Study on the Physiological Mechanism of Mitochondrial tRNA-Lys(T7719G)Gene Variation Affecting Apoptosis of Ovine Granulosa Cell
The previous studies have found that mitochondrial tRNA-Lys(T7719G)variation was associated with litter size,which decreased the apoptosis of granulosa cells in Small-tailed Han sheep.Therefore,the physiological mechanism of mitochondrial tRNA-Lys(T7719G)variation on granulosus cell apoptosis was explored.Three sheep were selected with G and T genotypes respectively,and slaughtered for ovarian tissue culture of granulosa cells.The detection experiment of granule cells physiological function were divided into two groups with 3 replicates in each group.Mitochondrial genome copy number,mitochondrial respiratory enzyme complex Ⅰ,complex Ⅱ,complex Ⅲ,complex Ⅳ and complex Ⅴ activities were detected,respectively.Mitochondrial oxygen consumption and extracellular acid production capacity,ROS production,ATP level and membrane potential,as well as total tRNA-Lys and aminoacylation level,expression level of 13 mtDNA-encoded polypeptide proteins were detected.The results showed that the mitochondrial copy number of G genotype granulosa cells was significantly different from that of T genotype(P＜0.01),which was 1.78 times that of T genotype.The activity of mitochondrial complex Ⅲ in G genotype cells was 1.25 times higher than that in T type cells(P＜0.05),indicating that the oxidative respiratory function of G genotype cells was enhanced.The mitochondrial oxygen consumption and ATP-coupled oxygen consumption of G genotype granulosa cells were 6.84％(P＜0.05)and 15.46％(P＜0.01)higher than those of T genotype cells,respectively.The glycolysis ability of G genotype cells was significantly decreased(P＜0.01),the level of reactive oxygen species was significantly decreased by 34％,the ATP content was significantly increased,which was 1.25 times that of T genotype(P＜0.05),and the mitochondrial membrane potential was significantly increased,which was 1.24 times that of T genotype(P＜0.01).Total tRNA-Lys was significantly increased by 50％(P＜0.01)in G genotype cells,and the proportion of amylated tRNA-Lys was increased by 33.4％(P＜0.01)compared to T genotype cells.The expression levels of all 13 polypeptides encoded by the mitochondrial genome(ND1,ND2,ND3,ND4,ND4L,ND5,ND6,CtyB,CO Ⅰ,CO Ⅱ,COⅢ,ATP6,ATP8)were significantly increased(P＜0.05).The physiological mechanism of mitochondrial tRNA-Lys(T7719G)variation affecting ovine granulosa cell apoptosis was initially analyzed,that is,tRNA-Lys(T7719G)variation affected tRNA-Lys homeostasis and aminoacylation level,affected the translation efficiency of mtDNA-encoded peptides,and caused changes in copy number,ATP level,ROS content,and some enzyme activities,and then the energy metabolism function of mitochondria and apoptosis were changed.
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