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Fig. 6. Activation of granulosa cell SMAD2 and SMAD3 transducer molecules by GDF9 and oocytes. (A) Mural GCs were transiently transfected with a SMAD3-responsive CAGA-luciferase plasmid, and subsequently left untreated, treated with 0.5 ng/ml TGFß1, or treated with increasing doses of GDF9 (or 293H: the negative v/v control of conditioned medium from untransfected 293H cells) for 48 hours, and then relative luciferase activity was measured from cell extracts. (B) Mural GCs were cultured for 90 minutes either alone (control), co-cultured with oocytes (500 DOs/1-ml well), with human activin A (50 ng/ml), or mouse GDF9 (40 ng/ml), then extracted granulosa cell proteins were subjected to 10% SDS-PAGE and western blotting using SMAD2 and phospho-SMAD2 antibodies. Mural GC SMAD2 was phosphorylated by treatment with mGDF9, oocytes and, to a lesser extent, activin A.
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