| BackgroundCarbon disulfide (CS2) belongs to one of most important organic solvents and is widely used in industrial processes of rayon fibers manufacturing. China is a big textile country and the production of viscose fiber continues to grow rapidly. In the process of industrial production, CS2 is volatile and tends to spread to the production environment, which is harm to the exposed women employees. Some studies found that CS2 has obvious reproductive toxicity and exposure to CS2 would result in gonadal dysfunction, abortion and birth defects of child generation in female employees. With the animal studies, our group further found that exposure to CS2 at the peri-implantation resulted in the reduced number of implanted embryos, and the oxidative stress and DNA damage were detected. But its mechanism has not been elucidated yet.The success of embryo implantation on the one hand to the womb receptive, on the other hand depends on the ability of implantation of embryonic normal growth and development. What’s more, the consistent of the two processes is also very important. In recent years, the role of autophagy in the reproduction has become the focus. Autophagy is a conserved cellular degradation process, which played an important role in the fertilized egg formation, embryo development, implantation and placenta formation. The changes of utophagy may lead to the occurrence of adverse reproductive outcomes. Several studies have found that the oxidative stress and DNA damage could influence autophagy. Oxidative stress and DNA damage that caused by CS2 may result in the unbalance of autophagy in mice uterus and embryos, thus lead to the embryo implantation disorders. Besides, the arginine supplement could reduce the level of autophagy, and induce the development of cell. NCG (N-carbamylglutamate) is a kind of supplymentation of arginine. It is commonly used in the research on reproduction. Die supplyment NCG may affect the reproduction outcomes by influence the autophagy of CS2 exposure mice.Previous study found that GD4 mice reflect the most sensitive embryo toxic effect after CS2 exposure. In this study, we built an animal model in which pregnant mice were exposed to CS2 at the GD4 to detect the autophagy in mice uterus and embryos, in order to explore the role of autophagy in the mechanism of embryo implantation disorders induced by CS2. And die supplement NCG, to observe the outcome of embryo implantation and the autophagy in uterus and embryos, further testify the role of autophagy in the embryo implantation disorders induced by CS2 exposureObjectivesTo built the animal model of embryo implantation disorders induced by CS2 exposure at GD4 in mice and observe the the maternal toxicity and embryo toxicity by CS2 exposure at different time points and ddtect the autophagy in mice uterus and embryos, in order to explore the role of autophagy in mechanism of embryo implantation disorders induced by CS2. Then diet supplement NCG with pregency mice to further verify the role of reversed autophagy in the mechanism of embryo implantation disorders induced by CS2MethodsAnimal treatment:After acclimating to the standard laboratory conditions for 1 week, male mice were allowed to mate with the females (1:1). The day of vaginal plug presented was designated as the first day of gestation (GD1). Gestational mice on GD1 were randomized into groups. Each endpoint consisted of twelve mice in which six had CS2 exposure and six olive oil exposure.Exposure points and endpoints:In this study, the exposure points of the two parts were the same. Depending on the exposure time, GD4 mice have received single intraperitoneal injection of CS2 or solvents (631.4 mg/kg; solvent control for olive oil; injection capacity of 0.1 ml/10g weight). The Diet supplement of NCG strated in GD1 and end in the observe point. The adding proportionof NCG is 0.1％of fodder (240-330mg/kg).Sample collection and detection of autophagy:Mice on GD9 were sacrificed and gravid uteri were excised and weighted. The number of implanted embryos in each litter was counted; furthermore, litter weight of embryos was noted. The weights of uterus, ovary, liver, spleen and kidney on GD9 were recorded. The gravid uteri were excised and preserved at-80℃. ELISA (enzymes linked immunosorbent assay) were used for detecting the expression of autophagy related protein in uterine tissues and embryos tissues. Real-time PCR was used for detecting the expression of mRNA in uterine tissues and embryos tissues.Statistical analysis:Results were analyzed by a computerized statistical program (SPSS 17.0). If the data conforms to normal distribution and variance, to compare exposure group and control group by t-test. If not, the t’-test was used to compare the difference between exposure group and control group.Differences were considered statistically significant if P<0.05. Values of the results are presented as means± standard deviation.Results1. Maternal toxicity and embryo toxicity after CS2 exposureMaternal toxicity:No significant difference was observed in GD9 body weight, total body weight gain and net body weight gain among all the groups (P>0.05). There was also no statistically significant difference in absolute organ weights and relative organ weights-organ coefficient (adjusted with body weight) among these groups.Embryo toxicity:When compared to the controls, there were statistically