The animals had ad libitum access to food and water. Diabetes was induced in one group of rats by an intraperito neal injection of 1 ml of 50 mM sodium citrate solution containing streptozotocin. Control female selleck chemicals llc rats were injected with 50 mM sodium citrate solution. One week after injection, plasma glucose levels were checked for each ani mal and diabetes was confirmed. Then, control and STZ treated rats were killed at die strus stage. One sample of blood was taken and then tis sues were removed and frozen for western blotting. Isolation and culture of rat granulosa cells Immature female Wistar rats were injected sub cutaneously with DES every day for three days to increase the amount of granulosa cells as previously described.

On the third day of DES treatment the animals were killed and the ovaries removed aseptically and transferred to culture medium. Granulosa cells were Inhibitors,Modulators,Libraries harvested by puncturing the follicles allowing expulsion of the cells. Cells were recovered by centrifugation, washed with fresh medium and counted in Inhibitors,Modulators,Libraries a hemocytometer. The culture Inhibitors,Modulators,Libraries medium used was McCoys 5A supplemented with 20 mmol L Hepes, penicillin, streptomycin, L glutamine, 0. 1% BSA, 0. 1 mol l androstenedione, 5 mg l trans ferrin, 20 g l selenium and 5% FBS. The cells were first cultured for 48 h with no other treatment and then incu bated in DMEM medium without glucose and serum but containing penicillin, streptomycin, L glutamine and 0. 1 mol l androstenedi one with or without test reagents for the appropriate time as indicated in the legend of the fig ures.

All cultures were performed under a water saturated atmosphere of 95% air 5% CO2 at 37 C. Western blotting Lysates of granulosa cells or tissue were prepared on ice with an Ultraturax homogenizer in lysis buffer, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0. 5% Igepal containing various Inhibitors,Modulators,Libraries protease inhibitors and phosphatase inhibitors. Lysates were centrifuged at 13,000 g for 20 min at 4 C, and the protein concentration in the supernatants was determined using a colorimetric assay. Cell extracts were subjected to electrophoresis on 10% SDS polyacrylamide gel under reducing conditions. The proteins were then electrotransferred onto nitrocellu lose membranes for 2 h. The membranes were incubated for 1 h at room temperature with Tris buffered saline, containing 5% nonfat dry milk powder and 0. 1% Tween 20 to saturate non specific sites.

Then, the membranes were incubated overnight at 4 C with appropriate antibodies, in TBS containing Inhibitors,Modulators,Libraries 0. 1% Tween 20 and 5% NFDMP. They were washed in TBS 0. 1% Tween 20, incubated for 2 h at room temperature with a horseradish selleck peroxidase conjugated anti rabbit or anti mouse IgG in TBS, 0. 1% Tween 20 5% NFDMP, and washed again in TBS 0. 1% Tween 20. The signal was detected by ECL. The films were analyzed and signals quantified with the software MacBas V2. 52.