Effect of paternal aging and vitrification on mitochondrial DNA copy number and telomere length of mouse blastocysts

In this study, we examined the effects of paternal aging on the mitochondrial DNA copy number (mtcn), telomere length (TL), and gene expression in mouse embryos. The effects of vitrification on the mt-cn and TL of the embryos derived from young and aged male parents (YF and AF, respectively) were examined. C57BL/6N male mice were used for embryo production at 13-23 and 50-55 weeks of age. Two -cell stage embryos were collected from the oviducts of superovulated female mice (8-15 weeks old) and cultured for 24 h until the 8 -cell stage, followed by embryo vitrification. Fresh and vitrified -warmed embryos were incubated for 2 days until the blastocyst stage, and mt-cn and TL were investigated. The cell -free mitochondrial DNA copy number (cf-mt-cn) in the spent culture medium (SCM) of the embryos was then investigated. RNA sequencing of blastocysts revealed that metabolic pathways, including oxidative phosphorylation and mTOR pathways, were enriched in differentially expressed genes. The mt-cn and TL of AF -derived blastocysts were lower and shorter, respectively, than those of YF-derived blastocysts. Paternal aging did not affect the blastocyst rate after vitrification. Vitrification of the 8 -cell stage embryos did not affect the mt-cn of the blastocysts. However, it increased the cf-mt-cn (cell -free mt-cn) in the SCM of both YF- and AF -derived embryos. Vitrification did not affect the TL of either YF- or AF -derived embryos. Thus, paternal aging affected the mt-cn and TL of the embryos, but vitrification did not affect these parameters in either age groups.