Normal rat uterine stromal cells in continuous culture: characterization and progestin regulation of growth.

ABSTRACT

Stromal cells were isolated from rat uterus by sequential enzymatic digestion and density fractionation on Percoll gradient and subcultured by trypsinization. Two stable subcultures, named UII and UIII, were obtained. UII cells exhibited a spindle-shaped, elongated, fibroblast-like morphology, while UIII cells were rounded and polygonal. Both cell types expressed the intermediate filament vimentin but not cytokeratin, nor desmin, suggesting that both were of stromal origin. In UIII cells, the presence of progesterone and prolactin (PRL) receptors was demonstrated by immunocytochemical and binding studies. Cross-linking and Western blotting showed that PRL receptor in UIII cells corresponded to 3 molecular forms of 54, 42 and 32 kDa. The growth properties of these cells were studied under different conditions of culture. In fetal calf serum (FCS) supplemented medium, proliferation of UIII cells was dependent on serum concentration and was not affected by estradiol and progesterone. In 10% FCS supplemented medium, the doubling time was 41.5 +/- 0.8 h. When cultured in 10% dextran-charcoal-treated FCS, cells were maintained in a viable but quiescent state. Under these conditions, progesterone was able to induce growth of these cells in a dose-dependent manner. A 3-fold increase in DNA content was measurable in 10(-7) M progesterone-treated versus control cultures after 5 days. Reduction of serum concentration from 10% to 2% abolished the effect of progesterone suggesting that this effect requires the presence of serum factor(s). In conclusion, this study showed that uterine stromal cells, in continuous culture, retained progesterone and prolactin receptors and progesterone regulation of growth.