significant changes in litter weight of embryos and the coefficient of litter weight of embryos at GD4 CS2 exposed groups. In comparison with the control, the number of implanted embryos was clearly reduced in GD4 CS2 exposed groups (P<0.05). The rate of embryo implantation was decreased by 42.41% after CS2 exposure.2. Maternal-fetal interface autophagy changes after CS2 exposureCompare to the controls, autophagy of uterus tissue was lower at GD5 after CS2 exposure at GD4. P62 expression increased 38.67%(P﹤0.01), mRNA of P62 increased 113%(P<0.01), while mRNA of BECN1 decreased 24%(P<0.01).3. Effects of CS2 exposure on the expression of protein and mRNA of autophagy related gene in uterus tissue of miceAutophagy decreased in mice uterus tissue at GD6 and GD7 endpoints after CS2 exposure at GD4, compared to controls.1. GD6 endpoint:P62 mRNA expression increased 20.59%(P﹤0.01), BECN1 mRNA expression decreased 33.91%(P﹤0.01), and ATG5 mRNA expression decreased 20.04％(P﹤0.01) in uterus tissues of CS2-exposed group, comparing to the controls.2. GD7 endpoint:P62 mRNA expression increased 43.87％(P﹤0.01), BECN1 mRNA expression decreased 62.08% (P﹤0.01), and ATG5 mRNA expression decreased 19.23％(P﹤0.01) in maternal tissues of CS2-exposed group, comparing to the controls.4. Effects of CS2 exposure on the expression of mRNA of autophagy related gene in embryos tissuesAutophagy increased in mice embryos tissues at GD6 and GD7 endpoints after CS2 exposure at GD4, compared to controls.1. GD6 endpoint:P62 mRNA expression decreased 43.24％(P﹤0.01), BECN1 mRNA expression increased 68.73％(P﹤0.01), and ATG5 mRNA expression increased 22.37％(P﹤0.01) in embryos tissues of CS2-exposed group, comparing to the controls.2. GD7 endpoint:P62 mRNA expression decreased 67.96％(P﹤0.01), BECN1 mRNA expression decreased 62.08% (P﹤0.01), while BECN1 mRNA expression increase 67.36％(P﹤0.01) in embryo tissues of CS2-exposed group, comparing to the controls.5. Maternal toxicity and embryo toxicity after NCG supplementMaternal toxicity:When compared to the controls, total body weight gain and net body weight gain were statistically significant changes in NCG Supplement pregnant mice (P﹤0.01). When compared to simple CS2-exposed groups, total body weight and net body weight gain were statistically significant changes in NCG supplement pregnant mice (P﹤0.01). When compared to the controls, there were statistically significant changes in uterus coefficient of NCG supplement pregnant mice, however, there were no statistically significant changes in uterus coefficient between NCG supplement group and simple CS2-exposed groups.Embryotoxicity: When compared with simple CS2-exposed groups, the number of implanted embryos was increased 130.89％in NCG supplement groups (P﹤0.05) and the weight of uterus were statistically significant highter. There were no statistically significant changes in the average weight of uterus between simple CS2-exposed groups and NCG supplement groups.6. Effects of NCG supplement on expression of autophagy related mRNA in uterus tissue of miceThe autophagy was changed between NCG supplement groups compared to the simple CS2 exposure groups.1. In GD6 endpoint:Compared with simple CS2 exposure groups, P62mRNA expression levels decreased 24.37％and ATG5mRNA expression levels decreased 59.16%(P﹤0.01) in uterus of NCG supplement (P<0.01). There were no statistically significant changes of expression of BECN1mRNA in pregnant mice uterus between simple CS2 exposure group and NCG supplement group (P>0.05).2. In GD7 endpoint:Compared with simple CS2 exposure groups, P62mRNA expression level decreased 24.37%(P﹤0.01) and ATG5mRNA expression level decreased 59.16%(P<0.01) in uterus of NCG supplement group (P﹤0.01). There were no statistically significant changes in BECN1mRNA in uterus between NCG supplement group and simple CS2-exposure group (P>0.05).7. Effects of NCG supplement on expression of mRNA of autophagy related gene in embryo tissuesCompared with the simple CS2 exposure group, the autophagay decreased in NCG supplement groups. In GD6 endpoint: Compared with simple CS2 exposure groups, BECN1mRNA expression levels decreased 96.75%(P<0.01) and ATG5mRNA expression decreased 51.20%(P﹤0.01) in embryo of NCG supplement groups. In GD7 endpoint: Compared with simple CS2 exposure groups, P62mRNAexpression levels increased 27.95% and BECN1mRNA expression levels decreased 25.70%(P<0.01) in embryos of NCG supplement. There were no statistically significant changes in BECN1mRNA expression between NCG Supplement GD7 exposed groups and simple CS2 exposure groups (P>0.05).Conclusion1. Exposure to CS2 at the implantation period could induce the decreased level of autophagy in uterus and increased level of autophagy in embryo. The change of autophagy in uterus and embryo of pregnant mice might contribute to embryo loss after CS2 exposure.2. Exposure to CS2 at the implantation period conbine diet supplement NCG could relieve the embryo implantation disorders induced by CS2.3. Diet NCG supplement during pregnancy could affect the autophagy in uterus and embryo of mice. And the autophagy in embryo tissue of NCG supplement mice was lower than the mice in CS2 exposure group. The changes of autophagy after mice diet supplement NCG might be an important mechanism in the relief of implantion disorder caused induced by CS2. |
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