| BackgroundCarbon disulfide (CS2) is an organic solvent and important chemical raw material, which is widely used in the production of viscose fiber and the main workers are women. Epidemiological studies showed that women workers who had been exposed to CS2 had significantly higher incidences of early fetal loss, spontaneous abortion and birth defects in offspring than those in the control group, suggesting that female workers chronically exposed to CS2 would affect the early process of embryonic development. Our preliminary studies found that the number of embryos obviously reduced when mice exposed to CS2 during the process of embryo implantation. However, the mechanism of embryotoxicity caused by CS2 remains unidentified.Embryo implantation is a highly regulated event in which the trophoblast invades maternal endometrium and makes contact with the maternal blood supply. It involves a variety of substances, and integrins, one of the adhesion molecules, play an important role in the process of implantation. Results from embryology show that integrinαvβ3 is a molecular marker for endometrial receptivity, facilitating the endometrium to be in the adhesive state which prepares for embryo implantation, and its expression is regulated by estrogen and progesterone. Leukemia inhibitory factor (LIF) is expressed in endometrium specifically during the "implantation window". LIF can promote the expression of integrinαvβ3 and thus improve the adhesion of endometrium to blastocyst. Expression of survivin in endometrium is consistent with the process of implantation and it is involved in this process through inhibiting apoptosis of cells in the endometrium. In addition, integrinαvβ3 can increase the expression of survivin. These results indicate that LIF and survivin are the upstream and downstream regulatory proteins respectively during the expression of integrinαvβ3. Therefore, this study was designed to detect the expression of adhesion molecule-related proteins in order to explore the mechanism of embryotoxicity caused by CS2.ObjectiveMice were exposed to CS2 at different time points during embryo implantation and sacrificed on the corresponding endpoints, to establish the animal models of embryo development and implantation defects. We use the models to observe changes in the situation of embryo development and the number of embryos, to detect expression of adhesion molecule-related proteins (integrinβ3, LIF, survivin) in uterus, to determine concentration of progesterone (P4) and estradiol (E2) in serum, and to analyze the relationship between implantation defects and the protein expression and hormone levels, in order to explore the mechanism of embryotoxicity caused by CS2 and provide scientific basis for the protection of the occupational employees.Methods1. Establishment of animal modelDesign for the exposure:Pregnant mice were intraperitoneally exposed to CS2 at different time points (GD3, GD 4, GD 5, GD 6) during embryo implantation for one time and sacrificed on the corresponding endpoints (GD4, GD 5, GD 6, GD 7, GD 9) respectively. The day on which the female mice mated successfully was identified as the first day of gestation (GD1). Dosage of CS2 was designed to be 631.4mg/kg (O.4LD50) and the injection volume was O.1ml/10g body weight. Mice in the control group were injected with the corn oil.Animal disposal:Sexually-matured Kunming female mice (aged 8-12 weeks, weighted 29-33g) and male mice (weighted 35-37g) were bought from Laboratory Animal Research Center of Shandong University (Batch No. SCXK (LU) 20090001) and they were fed on free diet, under natural light. Mice were allowed to acclimatize for 1 week prior to the experiment. Then female mice were pretreated with 10U PMSG, 48h later they were injected with 10 U HCG, and afterwards mated with the male mice. The pregnant mice were randomly divided into different groups.Collection of the observation indexes:Mice were sacrificed on each endpoint. The body weights during the pregnancy were recorded. The embryo and uterus from each litter were harvested and weighted and the number of embryos on GD9 in each litter was counted. The weights of uterus, ovary, liver, spleen and kidney were taken down and then these tissues were frozen and preserved in-80℃refrigerator for further analysis.Statistical analysis:A computerized statistical program (SPSS 16.0) was used for statistical treatment. The datas were first analyzed by homogeneity test for variance, followed by one-way analysis of variance (ANOVA) if the variances were homogenous, and then the different toxic effects between groups were determined by Dunnett's t test. If the variance were not equal, the data would be analyzed by a non-parametric analysis of variance (Kruskal-Wallis H test). Statistical significance was determined at level of a=0.05.2. Detection of protein levels in murine uterusUterine tissues were manually homogenized and the supernatants were collected for detection of protein concentration by Bradford method. Then expression of adhesion molecule-related proteins (integrinβ3, LIF, survivin) in uterus of mice