Proliferation of mouse uterine epithelial cells in vitro

Although estrogens can stimulate the growth of uterine epithelial cells in vivo, there is no clear effect of estrogens on the in vitro growth of epithelial cells from reproductive tract tissues; thus, we have established a defined culture system for mouse uterine epithelial cells. Pieces of uteri from immature CD-1 mice (21-23 days of age) were treated with trypsin, and the epithelial fragments were separated, enriched by Percoll gradient centrifugation, and seeded on collagen gels prepared from rat tail tendon. Initially, the cells were cultured in a 1:1 mixture of Ham's F-12 and Dulbecco's Modified Eagle's Medium supplemented with epidermal growth factor (EGF; 10 ng/ml), insulin (10 micrograms/ml), transferrin (10 micrograms/ml), hydrocortisone (0.1 micrograms/ml), and vitamin A (10 ng/ml). The cells formed a monolayer on the collagen gel within 1-2 days, but with time, cells began to detach from the gel. Further studies revealed that the attachment and growth of these cells on collagen were markedly influenced by the calcium concentration. It was found that lowering the calcium concentration from 1.05 to 0.05-0.1 mM dramatically suppressed cell detachment; the number of cells doubled after 7 days of culture. Proliferation of uterine epithelial cells was enhanced by EGF, but not by fibroblast growth factor, platelet-derived growth factor, nerve growth factor, multiplication-stimulating activity, or somatomedin-C. The uterine epithelial cells exhibited a single class of high affinity binding sites for [125I]iodo-EGF (Kd, approximately 1.8 nM), with approximately 5 X 10(4) receptors/cell; binding was inhibited by EGF but not by the other polypeptides. This cell culture system will aid in our investigations on hormonal effects on the growth and differentiation of estrogen target cells.