| Objection:In this study, mice were used as experimental subject, to discuss whether the quality of the embryos was affected by the dose of hormone and interval time of repeat superovulate,as well as preliminary study of whether septin2 protein affected the quality of embryos.Method:Experiment 11.Choosing 120 mice of 8-week-old, randomly divided into four groups, respectively were natural estrus group(0IU), 5 IU group, 7.5 IU group and10 IU group, each group containing 30 mice, the natural estrus group was given no hormone,and the other groups will be respectively injected PMSG(5 IU)+ h CG(5 IU), PMSG(5 IU)+ h CG(7.5 IU), PMSG(10 IU) + h CG(10 IU).2.The female mice were copulated with male mice in a ratio of 1:1 after superovulation, and then killed the pregnantthe mice and picked 1- cell stage embryos on the next morning. Counting the total number of embryos and malformed embryos, and calculating the rate of deformity. Puting the 1- cell stage embryos into 37℃,5% CO2 incubator and culturing until to blastocyst, Counting the number of 2- cell embryos, 4-cell embryos, 8-cell embryos, morulas and blastaeas, and calculating the percentage during each period.3. Collecting the nutrient solution, and detecting the content of IGF-II by ELISA kit.4. Using confocal microscopy to observe blastaeas after stained by H33258.Experiment 2In this study, 40 mice of 8-week-old were used, and randomly divided into four groups, respectively were group 0 which the female mice were copulated with male mice in a ratio of 1:1 after superovulation; group 1 was repeated superovulation after 5 days from the first time superovulation;group 2 was repeated superovulation after 10 days from the first time superovulation;,group 3 was repeated superovulation after 15 days from the first time superovulation, each group containing 30 mice. Each mouse was injected PMSG(7.5 IU) + h CG(7.5 IU) everytime. The rest steps are same as the experiment one.Experiment 3:1. In this study, 30 mice of 8-week-old were used, and induceing superovulation by injecting PMSG(7.5 IU) + h CG(7.5 IU), and then copulated with male mice in a ratio of 1:1, the pregnantthe mice were killed the next morning, collecting 1-cell stage embryos and selecting 400 normal embryos that dividing into experimental group 1 and control group 1,each group included 200 embryos. Experimental group 1 should be cultured in FCF medium, control group 1 should be cultured in DMSO medium, placed in 37 and 5% CO2 ℃ incubator and cultured to blastocysts, statistical the number of blastocysts, and used for immunofluorescence of α-tubulin.2. The surplus embryos should be averagely divided into experiment group 2 and control group 2, separately culture in these above two kinds of medium, used for immunofluorescenc staining of septin2 protein.Result:Experiment 1:1. Compared with the natural estrous group, 1-cell embryos of 5IU group, 7.5 IU group and 10 IU group were significantly increased(P<0.01). The average embryo number at 7.5 IU group was the most and significantly more than 5 IU group(P<0.05); as for deformity rate 10 IU group was the highest, significantly higher than that of group 0 IU and 5IU(P<0.05); 2-cell embryo developmental rates were all above 95% and 4-cell embryo embryonic development rates are decreased to below 90%. Among the three groups there had no significant difference;8-cell embryos developmental rate of 10 IU group was the lowest, which was significantly lower than that of the 5IU(P<0.05); morula developmental rate, compared with 5IU group, of 7.5 IU group and 10 IU group was significantly reduced(P<0.05); blastocyst developmental rate was followed by 0IU>5IU>7.5IU>10IU, and 10 IU group was significantly lower than other groups(P<0.05).2. ELISA experiment results showed that the IGF-II content in every culture medium was 0IU>5IU>7.5IU>10IU, and 10 IU group was significantly lower than other groups(P<0.05).3. H33258 staining showed that the number of blastomeres at 10 IU group slightly reduced compared with 0IU group.Experiment 21. Repeated superovulation interval time is shorter; the embryo deformity rate is higher. The abnormality rate of one sexual cycle was significantly higher than that of three sexual cycle group and one superovulation group, and the number of embryos available is less. With the interval of time, the deformity rate became smaller and the number of embryos available increases. When it came to the interval of three sexual cycles, each group restored to a level of superovulation. The superovulation embryo rate of 2- cells, 4- cells and 8- cell embryo rate and the morula rate had no significant difference, but with longer intervals, each stage of embryo cleavage rate increased. After the interval of three sexual cycles, each group restored to a level of superovulation. As for blastocyst rate, interval of one sexual cycle was significantly lower than one superovulation and that of three sexual cycles(P<0.05).2. ELISA experiment results showed that the content of IGF-II in the culture medium was followed by groups0 > group3 >group2 > group1, which was significantly lower than that in group 0 and group 3(P<0.05).3. The result of H33258 staining showed that the number of blastomeres at group 1 and group 2 decreased.Experiment 31. The rate of blastocyst was significantly decreased after FCF treatment(P<0.05).2. The picturel of confocal microscope can be clearly observed that septin2 in the control group embryos was mainly expressed in and after every stage of embryo blastomeres, concentrated in the egg and it has strong fluorescence signal. In the experimental group 2, septin2 in 2- cell stage blastomere nuclei, and 4-cells, 8-cell embryos granular blastomere has a weak expression. After morulas there is almost no expression, and 4-cells, 8-cell embryos have particle phenomenon.3. Compared with the control group, the microtubule in experimental groups were irregular. There was abnormal spindle morphology and chromosome was disordered. 7. Septin2 protein in mouse embryos 2- cell stage to blastocyst stages was expressed, and it located in blastomere nuclei.Conclusions:Experiment 11. PMSG/HCG can increase the number of available embryos of mice;2. PMSG/HCG has a certain threshold for the super Ovulation of mice and the dose is too high or too low can not achieve the desired results;3. High dose of PMSG/HCG can affect the quality of embryos and reduce the development rate of embryo;Experiment 21. Interval time of repeated superovulation is shorter, the effect of superovulation and the embryo quality are worse;2. Interval time of repeated superovulation is shorter, the probability of ovarian cyst is higher.3. Interval time of repeated superovulation more than three sexual cycles will make mice restore to a level of one time superovulation.Experiment 31. Septin2 protein was located in the blastomere nuclein of embryos from 2- cell stage to blastocyst stages in mouse.2. FCF inhibitors made Septin2 protein express lower.3. Low expression of Septin2 protein can lead to embryonic develop abnormally; cytokinesis is uneven; the size of blastomeres is not uniform and the number is decreasing; chromosomes arranged is disorderly, and may cause "particles" phenomenon in the embryo.4. Low expression of Septin2 protein can lead to the distribution of microtubule in blastocyst confuse, the number of the blastomere decrease, and the shape of spindle and the arrangement of chromosomes develop abnormally. |
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