Culture of BLT-1 Cells
BLT-1 cells are an immortalized Leydig cell line derived from a testicular tumor of a transgenic mouse expressing the SV40 T-antigen under the murine inhibin a-subunit promoter . One day before stimulation, the cells were plated into 24-well culture dishes (Greiner, Frickenhausen, Germany) at a density of 105 cells/well. The next day the medium was changed, and the cells were incubated in 1.0 ml culture medium in the presence and absence of 10-6 M VIP, 10 6 M PACAP-27, or 30 |xg/L hCG, with a final concentration of 0.2 mM of MIX. The cells were incubated for 4 h at 37°C in an atmosphere of 5% C02 in air; then the medium was collected, heated at 100°C for 5 min, and stored at — 20°C until required for cAMP and testosterone measurements (see below). buy flovent inhaler
Stimulation of Intact Fetal Testes In Vitro by hCG and LH
Fetal testes were collected as described above. One of each pair of testes was cultured in 0.5 ml culture medium in the presence and absence of 1.0 |xg/L hCG in a shaking water bath at 34°C, in an atmosphere of 5% C02 in air. After 1, 2, 3, and 5 h, aliquots of 100 рЛ of each culture medium was collected for testosterone measurement and reconstituted to the original volume with the same medium. The media collected were heated at 100°C for 5 min and then stored at — 20°C until analyzed. In the same way, we stimulated the E17.5 intact fetal testis for 4 h using 0.005 and 2.0 |xg/L of recombinant rat LH (rec. rLH), which is equivalent in biopotency to 0.025 jxg/L and 10 jxg/L of the NIDDK RP-2 standard, respectively . The media were collected as described above.
Stimulation by hCG was assessed under identical culture conditions using dispersed fetal Leydig cells, and the results were compared.