关键词: 线粒体, tRNA-Lys基因, 颗粒细胞, 生理功能

Abstract: 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, COⅠ, COⅡ, 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.

Key words: mitochondrion, tRNA-Lys gene, granulosa cells, physiological functions