Epithelial and stromal cells were isolated from endometrium of Day 1 pseudopregnant rabbits by enzymatic digestion with trypsin or trypsin:collagenase:deoxyribonuclease. Dispersed cells were grown in RPMI 1640 supplemented with 10% whole or steroid-depleted fetal bovine serum (FBS). Epithelial and stromal cells reached confluency after 6 to 7 days in culture and showed specific characteristics. Cells could be differentiated according to morphology, growth patterns, electrophoretic patterns, and response to estrogen or progesterone. Hormonal stimulation of adenylate cyclase activity was measured in broken cell preparations by catalytic transformation of alpha-32P-adenosine triphosphate into 32P-adenosine 3'-5' cyclic monophosphate (cAMP). Adenylate cyclase activity was present in fresh endometrial tissue and in dispersed cells after 7 days in culture. The enzyme activity was significantly higher in stromal than in epithelial cells at all stimulation levels: basal (9.2 +/- 1.0 vs. 2.3 +/- 0.6, p less than 0.001) and guanosine triphosphate (GTP, 300 microM) (25.4 +/- 2.9 vs. 7.0 +/- 1.6, p less than 0.001). Net response to prostaglandin E2 (PGE2, 10 microM) was three times higher (p less than 0.001) in stromal (17 +/- 2) than in epithelial (5.0 +/- 1) cells. These results suggest that PGE2 can stimulate adenylate cyclase in rabbit endometrium and that the enzyme is preferentially localized in the stroma. Our results are in agreement with the hypothesis that cAMP formed in endometrium in response to PGE2 might be involved in the decidual reaction.
Myometrial cells were obtained following a three-step enzymatic digestion of uterine horns from Day 1 pseudopregnant rabbits. Isolated cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), whole or steroid depleted (FBS-DC) at a plating density of 0.5 X 10(6) cells/ml. The cells reached confluency on Day 6 to 7 with whole serum and on Day 7 to 8 with DC serum. The process yielded myometrial cells at a purity level of at least 80% as assessed by indirect immunofluorescence using desmin antibody on confluent cultures. The addition of increasing doses of 17 beta-estradiol (E2) (0.1 nM to 1 microM) to the culture medium resulted in an increase in total protein and DNA content (1.5-fold at 1 nM). Similar treatment with progesterone (P) resulted in a 25% inhibition of protein and DNA content at 10 nM. Pretreatment of cells with E2 (1 nM) for 3 d followed by P (10 nM) for 3 d resulted in a 1.8-fold stimulation of protein with a higher protein: DNA ratio indicating that the increase was due to cellular hypertrophy. Analysis of desmin by polyacrylamide gel electrophoresis showed that this cytoskeleton protein was not affected by steroid treatment. Our results indicate that PR can generate two different responses depending on cell pretreatment. In as much as myometrial cells grown in primary culture respond differentially to E2 and P they should provide a useful model to study the regulation of myometrial contractility.
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