Department of Obstetrics and Gynecology, Yamaguchi University School of Medicine, Minamikogushi 111, Ube 755-8505, Japan
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Abstract |
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Key words:
endometrial stromal cell/human/hydrogen peroxide/prostaglandin F2/reactive oxygen species
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Introduction |
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PGF2 can be produced by endometrial stromal cells (Lumsden et al., 1984
; Chen et al., 1995
; Skarzynski et al., 2000
), and cyclooxygenase, the rate-limiting enzyme in the biosynthesis of prostaglandins, is also expressed in endometrial stromal cells (Han et al., 1996
; Jones et al., 1997
). We recently reported close association of ROS or SOD with PGF2
in decidual cells (Sugino et al., 2000b
). Therefore, we focused on endometrial stromal cells and studied whether ROS could stimulate PGF2
production to influence endometrial function.
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Materials and methods |
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Materials
Phenol Red-free Dulbecco's modified Eagle's medium (DMEM) and glutamine were purchased from ICN Biomedicals Inc. (Aurora, OH, USA). Streptomycin, penicillin and trypsin-EDTA were from Life Technologies Inc. (Grand Island, NY, USA). Collagenase, indomethacin, 6--methyl-17OH-hydroxyprogesterone acetate (MPA), and oestradiol were from Sigma Chemical Co. (St Louis, MO, USA). Hydrogen peroxide was from Wako Pure Chemical Industries Ltd (Osaka, Japan). High performance liquid chromatography grade acetonitrile was obtained from Nacalai Tesque Co Ltd (Kyoto, Japan). Tissue flasks, culture plates and nylon mesh were from Becton Dickinson Co. (Franklin Lakes, NJ, USA).
Endometrial stromal cell isolation
Human endometrium was obtained at hysterectomy from normally cycling premenopausal women, aged 4042 years, who underwent surgery for myoma uteri. Endometrial samples were histologically diagnosed as late proliferative phase according to the criteria of Noyes et al. (Noyes et al., 1950). Tissue samples were washed with Phenol Red-free DMEM containing 200 mmol/l glutamine, 100 mg/ml streptomycin and 50 IU/ml penicillin, and minced into small pieces of <1 mm3. Endometrial stromal cells were isolated as reported previously (Sugino et al., 2000a
). In brief, after the enzymatic digestion of minced tissues with 0.2% collagenase in a shaking water bath for 2 h at 37°C, stromal cells were separated by filtration through a 70 µm nylon mesh. The filtrates were washed three times, and the number of viable cells was counted by Trypan Blue dye exclusion. The homogeneity of the stromal cell preparation was verified by immunocytochemistry for the stromal cell-reacting antibody (vimentin) (data not shown). Cells were seeded at 105 cells/cm2 in 75 cm2 tissue culture flasks and incubated in Phenol Red-free DMEM containing glutamine, antibiotics and 10% dextran-coated charcoal-stripped fetal calf serum (FCS) at 37°C, 95% air and 5% CO2. At confluence, cells were treated with 1xtrypsin-EDTA and subcultured into 25 cm2 tissue culture flasks. At ~80% confluence after the first passage, the cell culture medium was changed to the treatment medium.
Cell culture
To examine the effect of lipid peroxidation on PGF2 production by endometrial stromal cells, cells were incubated with hydrogen peroxide (10, 50, 100, 200 µmol/l) in the medium (Phenol Red-free and serum-free DMEM supplemented with glutamine and antibiotics) for 6 h at 37°C, 95% air and 5% CO2.
To study the time course, endometrial stromal cells were incubated with hydrogen peroxide (200 µmol/l) for 3, 6, or 24 h under the same condition as described above.
To examine whether hydrogen peroxide is also effective in decidualized endometrial stromal cells, endometrial stromal cells were decidualized by incubation with phenol red-free DMEM supplemented with glutamine, antibiotics, 2% stripped FCS, MPA (106 mmol/l) and oestradiol (108 mmol/l) for 18 days at 37°C, 95% air and 5% CO2, and then incubated with hydrogen peroxide (200 µmol/l) for 6 h under the same condition as described above. Decidualization was confirmed by the mRNA expression of insulin-like growth factor-binding protein-1 (IGFBP-1), which is a specific marker of decidualization (Giudice et al., 1992; Kim et al., 1998
; Sugino et al., 2000a
).
