21 Quantification of mitochondrial DNA copy number in interspecies somatic cell nuclear transfer embryos

Interspecies somatic cell nuclear transfer (iSCNT) may be used to assist in the rescue of endangered mammalian species. In reconstructed iSCNT embryos, mitochondrial DNA (mtDNA) from the donor cell and oocyte coexist, causing concerns regarding the mitochondrial heteroplasmy that is present throughout embryonic and fetal development. Here, two groups of early pre-implantation iSCNT embryos were analysed to determine bovine and caprine mitochondrial DNA contributions. Group 1 consisted of embryos produced via handmade cloning: mature bovine oocytes were bisected following centrifugation and removal of the zonae pellucidae to generate enucleated ooplasts with reduced mtDNA copy numbers (MR group). Following a 30 m rest period, two spherical ooplasts were fused with a caprine somatic cell. Group 2 embryos were reconstructed via traditional micromanipulation-based protocols (MM group) with enucleated bovine oocytes and caprine somatic cells. Both groups were chemically activated and cultured in vitro for up to four days. DNA was extracted from 1-cell, 4–6 cell, and 8–10 cell samples (11–15 embryos per stage per group, collected across 6 sessions). Most MR embryos arrested by the 8–10 cell stage. Standard curves were generated through quantitative PCR (qPCR) based on logarithmic somatic cell concentrations. qPCR reactions were designed to amplify the 12S region of each species’ mtDNA, with separate reactions run for each species. Formulas based on the standard curves, sample volumes, amplicon lengths, and cycle threshold values were used to determine relative mtDNA copy numbers; one-way ANOVA was used to analyse bovine copy numbers based on the validation of normality assumptions, while a general linear model ANOVA was used to analyse caprine copy numbers due to the violation of normality assumptions (Jamovi 1.6). The average bovine mtDNA copy number in MM 4–6 cell embryos (115,513 ± 51,536) was significantly greater than all MR embryos and MM 1-cell embryos (63,863 ± 32,976) (P < 0.05). The copy number is likely greater in MM 4–6 cell embryos compared to 1-cell embryos in the same group due to variation in respective sample quality. Though the average caprine mtDNA copy number tends to be higher in MR 8–10 cell embryos compared to all other groups, the difference is not significant (P = 0.102). These results demonstrate that intentionally reducing the native mtDNA copy number in a bovine oocyte before iSCNT may allow for increased proliferation of the somatic cell species’ mtDNA within the reconstructed embryo. However, since the total mtDNA copy number in these iSCNT embryos is relatively low compared to those of developmentally successful SCNT embryos (∼60,000 copies are present in the MR iSCNT embryos compared to a previously determined minimum requirement of over 100,000 copies in SCNT embryos), supplementation with the somatic cell species’ mitochondria is likely necessary to provide the embryo with adequate energy for successful growth and development.