Comparative analysis of cell-free DNA content in culture medium and mitochondrial DNA copy number in porcine parthenogenetically activated embryos

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  • KOBAYASHI Mitsuru
    Department of Animal Science, Tokyo University of Agriculture, Kanagawa 243-0034, Japan
  • ITO Jun
    Department of Animal Science, Tokyo University of Agriculture, Kanagawa 243-0034, Japan
  • SHIRASUNA Koumei
    Department of Animal Science, Tokyo University of Agriculture, Kanagawa 243-0034, Japan
  • KUWAYAMA Takehito
    Department of Animal Science, Tokyo University of Agriculture, Kanagawa 243-0034, Japan
  • IWATA Hisataka
    Department of Animal Science, Tokyo University of Agriculture, Kanagawa 243-0034, Japan

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<p> We examined the effect of ploidy on mitochondrial DNA (mtDNA) copy number in embryos and the amount of cell-free mitochondrial and nucleic DNA content (cf-mtDNA and cf-nDNA) in spent culture medium (SCM). Oocytes collected from the ovaries were matured, activated, incubated in medium containing cycloheximide (CHX) or CHX and cytochalasin B (CB) for 4.5 h to produce haploid or diploid embryos (H-group and D-group embryos). These embryos were cultured for 7 days, and the blastocysts and SCM were examined. The amount of mtDNA and nDNA was determined by real-time PCR. The rate of development to the blastocyst stage was higher for the D-group than for the H-group. Moreover, D-group blastocysts had less mtDNA compared to the H-group blastocysts. After activation, the mitochondrial content was constant before the blastocyst stage in D-group embryos, but increased earlier in H-group embryos. The amount of cf-mtDNA in the SCM of D-group blastocysts was greater than that of H-group blastocysts. However, when the cf-mtDNA in the SCM of 2 cell-stage embryos (day 2 post-activation) was examined, the amount of cf-mtDNA was greater in the H-group than in the D-group embryos. When D-group embryos were cultured for 7 days, a significant correlation was observed between the total cell number of blastocysts and cf-nDNA content in the SCM. Hence, although careful consideration is needed regarding the time point for evaluating mtDNA content in the embryos and SCM, this study demonstrates that mtDNA in the embryos and SCM was affected by the ploidy of the embryos.</p>

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