To study whether the effect of hydrogen peroxide on PGF2 production is mediated by cyclooxygenase, endometrial stromal cells were incubated with indomethacin (0.5 µg/ml), an inhibitor of cyclooxygenase, in the presence of hydrogen peroxide (200 µmol/l) for 6 h under the same condition as described above. After cell incubation, PGF2
concentrations in the medium and cells were determined. A single incubation was performed in triplicate on cells from a single hysterectomy sample. The samples from three individuals were used in a single experiment. Therefore, three different incubations were performed in a single experiment. To examine the effect of hydrogen peroxide and/or indomethacin on cell viability, cells were seeded at 105 cells/ml into each well of a 24-well culture plate and incubated. After incubation, cell viability was tested by the Trypan Blue dye exclusion method. Three different incubations were performed in triplicate. Hydrogen peroxide (200 µmol/l) and/or indomethacin had no effect on cell viability after 6 h incubation: control: 75.3 ± 3.3%, H2O2: 73.5 ± 3.7%, indomethacin: 72.9 ± 2.3%, H2O2 + indomethacin: 71.3 ± 5.3%; mean ± SEM of three different incubations.
PGF2 assay
After incubation, the cells were washed twice, resuspended in PBS (0.01 mmol/l, pH 3.0) and sonicated. Prostaglandins were extracted as reported previously (Sugino et al., 2000b) based on a previously reported method (Olofsson et al., 1990
). In brief, sonicated samples and the medium were applied to a C18-LRC solid phase extraction cartridge (Bond-Elut, Varian Co., Harbor City, CA, USA), and the cartridge was rinsed with distilled water and 10% acetonitrile. Prostaglandins were then eluted with methanol and evaporated under nitrogen. The dried extract was dissolved in ethanol and the kit assay solution, and PGF2
concentrations were determined by a PGF2
enzyme immunoassay kit (Assay Designs, Inc., Ann Arbor, MI, USA). The sensitivity of the assay was 4.6 pg/ml. The intra- and inter-assay coefficients of variation were 7.8 and 7.0% respectively. The results were expressed as ng PGF2
per mg protein for cellular concentrations and as ng per ml for medium concentrations. Protein concentrations in the sonicated samples were determined by the method described by Lowry et al. (Lowry et al., 1951
).
Statistical analysis
Data were examined by analysis of variance (ANOVA) and Duncan's new multiple range test. Where appropriate, Student's t-test was employed. Differences were considered significant at P < 0.05.
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Results |
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Discussion |
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In contrast, it has been reported that synthetic capacity of prostaglandins is very low after conception in human decidua (Maathuis and Kelly, 1978; Ishihara et al., 1986
). We also found that ROS were low in the decidua of early pregnancy (Sugino et al., 1996
). These findings are in agreement with the present result and seem reasonable for the maintenance of pregnancy. In spontaneous abortion, expulsion of the uterine content occurs to terminate pregnancy, usually accompanied by uterine contraction. However, in the case of the missed abortion, dead products of conception are retained in the uterus without bleeding for several weeks. Thus, the exact mechanism responsible for spontaneous expulsion is not precisely clarified. We recently reported that the concentrations of lipid peroxide and PGF2
of the decidua of missed abortion were the same as those of normal pregnancy and much lower than those of incomplete abortion with uterine contraction and uterine bleeding (Sugino et al., 2000b
). Therefore, increased lipid peroxides in the decidua could be responsible for the increased PGF2
production, eventually to induce uterine contraction and expulsion of the uterine content (Cherouny et al., 1988
; Norman et al., 1991
).
In conclusion, the present study has shown that ROS might influence endometrial function by regulating PGF2 production in human endometrial stromal cells.
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Acknowledgements |
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Notes |
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References |
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Submitted on November 14, 2000; accepted on June 5, 2001.