Induction of ChM-I expression during the decidualization of mouse endometrial stromal cells in vitro. Mouse endometrial stromal cells were enzymatically isolated from the uteri of 4 week-old non-pregnant mice and cultured in medium containing 10% charcoal-stripped FBS. The in vitro decidualization of endometrial stromal cells was induced by the addition of E2 (0.1 nM) and P4 (100 nM) to the culture media. (A) Representative microphotographs of the endometrial stromal cells cultured for nine days in the presence (lower panel) or absence (upper panel) of E2 + P4. Arrowheads indicate enlarged or multi-nucleated cells that are characteristic of decidual cells. Bars, 50 μm. (B) Expression of ChM-I and Prl8a2 in decidualized cultures of endometrial stromal cells. Total RNA was extracted from the cells at the indicated time points and from mouse decidua (7.5 days p.c.). One microgram of each total RNA preparation was reverse-transcribed and analyzed by RT-PCR (35 cycles) for comparison. GAPDH was used as an internal control. (C, D) Expression of Prl8a2 gene in decidualized cells and immunostaining of ChM-I protein. The endometrial stromal cells were cultured for six days with or without E2 + P4, fixed, and subjected to in situ hybridization for Prl8a2 (C) and immunostaining with anti-ChM-I antibody (D). Note that intense signals for ChM-I protein (arrowheads) were observed in enlarged cells that also expressed Prl8a2. Bars, 30 μm. All images are representative of three independent experiments.