Reagents and Hormones
Reagents were Dulbecco’s Modified Eagle’s Medium (DMEM), Ham’s F-12, insulin (bovine; 25.7 U/mg), enzymes, and fetal calf serum (FCS), all obtained from Sigma Chemical Company (St. Louis, MO); bovine LH (L1914, LH activity 2.0 X NIH-LH-S1 U/mg) obtained from Scripps Laboratories (San Diego, CA); recombinant mouse leptin obtained from Pepro Tech Inc. (Rocky Hill, NJ); and [125I]insulin (porcine, specific activity 80-135 (xCi/jxg) obtained from ICN Pharmaceuticals Inc. (Costa Mesa, CA). buy prednisone
Ovaries were obtained at a nearby commercial abattoir from beef and dairy cattle after slaughter. After transport to the laboratory on ice (< 120 min), the ovaries were processed and thecal cells were isolated as described previously. Briefly, large (s 8 mm) follicles were dissected from the ovary, follicular fluid was aspirated, follicles were bisected, the theca interna layer was microdissected from the follicle wall, granulosa cells were removed, and the theca was enzymatically digested for 1 h at 37°C. After incubation, undigested tissue was removed from the cell suspension by filtration and washed in serum-free medium. Contamination of thecal cells by granulosa cells is minimal (i.e., < 10%) using this procedure. Cells were resuspended in serum-free medium, and the number of viable cells was determined using the trypan blue dye exclusion method. Thecal cell viability averaged 83 ± 5% at the time of plating.