Nongenomic action and mechanism of 17β-estradiol in cytosolic calcium concentration in delayed implantation mouse endometrial stromal cells / 生理学报

ABSTRACT

To investigate the existence of nongenomic action of 17β-estradiol (E(2)) in the delayed implantation mouse endometrial stromal cells, the changes in intracellular calcium concentration ([Ca(2+)](i)) and the upstream of calcium signal in vitro were detected. The experiment was composed of two parts. Firstly, the change in [Ca(2+)](i) in endometrial stromal cells induced by E(2) under different conditions was detected. The mice were divided into 6 groups as follows: 0.1% dimethylsulfoxide (DMSO) control group, 1×10(-8) mol/L bovine serum albumin (BSA) control group, 1×10(-8) mol/L E(2) group, 1×10(-8) mol/L E(2) conjugated with BSA (E(2)-BSA) group, 1×10(-8) mol/L E(2) + calcium-free medium group, 1×10(-8) mol/L E(2) + 5 mg/mL tamoxifen group, with 4 mice in each group. The endometrial tissue was obtained from delayed implantation mice at pregnant day 7, and digested by incubation of tissue minces in Hankos balanced salts (HBSS, pH 7.2), which contained glucose (1 g/L), and collagenase I (0.125%), for 1 h at 37 degrees C. The stromal cells were preloaded with 2.5 mmol/L Fluo-3/AM, a fluorescent probe of calcium, for 30 min. A confocal laser scanning microscope, which fixed the wave length of excitation and emission at 488 nm and 526 nm, respectively, was used to detect the change in [Ca(2+)](i). Secondly, the mechanism of E(2) effects in endometrial stromal cells was investigated. Immunofluorescent method was used to detect the change in phosphorylation of phospholipase C (PLC) before and after the stromal cells were treated with E(2) for 5 min, 15 min, and 30 min. Seven delayed implantation mice were used. The results were as follows. [Ca(2+)](i) increased immediately and reached the maximum at 15 min after the stromal cells were treated with 1×10(-8) mol/L E(2) and returned to the normal level at 30 min. In E(2)-BSA group and E(2) + calcium-free medium group the same results were obtained as that in E(2) group, but there was no increase of [Ca(2+)](i) in DMSO and BSA groups. Tamoxifen, a traditional antagonist of estrogen receptor, did not inhibit the increase in [Ca(2+)](i) induced by E(2). Immunofluorescent results showed that the change in phosphorylated-PLC level had the same trend as [Ca(2+)](i) after the cells were treated with E(2). Compared with that in the control group, the immunofluorescent intensity increased at the beginning and achieved the maximum at 15 min (P<0.001), then declined to the normal level at 30 min. These results suggest that the rapid response of [Ca(2+)](i) induced by E(2) in the endometrial stromal cells in delayed implantation mice is possibly carried out through a nongenomic pathway, and the transmembrane signal transduction is related to the phosphorylation of PLC in this process.