Time-Dependent Effect of PRL on PAI-I mRNA and Activity in eCG-Primed Granulosa Cells
To examine whether PRL stimulates PAI-I expression in cultured granulosa cells, 2 X 106 cells obtained from eCG-treated ovaries were preincubated for 6 h in the presence of 2% calf serum, and then incubated in fresh serum-free medium for the indicated times with or without LH (100 ng/ml) and PRL (1000 ng/ml) alone or in combination. The conditioned media were collected for PAI-I assay, and the cells were frozen in liquid nitrogen for mRNA preparation. As shown in Figure 1, LH and PRL alone slightly induced PAI-I mRNA production in a time-dependent manner. After 48-h incubation, 1.6- and 4.5-fold increases in the mRNA production were observed in the presence of LH and PRL, respectively, as compared with the control. birth control pills
Cotreatment of LH with PRL had a synergistic effect on PAI-I mRNA production, with an 8.2-fold induction in relation to LH alone after 48-h incubation. The increase in the cell PAI-I mRNA levels in the culture was well correlated with the increase in PAI-I activity in the conditioned media. After 12-h culture, a small amount of PAI-I activity could be detected in the groups incubated with PRL alone and with LH plus PRL.
FIG. 1. Time-dependent induction of PAI-I mRNA levels by PRL and LH in cultured eCG-primed granulosa cells. Immature female rats (21-22 days old) received injections of 10 IU eCG to stimulate follicular growth; 46 h later, the animals were killed, and the ovaries were removed for preparation of granulosa cells. Granulosa cells (3 X 106) were preincubated for 6 h in poly-D-lysine-coated polypropylene dishes (10 X 35 mm, 5 ^g/cm2) in 1 ml of McCoy’s 5a medium containing 2% calf serum. After being washed, the cells were further incubated for the designated times in 1 ml fresh serum-free McCoy’s 5a medium in the presence or absence of the indicated hormones. The cells in the dishes were quickly frozen in liquid nitrogen and processed for PAI-I mRNA measurement as described in Materials and Methods. Mean ± SEM of three experiments with duplicate incubations per experiment; analyzed by ANOVA followed by Tu-key’s multiple comparison test. Groups with at least one identical letter not significantly different.