Progesterone-Growth Factor Interactions in Uterine Stromal Cells: A NEW MODEL FOR STUDYING PROGESTERONE(1)

In: Progesterone

6 Feb 2013

In humans and in rodents, stromal cells proliferate and differentiate into large, polyploid cells with characteristic morphology. A variety of markers exist that are associated with differentiation of rat and human decidual cells. While these markers have proved useful, their expression does not correlate strictly with the fully differentiated phenotype. Cultured stromal cells expressing ‘‘decidual markers’’ continue to proliferate. Differentiation programs are normally initiated as cell proliferation and growth arrest occur. Therefore, activation of genes that are closely linked to molecular mechanisms involved in cell cycle arrest are expected to initiate a differentiation program. cialis professional

In the presence of charcoal-stripped calf serum (i.e., no steroids), stromal cells do not proliferate significantly. Tessier et al. showed that stromal cells cultured in serum incorporate significantly more [3H]thymidine than cells cultured in charcoal-stripped serum. Progesterone restored the proliferative response when added to charcoal-stripped serum.

We have extended those earlier observations of progesterone-dependent stromal cell proliferation and developed a serum-free culture system. It is essential to use a chemically defined culture system in order to identify growth factor-directed versus hormone-dependent cell cycle regulation. Stromal cells are cultured in Dulbecco’s Modified Eagle’s medium containing supplements as described. Under these conditions the cells become quiescent 48-72 h after the start of culture. Even though no net increase in cell number is seen, there may be some proliferation that is balanced by cell loss. Analysis of stromal cells cultured for 72 h in serum-free medium using flow cytometry showed that approximately 80% are in G1 phase of the cell cycle (Fig. 1). The percentage of cells in Gl phase in different experiments ranged from 73% to 83% with use of these same serum-free culture conditions. Stromal cells propagated in medium containing 10% fetal bovine serum (FBS) were asynchronous, and the percentage of cells in G1 72 h after plating was 32% (Fig. 1). However, cell cycle phase parameters in asynchronous cultures depended on the time after plating. At 48 h after plating, 50% of the cells were at G1, while as the cells approached confluence at 96 h after plating, 25% were at Gl.
Fig1Progesterone-Growth Factor
FIG. 1. Synchronization of uterine stromal cells at G1 phase of the cell cycle. Uterine stromal cells (UII, passage 17) were cultured in serum-free medium (serum starved) or medium 199 containing 10% FBS (asynchronous) for 72 h. Between 1-2 X 106 cells from each culture were analyzed. Cells were collected and stained for at least 4 h with a one-step propidium iodide solution (0.005% propidium iodide, 0.02% ribonuclease, 0.3% sodium phosphate, and 0.1% sodium citrate in distilled water) using standard methods. The cells were analyzed on an EPICS 752 flow cy-tometer (Coulter Electronics, Hialeah, FL) with a laser output of 400 nW at 488 nm. Red fluorescent data were collected and DNA histograms analyzed with the Modfit program (Verity Software, Topsham, ME).


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