Prolactin Regulation of Tissue: RESULTS(3)

Time- and Dose-Dependent Inhibition of LH-Induced tPA mRNA and Activity Levels by PRL in Cultured Granulosa Cells

To examine whether PRL inhibits LH-induced tPA gene expression in cultured granulosa cells, 2 X 106 cells obtained from eCG-primed ovaries were preincubated for 6 h in the presence of 2% calf serum, and then further incubated in serum-free medium for the indicated times. As shown in Figure 4, LH alone dramatically stimulated tPA mRNA levels in a time-dependent manner. Cotreatment with PRL time-dependently decreased the LH-induced tPA mRNA levels. Significant inhibition could be observed at 3-, 6-, and 12-h culture, with 36.4%, 51.4%, and 66.7% decreases, respectively, of the LH-induced tPA mRNA levels.

To analyze the dose-dependent effect of PRL on LH-induced tPA activity levels, eCG-primed granulosa cells (5 X 105 cells) were preincubated in 0.5 ml of McCoy’s 5a medium containing 2% serum for 6 h, and further incubated in serum-free medium with the indicated hormones for 48 h. The tPA activity in the medium is shown in Figure 5. LH stimulated synthesis of tPA in the culture, and the increase of tPA activity was diminished markedly by cotreatment with PRL in a dose-dependent fashion. The presence of high-molecular weight PA-PAI-I complexes indicated the neutralization of LH-induced tPA activity by the increased PAI-I activity. levitra super active plus

In Situ Hybridization of tPA and PAI-I mRNA in the Cultured Granulosa Cells

To examine whether PRL in culture in combination with LH stimulates PAI-I and inhibits tPA mRNA expression, 1-2 X 106 eCG-primed granulosa cells were cultured for 6 h in the presence of LH with or without PRL. The tPA and PAI-I mRNAs in the cells were localized by in situ hybridization. As shown in Figure 6, PRL was capable of decreasing LH-induced tPA mRNA expression (Fig. 6, A and B), while increasing PAI-I mRNA level, in the cells (Fig. 6, C and D).
Fig4Prolactin Regulation
FIG. 4. Time-dependent inhibitory effect of PRL on tPA mRNA levels in the cultured granulosa cells. Granulosa cells (2 X 106 cells) were prepared and precultured as indicated in Figure 1. After being washed, the cells were further incubated for the indicated times in serum-free medium in the presence or absence of LH (100 ng/ml), PRL 1000 ng/ml), or LH plus PRL. The cells in the dishes after removal of media were quickly frozen in liquid nitrogen and processed for tPA mRNA measurement as described in Materials and Methods. Mean ± SEM of three experiments with duplicate incubations per experiment; analyzed by ANOVA, followed by Tukey’s multiple comparison test. Groups with at least one identical letter were not significantly different. CPX, PA-PAI-I complexes.

Fig5Prolactin Regulation
FIG. 5. Dose-dependent inhibitory effect of PRL on LH-induced tPA activity in the cultured granulosa cells. 5 X 105 viable granulosa cells per well were preincubated for 6 h in 0.5 ml of McCoy’s 5a medium containing 2% serum. At the end of incubation, the cells were washed and further incubated for 48 h in the presence or absence of LH (100 ng/ml), and various doses of PRL. Media were collected at the end of incubation. Activity of tPA in the media was determined by fibrin-overlay technique as described in Materials and Methods. Representative experiment from three similar experiments is shown.

Fig6Prolactin Regulation
FIG. 6. Effects of PRL on LH-induced tPA and PAI-I mRNA levels in cultured granulosa cells. Granulosa cells (2 X 106 cells) were prepared and precultured as indicated in Figure 1. After being wash, the cells were further incubated for the indicated times in serum-free medium in the presence or absence of LH (100 ng/ml), PRL (1000 ng/ml), or LH plus PRL. The cells were fixed and processed for tPA and PAI-I mRNA localization by in situ hybridization as described in Materials and Methods. A, C) Granulosa cells cultured with LH alone. B, D) Granulosa cells cultured with LH plus PRL. E) Sense probe of hybridization of the cultured granulosa cells. A, B) tPA mRNA level. C, D) PAI-I mRNA level. Incubation time: tPA based on time course, PAI-I based on time course. X400.

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