Comparative Studies on the In Vitro: RESULTS(2)
In order to induce decidualization in vitro in stromal cells of the baboon and human endometrium, cells were treated with estradiol, MPA, relaxin, and dbcAMP. Prolactin and IGFBP-1 were measured in culture media as indicators of decidualization. Human stromal cells produced both IGFBP-1 and prolactin in response to hormones alone. Both proteins were detectable under our culture conditions by Day 6 of treatment and increased thereafter (Fig. 3, B and D). Baboon stromal cells, on the other hand, produced minimal levels of IGFBP-1 in response to hormones alone (Fig. 3A). These levels were too low to be detected by Western blot analysis (data not shown). These cells did not produce detectable quantities of prolactin in response to hormones either (Fig. 3C).
It has been demonstrated previously that agents that stimulate the cAMP second messenger system increase the production of prolactin and IGFBP-1 in hormone-treated human stromal cells in culture. Human stromal cells produced higher levels of IGFBP-1 and prolactin protein in response to dbcAMP and hormones as compared to the levels obtained with hormones alone. Significant levels of IGFBP-1 were observed as early as 2 days after initiation of dbcAMP and hormone treatment (see Fig. 5B; p < 0.01). buy prednisone
FIG. 2. In vitro induction of aSMA production in endometrial stromal cells. Stromal cells from baboon endometrium (A) were lysed at the time of isolation (before plating), lane 1; at passage, lane 2; at day before confluence, lane 3; and at confluence, lane 4. Stromal cells from human endometrium (B) were lysed at the time of isolation, lane 1; at 24 h after plating, lane 2; and at confluence, lane 3. Cytosol extracts were subjected to Western blot analysis and probed for aSMA using a monoclonal antibody.
FIG. 3. Time course of IGFBP-1 and prolactin protein secretion by endometrial stromal cells. Confluent stromal cells from baboon and human endometrium were treated with medium only (RPMI + 2% stripped FBS; — /—), 0.1 mM dbcAMP only (—/+), hormones only (36 nM estradiol, 1 ^M MPA, and 100 ng/ml relaxin; +/—), or hormones and dbcAMP (+/+) for 2, 6, 12, and 17 days. Fresh medium was added at 24 h prior to each sampling time point, after which IGFBP-1 from baboon (A) and human (B) cells and prolactin from baboon (C) and human (D) cells were measured using RIAs. Data are expressed as least-squares means ± SEM of 4 baboon or 3 human tissues.