ALTERED STEROIDOGENIC ACTIVITY OF HUMAN GRANULOSA-LUTEIN CELLS AT DIFFERENT CELL DENSITIES IN CULTURE

In the present study, the relationship between human granulosa-lutein cell (hGLC)-plating density and steroidogenic activity was evaluated. Increasing hGLC-plating density 32-fold, from 0.25 x 10(4) to 8 x 10(4) cells/well, was associated with a concomitant increase in the total amount of progesterone (P4), testosterone (T), and estradiol-17beta (E2) secretion. The daily amount of each steroid (P4, T, and E2) secreted by hGLC at different cell-plating densities was further normalized per 10(3) cells. Thus, an increase in hGLC-plating density from 0.25 X 10(4) to 1 X 10(4) cells/well was associated with approximate increases of 1.3-fold in P4 and 3-fold in T and a 50% decrease in E2 secretion, per 10(3) cells. A further increase in hGLC-plating density, from 1 X 10(4) to 8 x 10(4) cells/well, was associated with a significant decrease of approximately 3.7-fold in P4 and 6-fold in T per 10(3) Cells. A similar increase in hGLC-plating density was associated with no change or a 2-fold decrease, per 10(3) cells, in E2 secretion during days 0-3 or days 3-5 of culture, respectively. The P4/E2 ratio was increased and the E2/T ratio decreased with extension of the culture period. These two ratios had a tendency to be altered inversely, concurrent with the increase in cell-plating densities. At 1-2 x 10(4) cells/well, P4/E2 was maximal, whereas E2/T was minimal. Addition of human follicle-stimulating hormone (hFSH) or human chorionic gonadotropin (hCG) to the culture medium caused a significant,increase in E2 secretion, mainly at the lower cell-plating density of 0.25-1 x 10(4) cells/well. Furthermore, addition of hFSH or hCG increased hGLC-P4 secretion at 1-2 x 10(4) cells/well and at higher cell-plating densities. This study indicates that basal as well as hFSH- or hCG-stimulated secretion of P4 and E2 per 10(3) cells is altered at different hGLC-plating densities. Collectively, it seems that under the culture conditions employed in this study, at 2-4 x 10(4) Cells/well, hGLC manifests a more luteinized state than at lower (0.25 X 10(3) and 0.5 X 10(3) cells/well) or higher (8 x 10(3) cells/well) cell-plating densities.