tained 1 106 cells per mouse. purity was assayed by RT PCR Sorafenib Tosylate and immunocytochemistry against CYP11A protein. To e clude contamination with granulosa, the e pression of the FSH receptor transcript and the responsiveness of CREB phosphorylation to hCG or FSH were assayed. Reverse Transcription Polymerase Chain Reaction Total RNA of TIC cultures or from the indicated organ was purified using the guanidine isothiocyanate method. First strand cDNA was synthesized using 2 ug of DNase treated RNA as template, 1 mg of oligo, ran dom he amers, and reverse transcriptase. The cDNA was used as template in a polymerase chain reaction to amplify cDNA fragments for B actin, p2y2r, p2y4r, and p2y6r transcripts, and for cyp11A, cyp17A, star, and fshr as positive and negative theca cell markers, respectively.

All the PCR programs started at 96 C for 3 min and finished at 72 C for 1 min. The amplification cycles consisted in 40 s at 96 C, 40 s at the specific annealing temperature for each primer set, and 40 s at 72 C. The amplified products were gel isolated, phenol chlo roform purified, and subcloned into the pCR4 TOPO vector. Their nucle otide sequences were confirmed by automatic sequenc ing. Fluorescence microscopy Mouse ovarian TIC were grown on 12 mm diameter cover slides. Semi confluent cultures were loaded for 15 min with 5 mM fluo 4 AM and 0. 1% pluronic acid in Krebs solution. The cells were washed with Krebs solution for 10 min to elim inate e cess dye and then placed in a constant flow recording chamber that allowed them to be visualized with an inverted fluorescence microscope.

Drugs were applied by superfusion and responses were recorded with an Evolution QEi cam era. Sequences of images were analyzed using the Image Pro Plus software and Imagenes soft ware, a program developed specifically for this analysis. In the Ca2 free Krebs solution, CaCl2 was replaced by 3 mM MgCl2. Western Cilengitide blot For MAPK p42 and p44 or CREB phosphorylation e per iments, cultured TIC were harvested 24 h before the e periment to reduce serum dependent kinase activ ity. After that, cells were stimulated with the indicated drugs, scraped in Laemmli buffer, and boiled for 5 min. For electrophoresis, samples were fractionated in a 10% SDS polyacrylamide gel and transferred to a nitrocellu lose membrane. Membranes were blocked for 1 h at room temperature in 150 mM NaCl, 20 mM Tris, pH 7.

4, and 0. 1% Tween 20 containing 5% nonfat dry milk and then incubated over night at 4 C with the appropriate rabbit primary antibody directed against the phosphorylated form of MAPK p44 and p42 or CREB. After washing with TBS T, membranes were sellekchem incu bated 1 h at 37 C with HRP conjugated goat anti rabbit antibody in TBS T. The immunoreactive proteins were detected by chemiluminescence, and images were analyzed by pi el density with ImageJ Software, the results were e pressed in terms of optic density normalized against the basal condition, a parameter that is propor tional to the change in protein phos