| To explore the effect of characteristics with physical and biochemical on oocyte zona pellucida by vitrification, understand the factors with fertilization rate reduction of oocytes after vitrification, and provide ideas and theoretical basis for improving vitrification methods and fertilization rate after vitrification, the four vitrification cryoprotectants with most widely applied were selected to observe the mouse oocyte development. The vitrification method with smaller injury on the development of mouse oocytes has been chosen and the changes on physical and biochemical characteristics of zona pellucid before and after vitrification have been observed. The injury situation and recovery effect of transmembrane protein CD9 after vitrification by fluorescence immunohisto-chemistry also has been observed. The results showed that:1. Four groups' cryoprotectants all have low toxic effect on mouse oocytes. resuscitation survival rate and fertilization rate of group 1?4 were significantly lower than control group (P< 0.05), cleavage rates of group 3 and blastocyst rate of group 4 were significantly different to control group (P<0.05). As a result, in the experiments, cryoprotectant with the smallest injury on mouse oocytes developmental competence from group 1 was selected. The formula was HM+7.5%(DMSO+EG), HM+15%(DMSO+EG)+5mol·L-1 Su.2. Under the microscope, each observation point of oocyte zona pellucida in the frozen group, exposed group and control group by the Hoffman system has been observed and recorded, and oocyte zona pellucida thickness(ZPT) and thickness of variables (ZPTV) in each group have been calculated. The results showed that ZPT of frozen group (6.05?m) was significantly higher than control group (5.77?m) (P<0.05); the difference on ZPTV among groups is not significant. The vitrification has effect on oocytes ZPT but little on oocytes ZPTV.3. Under the polarized light microscope, the real-time display of biref-ringence score in each group by OCTAX Polar AIDE system has been obtained. The statistical analysis showed that the difference between frozen group (0.3) and control group (1.22), and exposed group (2.02) was significantly (P<0.05), but the difference between exposed group and control group was not significantly. The results showed that vitrification reduces zona pellucida birefringence (ZPB) significantly.4. The correlate analysis of the ZPT, ZPTV and ZPB showed that, ZPT and ZPB were significantly correlated (P<0.05). Therefore, ZPT can reflect ZPB to same degree.5. The protein concentration and soluble sugar concentrations from the same number of oocytes zona pellucida extract in frozen group, exposed group and control group were measured. The protein concentration in frozen group was 0.218mg·mL-1. Compared with control group (0.239 mg·mL-1) and exposed group (0.223 mg·mL-1), the differences were not significant (P< 0.05). Frozen group with 0.041 mg·mL-1 soluble sugar concentration was significantly higher than control group (0.008 mg·mL-1) and exposed group (0.018 mg·mL-1) (P< 0.05). The results showed that the vitrification has little effect on the protein content of oocytes zona pellucida, but much on the soluble sugar content.6. By immunofluorescence, the oocytes transmembrane protein CD9 expression situation of frozen group, exposed group and control group has been observed. In frozen group, level IV cells was found, and level I cells and surface expression of oocyte CD9 protein were significantly reduced (P<0.05). Further, the oocytes of recovery group have been prolonged culture with IBMX. Compared with frozen group, the number of level I cells in recovery group and culture group increased significantly (P<0.05). The results showed that CD9 expression can be restored by prolonging culture after vitrification, and the effect is more significant with IBMX. |
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