NORMAL RAT UTERINE STROMAL CELLS IN CONTINUOUS-CULTURE - CHARACTERIZATION AND PROGESTIN REGULATION OF GROWTH

Stromal cells were isolated from rat uterus by sequential enzymatic di gestion and density fractionation on Percoll gradient and subcultured by trypsinization. Two stable subcultures, named U(II) and U(III), wer e obtained. U(II) cells exhibited a spindle-shaped, elongated, fibrobl ast-like morphology, while U(III) cells were rounded and polygonal. Bo th cell types expressed the intermediate filament vimentin but not cyt okeratin, nor desmin, suggesting that both were of stromal origin. In U(III) cells, the presence of progesterone and prolactin (PRL) recepto rs was demonstrated by immunocytochemical and binding studies. Cross-l inking and Western blotting showed that PRL receptor in U(III) cells c orresponded to 3 molecular forms of 54, 42 and 32 kDa. The growth prop erties of these cells were studied under different conditions of cultu re. In fetal calf serum (FCS) supplemented medium, proliferation of U( III) cells was dependent on serum concentration and was not affected b y estradiol and progesterone. In 10% FCS supplemented medium, the doub ling time was 41.5 +/- 0.8 h. When cultured in 10% dextran-charcoal-tr eated FCS, cells were maintained in a viable but quiescent state. Unde r these conditions, progesterone was able to induce growth of these ce lls in a dose-dependent manner. A 3-fold increase in DNA content was m easurable in 10(-7) m progesterone-treated versus control cultures aft er 5 days. Reduction of serum concentration from 10% to 2% abolished t he effect of progesterone suggesting that this effect requires the pre sence of serum factor(s). In conclusion, this study showed that uterin e stromal cells, in continuous culture, retained progesterone and prol actin receptors and progesterone regulation of growth.