| Vitrification cryopreservation of oocytes and embryos is an ideal cryopreservation technology method,which can significantly improve the survival rate and development rate of cryopreserved oocytes and embryos compared with other freezing technologies.Existing studies have shown that after vitrification of oocytes,the level of reactive oxygen content increases,and the copy number of mitochondrial DNA and the activity of mitochondrial cytochrome C oxidase decrease.It is believed that there is a connection between the redox state of oocytes and glucose transport and metabolism.In this study,mouse oocytes were used as the object,using fluorescence quantitative PCR,immunofluorescence staining,western blotting and other technical methods to measure the glucose transport capacity,NAPDH,ROS,GSH,ATP and GLUT1 of vitrified vitrified-thawed oocytes.Perform tests to study the effect of vitrification on the glucose transport level of oocytes,explore the reasons for the abnormal glucose uptake and metabolism of mouse oocytes caused by vitrification,and conduct research on methods to improve the glucose transport and metabolism levels of vitrified oocytes.The following research results have been obtained:1.The results of the study on the effect of vitrified oocytes on glucose transport and metabolism showed that the glucose uptake capacity of mouse oocytes was significantly reduced after vitrification(p < 0.01),the intracellular energy production was blocked,and the detection of ATP content decreased(p < 0.01).The redox state was abnormal,the levels of NADPH and GSH were significantly reduced(p < 0.01),and the level of ROS was significantly increased(p < 0.01).It shows that after vitrification of oocytes,the glucose transport capacity is reduced,and the energy production and redox state of the oocytes are affected.2.The expression level of glucose transporter GLUT1 in vitrified oocytes showed that the m RNA level of GLUT1 decreased after vitrification of oocytes(p < 0.01),and the expression of GLUT1 protein and GLUT1 on the cell membrane decreased(p < 0.01).3.In order to study the function of oocyte glucose transporter GLUT1,GLUT1 inhibitor was added to the unvitrified oocyte culture medium.As the result,GLUT1 protein expression decreased and AMPK phosphorylation increased,indicating successful inhibition of GLUT1.At the same time,the glucose transport capacity decreased,the NADPH and GSH levels decreased,the ROS level increased,and the ATP level decreased(p < 0.01),which is consistent with the related abnormalities detected in oocytes after vitrification.It indicates that the glucose transport level and abnormal metabolism of oocytes after vitrification were closely related to the decrease of GLUT1 expression level.4.After adding vitamin C during the process of thawing and resuscitation of oocytes,the expression level of GLUT1 was detected and it was found that the m RNA level and protein level were not significantly different from those of the unvitrified group(p > 0.05).At the same time,the levels of NADPH,ROS and GSH in oocytes and the production of ATP in oocytes were the same as those in the unvitrified group(p > 0.05);the recovery rate,cleavage rate and blastocyst rate of vitrified oocytes were significantly higher than those of the non-vitamin C vitrified group(p < 0.05),and there was no significant difference from the unvitrified group(p > 0.05).Overall,vitrified frozen oocytes will cause decreased glucose transport capacity and abnormal metabolism.Adding vitamin C during the thawing and resuscitation process can improve the glucose transport ability of frozen oocytes and improve the quality of frozen oocytes.This research is highly instructive for the cryopreservation,utilization of oocytes and the improvement of embryonic development potential and safety of frozen oocytes. |
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