Quantitative Changes Of Mitochondrial DNA Copy Number In Various Tissues Of Pigs During Growth

Mitochondrial DNA (mtDNA), a circular double-stranded DNA molecule located in mitochondria, is approximately 16 kb in length, encoding only 37 genes. A somatic mammalian cell contains approximately 1000-10,000 copies of mitochondrial DNA (mtDNA). mtDNA content was frequently used as a marker of mitochondrial density, reflecting the oxidative or ATP-producing capacity of tissues and the energy requirements of tissues. Nuclear genes encode the enzyme systems responsible for mtDNA replication and transcription. TWINKLE is the only mitochondrial replicative DNA helicase that can catalyze 5’to 3’NTP-dependent unwinding of the mtDNA, so TWINKLE is essential for mtDNA copy number. Mammalian species accumulate mtDNA deletions in various tissues during development, especially with ageing. Large deletions would affect the replication and transcription of mtDNA through destroying the structural integrity. In recent reports, DNA methyltransferase 1 (DNMT1) was found in the mitochondria and a low level of DNA methylation was observed in mtDNA. Then it is proposed that expression of mitochondrial genes is regulated by methylation of mtDNA. Previous studies have revealed the variation of mtDNA copy number in different tissues in human and mice. However, a profile of mtDNA content across various tissues in pigs has been long overdue. In current study, With long PCR, we could not detect any evidence for the occurrence of large-scale mtDNA deletions. Q-PCR was performed to quantify the mtDNA copy number in 15 pig tissue types, across at five growth stages, from embryo to adults. mtDNA contents of the heart, longissimus doris muscle, psoas major muscle, kidney, brain, ovary and subcutaneous adipose, increased with growth. Expression of the replicative mitochondrial helicase (TWINKLE), which regulates the turnover of mtDNA content, was significantly associated with changes in mtDNA copy numbers across tissues during growth (r=0.33, P=0.01).And we also demonstrated that the expression levels of mitochondrial genes positively correlated with mtDNA copy number. Under bisulfite sequencing, we found mtDNA contain 5-methylcytosine at CpG dinucleotides with a low level of methylation, as earlier reported.