FIG. 6.

Induction of Dmrt1 mRNA by cAMP requires activation of protein kinase A. Sertoli cells were treated with the inhibitor of protein kinase A, H89, in the presence and absence of 8-bromo-cAMP and RNA assayed for Dmrt1 and actin transcripts by RPA. H89 treated cells were incubated for 4 h in the absence (+H89) or presence 8-bromo-cAMP (+cAMP & H89). Also included are cells cultured in the absence of additional treatments (no treatment) for the same period of time or ones treated with 8-bromo-cAMP alone for 4 h (+cAMP). B, Sertoli cells were treated with the inhibitors for PI3 kinase (wortmannin, 1 μm) and MEK1 (PD98059 50 μm) in the presence and absence of 8-bromo-cAMP and RNA assayed for Dmrt1 and actin transcripts by RPA. Cells were incubated with the inhibitors for either 1 or 4 h in the absence (1 h and 4 h inhibitor) or presence 8-bromo-cAMP (1 h and 4 h inhibitor + cAMP). Also included are cells cultured in the absence of additional treatments (no treatment) for the same period of time or ones treated with 8-bromo-cAMP alone for 1 and 4 h (1 h and 4 h cAMP). C, Sertoli cells were treated with the p38 kinase inhibitor SB203580 (1 μm) in the presence and absence of 8-bromo-cAMP and Dmrt1 and actin mRNA was measured using RPA. Cells were incubated with SB203580 for either 1 or 4 h in the absence (1 h and 4 h SB) or presence 8-bromo-cAMP (1 h and 4 h SB +cAMP). Also included are cells cultured in the absence of additional treatments (no treatment) for the same period of time or ones treated with 8-bromo-cAMP alone for 1 and 4 h 1 h and 4 h cAMP). Arrows mark the protected fragments and tRNA and RNA from freshly isolated Sertoli cells were added as negative and positive controls, respectively.