Effect Of Dimethyl α-Ketoglutarate On In Vitro Maturation And Early Embryonic Development Of Ovine Oocytes

Oxidative stress is an important factor that affects in vitro oocyte maturation and early embryonic development.Recent studies have found that,an intermediate product of the tricarboxylic acid cycle,α-ketoglutarate（α-KG）can reduce cellular oxidative stress.Dimethylα-ketoglutarate（DMKG）is an ester precursor ofα-ketoglutarate which can enter a cell through its membrane and be hydrolyzed intoα-ketoglutarate by the corresponding esterase diacids to function.In this thesis,the objectives were to determine the effects of DMKG on in vitro maturation of ovine oocytes collected in non-breeding season and breeding season and their early embryonic development.To achieve the objectives,three different concentrations of DMKG（1.5 mM,3 mM and 4.5 mM）were chosen respectively to treat these oocytes to find the optimal concentration.we determined the effect of DMKG on maturation was by examining the maturation rate of oocytes,whilst its effect on developmental competence of these oocytes was evaluated by cleavage rate and blastocyst rate.Based on these results optimal concentration of DMKG was selected.Secondly,Immunofluorescence staining was used to detect the level of H3K9me3 and H3K27me3 in the oocytes and embryos which treated with the optimal level of DMKG.Finally,the in vitro oxidative damage model was established for research in ovine oocytes such as,study the nucleus and cytoplasmic maturation of ovine oocytes,mitochondrial activity,reactive oxygen species（ROS）concentration,GSH level and cell apoptosis,analysis of antioxidant-related genes（SOD2,CAT,GPx,BCL2）,apoptosis-related genes（BAX,CASP3）,oocyte development related genes（BMP15,GDF9,OCT4）expression changes,to explore the mechanism of DMKG against oxidative damage in ovine oocytes.The results in this study showed that,compared with the control group,the treatment with 3 mM DMKG could significantly increase all developmental parameters of oocytes,namely maturation rate（58.51%±8.05 in the control VS 79.85%±6.69 in the treatment）,cleavage rate（71.59%±2.08 VS 84.18%±3.20）and blastocyst rate（26.22%±1.90VS 39.47%±1.34）of ovine oocytes in non-breeding season（P<0.05）.Likewise,the same treatment also significantly enhanced the maturation rate（70.38%±2.99 in the control VS 84.89%±5.08 in the treatment）,cleavage rate（70.58%±1.35 VS 82.46%±1.95）and blastocyst rate（30.71±2.22 VS 39.86±1.26）of ovine oocytes collected in breeding season（P<0.05）.Therefore,we found 3 mM to be the best concentration of DMKG.Immunofluorescence staining showed that treatment with3mM DMKG reduced the levels of histone H3K9me3 and H3K27me3 in ovine oocytes and embryos（P<0.05）.In addition,adding 3 mM DMKG to the in vitro maturation medium could effectively alleviate the oxidative stress injury of ovine oocytes induced by H₂O₂,and could significantly improve the efficiency of ovine oocyte nuclear and cytoplasmic maturation,mitochondrial activity and GSH levels（P<0.05）.Moreover,the expressions of antioxidant-related genes（SOD2,CAT,GPx,BCL2）,as well as oocyte development related genes（BMP15,GDF9,OCT4）were all up-regulated,and the ROS content in ovine oocytes was significantly reduced.In addition,treatment with 3 mM DMKG was shown to prevent cell apoptosis and reduce the expression level of apoptosis-related genes BAX and CASP3（P<0.05）.The results of this study provide basic knowledge for understanding the potential mechanism of DMKG on the in vitro maturation of ovine oocytes,and provide a new method to improve the efficiency of in vitro maturation of ovine oocytes and early embryonic development.