Developmental mechanisms regulating the formation of smooth muscle layers in the mouse uterus†

Uterine smooth muscle cells differentiate from mesenchymal cells, and gap junctions connect the muscle cells in the myometrium. At the neonatal stage, a uterine smooth muscle layer is situated away from the epithelium when smooth muscle cells are grafted near the epithelium, suggesting that the epithelium plays an important role in differentiation, proliferation, and/or migration of smooth muscle cells. In this study, developmental mechanisms regulating the formation of the smooth muscle layers in the mouse uterus were analyzed using an in vitro culture model. Differentiation of smooth muscle cells occurs at a neonatal stage because ACTA2 gene expression was increased at the outer layer, and GJA1 was not expressed in cellular membranes of uterine smooth muscle cells by postnatal day 15. To analyze the effects of the epithelium on the differentiation of smooth muscle cells, a bulk uterine mesenchymal cell line was established from p53-/- mice at postnatal day 3 (P3US cells). Co-culture with Müllerian ductal epithelial cells (E1 cells) induced repulsive migration of ACTA2-positive cells among bulk P3US cells from E1 cells, but it had no effects on the migration of any of 100% ACTA2-positive or negative smooth muscle cell lines cloned from P3US cells. Thus, uterine epithelial cells indirectly affected the repulsive migration of smooth muscle cells via mesenchymal cells. Conditioned medium by E1 cells inhibited differentiation into smooth muscle cells of clonal cells established from P3US cells. Therefore, the uterine epithelium inhibits the differentiation of stem-like progenitor mesenchymal cells adjacent to the epithelium into smooth muscle cells.