Prolactin Regulation of Tissue: RESULTS(2)

RESULTS(2)

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.
Fig2Prolactin Regulation
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).

Fig3Prolactin Regulation
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.

This entry was posted in Plasminogen and tagged Plasminogen Activator, Plasminogen Activator Inhibitor, Primed Rat, Prolactin.