The Effects Of Exogenous BDE-209 Exposure On The Development Of Early Embryos In Vitro And The DNA Damage Of Mice Sperm

Part I The effects of exogenous BDE-209 exposure on fertilization and the development of embryos in vitro in the mice[Objective]The prior research pointed out that exogenous BDE-209 can reduce the oocyte survival rate. In this study, we use the methods of in vitro fertilization and culture the early embryos, aiming to investigate the toxic effects and dose-response relationship of exogenous deca-brominated diphenyl ether (BDE-209) on in vitro fertilization in the mice.[Method]Divided the culture medium into four groups according to the final concentration of BDE-209:Control group A:0μg/ml; Group B:10μg/ml; Group C:20μg/ml; Group D:40μg/ml. Take sexually mature Kunming mice female and male respectively 20 and 8. Ovulate on the female mice and mature eggs with hormones. Collect mature oocytes and placed them in the culture medium randomly. Then take the sperm from epididymal tail of the male mice and make the sperm capacitation by swim-up. Make sperm and eggs in a droplet to culture. Put the disk into the incubator to fertilizate. Observations:According to the development of fertilized eggs in the mice in vivo, we observed the cells every 24-hour. Statistics:the forming rates of 2-cells, 4-cells,8-cells, morula and blastocyst. Meanwhile, observe the morphological changes in each stage.【Results】1. During the 2-cell stage, there is no statistical significance (P> 0.05) of the ratio between the group B and that of the control group A; while the rates of middle-dose group C and high-dose group D are significantly lower than that of control group A(P<0.01).2. The forming rates of 4-cells,8-cells, morale and blastocyst in the experimental group is lower than that in the comparison group, which has the remarkable statistics significance(P <0.01).3. The forming rates in Group B are totally lower than that in Group A except the stage of 2-cells. Group B VS Group A:①stage of 4-cells:χ2=4.927,P=0.026.②stages of 8-cells, morale and blastocyst:P<0.001.4,By comparison, the embryonic development rate during each stage of C group is lower than that of B group(P<0.01).5,By comparison, the embryonic development rate during each stage(2-cell stage not included) of D group is lower than that of C group(P<0.01).【Conclusions】1. Exogenous low-dose(10μg/ml) of BDE-209 did not have an apparent effect on fertilization rate in mice in vitro. However, middle and high dose(20μg/ml,40μg/ml) BDE-209 could decline the fertilization rate of the mice in vitro remarkable.2. A certain concentration (>10μg/ml)of exogenous BDE-209 could inhabit the early embryos from normal development in mice apparently.3. The genotoxicity of BDE-209 on the embryos in the mice could be interwoven in a dose-response relationship.Sum up, through this experiment, the data reviewed illustrate that the BDE-209 indeed contribute to toxic effect on the reproductive disorders and emphasize the sensitivity of early life-stage exposures. Fertilization and embryo development, and its mechanism needs further explore metabolism of cell development in accordance with principles of targeted lines for further study. Part II Detection of DNA damage induced by exogenous BDE-209 in mice sperm of epididymal tail with single-cell gel electrophoresis assay【Objective】In the first part, we know that BDE-209 could decline the rate of fertilization and inhibit the development of early embryos in Kunming mice. Based on the above, we will do a further research to find out some possible mechanism to be an explanation of the BDE-209's toxicity effect. In this part, first, make the epididymal sperm exposed to BDE-209 in different concentration in vitro. Then, to study the genotoxicial mechanism of BDE-209 by detecting the DAN damage in mice sperm with single-cell gel electrophoresis assay(SCGE).【Method】1. Mice sperm were obtained in epididymal in vitro, adding to HTF medium respectively. According the different concentration of BDE-209, divided the experiment to five groups: Group A:0μg/ml; Group B:2.5μg/ml; Group C:5μg/ml; Group D:10μg/ml; Group E:20μg/ml. Put the culture disks containing sperm into the incubator for two and a half hours.2. The SCGE of spermAfter be exposed to BDE-209, each sample was handled following the procedure of shopping rubber, cracking, removing the nuclear RNA and nuclear protein, helicase, electrophoresis, neutralization, dyeing. Finally, the slides were observed by fluorescence microscope and photographed.3. Statistics Each experimental group was repeated three times and counting 70 cells every time, a total of 200 cells each group. Comet analysis software was used to analysis each cell. The index of DNA damage, tail length, tail-head light intensity, the proportion of DNA in tail, Olive tail moment, tail length/head length and mobility were analyzed to evaluate the extent of DNA damage.【Results】1. The impact of distance and strength index on sperm DNA damage by BDE-209 in the SCGE test.In the SCGE test image of the control group, the nucleus can be seen clearly, most of which were round, with clear boundary and uniform density. There be rarely trailing cells and the average tail length was 1.15±1.27μm. As the BDE-209 dose increasing, when the dose reaches to 10 and 20μg/ml, some trailing cells were be observed and the average tail length was add to 2.13±1.29μm and 2.83.±2.46μm respectively. Moreover, the proportion of DNA in the tail of the cells was significantly increased. Withχ2 test, the tail length and proportion of DNA in the tail in Group D and Group E were significant different from that in control Group A (P<0.01). There is a significant changes in the index of the tail length, tail light intensity and proportion of DNA in the tail among experimental groups and that in control Group A by F test (P<0.01)2. the impact of relative index on sperm DNA damageThe results in this part will be better to explain the problem because that the results were relative index. The results showed that the Olive tail moment in Group A was 0.270±0.322, while that in Group D and Group E were 0.453±0.375and 808±0.822 respectively. The tail length/head length in Group C, D, E (0.077±0.093,0.112±0.068,0.191±0.207 respectively)were all higher than that in Group A(0.045±0.049). With x2 test, the Olive tail moment and tail length/head length in Group D and Group E were significant different from that in control Group A(P<0.01). There is a significant changes in the tail length/head length between Group C and that in control Group A (P<0.01).3. The correlation analysis between changes in each index of sperm DNA damage in mice and the concentration of exogenous BDE-209In the 6 index selected in the results, except mobility, the other five indicators were tested by the Person correlation analysis, indicating that all of the correlation coefficients surpassed 0.9.【Conclusions】1. Exogenous BDE-209 could damage the DNA in the mice, and lead to the sperm DNA chains breaking up.2. There is a dose-response relationship between the concentration of exogenous BDE-209 and the extent of damage in DNA chains in mice.