After 24-h incubation, an obvious synergistic increase in PAI-I activity could be observed in the culture of LH with PRL (data not shown).
PRL Dose-Dependent Stimulation of the Synthesis of PAI-I mRNA and Activity Levels in Cultured Granulosa Cells
To estimate the dose-dependent effect of PRL on PAI-I mRNA levels in the culture, granulosa cells (2 X 106 cells/ dish) were preincubated for 6 h in medium containing 2% calf serum, and then incubated for 24 h in serum-free medium. As shown in Figure 2, addition of various doses of PRL in the presence or absence of LH stimulated PAI-I mRNA induction in a dose-dependent manner. The presence of LH resulted in a synergistic induction of PAI-I mRNA levels. buy diabetes drugs
To analyze PAI-I antigen and activity levels in the conditioned medium, granulosa cells (5 X 105 cells/well) were preincubated for 6 h in medium containing 2% serum and further incubated for 48 h in fresh serum-free medium in the presence or absence of LH (100 ng/ml) with various doses of PRL alone or in combination with LH. As shown in Figure 3A, PAI-I antigen in the control group was hardly detected. Addition of LH (100 ng/ml) to the culture did not increase the PAI-I antigen level. Addition of PRL induced PAI-I antigen production in a dose-dependent manner. PRL induction of PAI-I antigen was further enhanced by the addition of LH to the culture. PAI-I activity in the sample was also assayed by RFA, as shown in Figure 3B. LH alone did not significantly increase the PAI-I activity. Addition of PRL to the culture increased PAI-I activity levels in a dose-dependent manner, and this increase was greatly enhanced by cotreatment with LH.
FIG. 2. Dose-dependent effect of PRL on PAI-I mRNA levels in the cultured granulosa cells. Granulosa cells (3 X 106 cells) were prepared and cultured as indicated in Figure 1 in the presence or absence of LH (100 ng/ml) and various concentrations of PRL (10-1000 ng/ml) alone or in combination. The cells in the dishes after removal of media were quickly frozen in liquid nitrogen, and processed for the PAI-I mRNA measurement as described in MaterialsandMethods. Mean ± SEM of three experiments with duplicate incubations per experiment; analyzed by ANOVAfollowed by Tukey’s multiple comparison test. A different letter denotes a significant difference (p < 0.05).
FIG. 3. Dose-dependent stimulation PAI-I antigen (A) and activity (B) in cultured granulosa cells. For determination of PAI-I activity and antigen in the conditioned media, granulosa cells (5 X 105 cells/well) obtained from the ovaries at 46 h after eCG injection were preincubated for 6 h in 0.5 ml McCoy’s 5a medium containing 2% serum and further incubated for 48 h in fresh medium in the presence or absence of LH (100 ng/ml) and various concentrations of PRL (10-1000 ng/ml) alone or in combination; PAI-I protein in the medium was fractionated by SDS-PAGE, and activity and antigen were determined by RFA and Western blotting, respectively. Representative experiment from three similar experiments is shown.