| Good quality oocytes are the prerequisites for biotechnology such as somatic cell nuclear transfer(SCNT).In vitro maturation is a commonly used method to obtain mature oocytes.Energy metabolism is critical for oocyte maturation,and glucose is a key metabolite of oocytes.So glucose concentration is very important for oocyte maturation.With the improvement of human living standards,the number of diabetic patients has increased dramatically,and studies on diabetic patients and animal models have revealed that abnormal glucose metabolism causes Abnormal nuclear maturation of oocytes,and Can delay embryonic development,reduced embryonic development potential,embryo degradation and debris Increasing incidence.As a possible source of xenotransplantation,pigs have explored the effects of high glucose concentration on porcine oocytes to improve the efficiency of SCNT and embryo transfer(ET).Although glucose is one of the energy sources necessary for maturation of oocytes,few studies have focused on the high grape concentration during the in vitro maturation of porcine oocytes.How does mitochondria,which is closely related to energy metabolism,produce ROS,and whether ROS levels have changed accordingly.1 Effect of high glucose on cumulus expansion and oocyte maturation and developmentThe porcine cumulus oocyte complexes(COCs)in the foaming phase were isolated in vitro and treated with 5.6 mM glucose(group C),10 mM glucose(group G1),and 15 mM glucose(group G-2).IVM treatment of COCs for 42 h,observation and statistics of cumulus cell spread and first polar body excretion rate,Gap connexin gene expression was then measured in cumulus cells;parthenogenetic activation of oocytes after 42 h of in vitro maturation,2-cell,4-cell,7th day blastocyst development.The results showed that the cumulus cell expand of the G-1 and G-2 groups was significantly lower than that of the C group(P<0.05);expressions of gap junction protein Cx43 and Cx32 were significantly reduced(P<0.05);The mortality and survival rate of MII oocytes in the-2 group were not significantly different from those in the C group(P>0.05),but the polar body rate in the G-1 group was significantly reduced(P<0.05).The rate was significantly lower than that in group C(P<0.01).After parthenogenetic activation,compared with group C,the 2-cell division rate was significantly reduced in the G-1 and G-2 groups(P<0.05),and the 4-cell division rate and blastocyst development rate were significantly reduced(P<0.01),but the number of blastocyst cells in the G-1 and G-2 groups was not significantly different from that in the C group(P>0.05).It shows that although high glucose reduces the maturation efficiency and development ability of oocytes,it will not cause too many oocytes to die during maturation,mtDNA replication was not completed normally.2 Effect of high glucose on mitochondrial distribution,copy number and ATP content in oocytesMitochondrial staining of MII oocytes in each group found that the mitochondria of the G-1 and G-2 groups were unevenly distributed compared to the C group,and the fluorescence intensity of the G-1 and G-2 groups was found by gray value analysis Significantly decreased(P<0.05).As an important measure of mitochondrial function,copy number is a relative quantitative analysis of mitochondrial copy number.There is no statistical difference between group G-1 and group C,but the relative copy number of G-2 group has a significant decrease(P<0.05).Further determination of ATP content,the ATP content of the G-1 group and the G-2 group showed a significant decrease compared with the C group(P<0.05).This shows that high glucose has an adverse effect on mitochondrial function.3 Effect of high glucose on oxidative stress in oocytesMitochondria are the source of ROS,so the oxidative stress-related indicators of MII oocytes were tested.Compared with group C,the fluorescence results showed that the ROS levels in the G-1 and G-2 groups were significantly increased(P<0.01),Although there was no statistical difference in the total glutathione content of the G-1 group,the total glutathione content of the G-2 group was significantly reduced,and the GSH content of the G-1 group and the G-2 group was significantly reduced(P<0.05)In addition,there was no significant difference in MDA in the G-1 group(P>0.05),but the MDA content in the G-2 group increased significantly(P<0.05).By analyzing this,it was found that oocytes produced oxidative stress during maturation in a high glucose environment.In summary,high glucose concentration leads to abnormal proliferation of cumulus cells,damaged gap junctions,resulting in abnormal mitochondrial distribution and function during maturation of oocytes,oxidative stress occurs in oocytes,which reduces the maturation efficiency and Early embryonic development potential. |