were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Bands of protein were scanned and the integral optical density (IOD) of the objective strip was quantified using Quantity One 4.4 software. The method of Independent-Samples T Test was applied to detect difference in IOD between the treated group and the control group.3. Detection of P4 and E2 concentration in murine serumConcentration levels of P4 and E2 in maternal serum were determined by enzyme-linked immunosorbent assay (ELISA) and the ELISA kits were purchased from Shanghai blue-gene biotechnology company. The detection of hormone levels was operated according to the instructions of the ELISAkits. Statistical analysis of the data was the same as the methods used in part 1.Results1. Embryotoxicity induced by CS2 exposure during implantationMaternal toxicity:Exposure to CS2 during embryo implantation can reduce the body weight on GD9 and the total body weight gain, but difference in the net body weight gain among groups was not significant (P=0.08). No effects were observed on the weights of uterus, ovary, liver, spleen, kidney and their coefficients (That is the value of tissue weight divided by body weight) in each treated group, suggesting that there was no significant maternal toxicity under this dose of CS2. Embryotoxicity:The number of embryos decreased significantly in mice exposed to CS2 during embryo implantation, especially on GD4 and GD5 exposure time (P values were 0.01 and 0.02.). Exposure on GD4 and GD5 also reduced the litter weight of embryos obviously (P values were both 0.02.), but no significant difference was observed in the mean weight of embryos, which suggested that the number of implanted embryos was a main indicator affecting the litter weight of embryo.2. Effects of CS2 exposure on expression of proteinsExposure to CS2 during embryo implantation (GD3,4,5,6) reduced the expression of LIF in uterine tissue on each first endpoint (P<0.05), and the LIF expression declined obviously on GD5 and GD6 endpoints when the exposure time was on GD3 (P<0.05). The expression of integrinβ3 decreased significantly on GD7 endpoint in all the treated groups, and the integrinβ3 expression declined markedly on GD4 and GD5 endpoints when the exposure time was on GD3 (P<0.01). Results indicated that the expression of LIF and integrinβ3 was much more sensitive to the toxic effects caused by CS2 when the exposure time was on GD3. However, no effect of CS2 exposure was observed on the expression of survivin in mice uterus.Correlation analysis was applied to evaluate the relationship between the number of embryos and the protein expression in maternal uterine tissues on GD9. Results showed that the correlation coefficient between the number of embryos and LIF expression was 0.327 (P=0.037), for integrinβ3 and survivin the correlation coefficients were 0.289 (P=0.025) and 0.188 (P=0.151) respectively, suggesting that there was a significant correlation between the number of embryos and expression of LIF and integrinβ3, while the relationship between the embryo number and the survivin expression was not statistically significant.3. Effects of CS2 exposure on concentration of hormonesExposure to CS2 during embryo implantation (GD3,4,5) significantly affected the concentration level of P4 in mice serum on GD6 endpoint (P<0.01), and the P4 level declined on GD9 endpoint when the exposure time was on GD5 and GD6 (P<0.05). The concentration of E2 obviously decreased on GD7 and GD9 endpoints in each treated group (P<0.05), and the serum E2 level was decreased on GD6 endpoint when the exposure time was on GD3 and GD5 (P values were 0.016 and 0.055.). Results indicated that serum P4 was much more sensitive to the effects of CS2 on GD6 and GD9, while for E2 it was on GD7 and GD9.Correlation analysis was applied to evaluate the relationship between the number of embryos and the concentration of P4 and E2 in maternal serum on GD9. Results showed that the correlation coefficients were 0.119 (P-0.365) and 0.102 (P=0.438) respectively, suggesting that there was no significant correlation between the number of embryos and the concentration of P4 and E2.Conclusions1. Exposure to CS2 during embryo implantation could produce embryotoxicity, and it was obvious when the exposure time was on GD4 and GD5.2. Exposure to CS2 during embryo implantation reduced the expression of LIF and integrinβ3, and there was a significant correlation between the number of embryos and expression of LIF and integrinβ3, which indicated that the lower level of protein expression may be one of the important mechanisms of embryotoxicity caused by CS2.3. Exposure to CS2 during embryo implantation reduced the concentration of P4 and E2 in mice serum, which occurred later than that of protein. But the relationship between the number of embryo and the level of hormone was not significant, suggesting that exposure to CS2 may induce implantation defects through decreasing expression of LIF and integrinβ3, it was not regulated by hormones and this phenomenon needs to be verified. |
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