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Nongenomic action and mechanism of 17#beta#--estradiol in cytosolic calcium concentration in delayed implantation mouse endometrial stromal cells

Wang Qiang, Yue Limin, Zhang Jinhu, Tian Jimei, He Yaping

Department of Physiology,West China School of Preclinical and Forensic Medicine,Sichuan University.Chengdu 610041,Sichuan

Abstract

To investigate the existence of nongenomic action of 17#beta#-estradiol (E_(2)) in the delayed implantation mouse endometrial stromal cells, the change of 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 of [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, 5 #mu#g/mL tamoxifen + 1×10~(-8) mol/L E_(2) 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 Hank’s balanced salts (HBSS, pH7.2), which contained glucose (1 g/L), collagenase I (0.125%), for 1 h at 37 oC. 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 light at 488 nm and 526 nm, respectively, was used to detect the change of [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) 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: the [Ca~(2+)]_(i) concentration 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 no increase of [Ca~(2+)]_(i) in DMSO group and BSA group. Tamoxifen, an antagonist of traditional estrogen receptor, did not inhibit the increase of [Ca~(2+)]_(i) induced by E_(2). Immunofluorescent results showed that the change in phosphor-PLC level had the same time-dependent result of [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 of delayed implantation mice is possibly carried out through a nongenomic pathway, and that the transmembrane signal transduction is related to the phosphorylation of PLC in this process.

Key words: Estrogen;Phospholipase C;Calcium;stromal cells

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Citing This Article：

Wang Qiang, Yue Limin, Zhang Jinhu, Tian Jimei, He Yaping. Nongenomic action and mechanism of 17#beta#--estradiol in cytosolic calcium concentration in delayed implantation mouse endometrial stromal cells. Acta Physiol Sin 2008; 60 (2): 169-174 (in Chinese with English abstract).