Fig. 4.

Testosterone induces CREB phosphorylation in Sertoli cells derived from wild-type but not AR-defective tfm mutant rats. (A) A portion of the sequencing results after PCR amplification of AR genomic DNA from wild-type and tfm rats is shown. The arrows denote the 1-bp difference in the sequences for AR. (B) A DIC image of Sertoli cells from 15-day-old tfm rats stained for peritubular cell-specific alkaline phosphatase activity after 3 days in culture shows that peritubular cells account for <5% of the culture. (C) The relative [³H]R1881-binding activities of wild-type (wt) and tfm Sertoli cells were determined by hormone-binding assay. The data shown represent the means (±SE) of three independent experiments. (D) Sertoli cell cultures from wild-type or tfm rats were stimulated with ethanol vehicle (V) or 100 nM testosterone (T) for 15 min. Whole cell extracts were subjected to Western analysis with antisera specific for P-CREB followed by reprobing with antisera against all CREB isoforms. (E) The relative mean fold inductions (±SE) of P-CREB levels for testosterone-stimulated cells versus ethanol-treated cells are shown for the studies performed in D (n = 4). (F) Sertoli cells derived from tfm rats were infected in duplicate with adenoviral vectors expressing β-galactosidase or AR and then stimulated 3 days later with EtOH (V) or 100 nM testosterone (T). CREB and P-CREB levels were determined by Western blot.