) Mural GCs were cultured for 24 hours and thymidine incorporation was stimulated with either 20 ng/ml GDF9, 0.5 ng/ml TGFß1, 16 DOs/well or 50 ng/ml BMP6, all of which, in turn, were antagonised by SB431542 at 1.0 µM. An equivalent dose of the SB431542 carrier DMSO did not affect ligand-stimulated mural GC DNA synthesis. Columns are means ± s.e.m. from triplicate wells and are representative of three replicate experiments; asterisks indicate significantly different to control (mitogen alone; P<0.001). (C) Oocyte (14/well)- and GDF9 (20 ng/ml)-stimulated mural GC DNA synthesis were inhibited in a dose-dependent manner with increasing concentrations of SB431542. Points are means ± s.e.m. from triplicate wells and are representative of three replicate experiments. Asterisks represent the lowest dose of SB431542 that is significantly less than the positive control (P<0.05).
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