18 Development of interspecies somatic cell nuclear transfer (iSCNT) caprine-bovine embryos injected with demethylase mRNA and mitochondrial extract

Interspecies somatic cell nuclear transfer (iSCNT) has the potential to play a significant role in the conservation of endangered species. The use of recipient oocytes from domestic species and somatic donor cells from endangered species would eliminate the need to obtain germ cells from the endangered animals. However, the overall efficiency of iSCNT is low: under 1%. The developmental arrest at embryonic genome activation (EGA) and the degree of mitochondrial heteroplasmy in the reconstructed embryo are the two primary challenges to enable in vitro and in vivo iSCNT embryo development. Here, we generated iSCNT caprine-bovine embryos using bovine oocytes and caprine fetal fibroblast cells and injected them with caprine mitochondrial (mt) extract (6–7 pl) 1-h postactivation (hpa) to reduce the degree of mitochondrial heteroplasmy. Additionally, KDM4E mRNA (an H3K9me2/me3 demethylase), shown in a previous study (Liu et al. 2018 Development 145, dev158261) to increase chromatin accessibility in embryos, was injected in the iSCNT embryos (6–7 pL of 139 ng/µL) either by itself or in combination with the mt extract 1 hpa. Following injection, reconstructed embryos were cultured in BO-IVC media (IVF Bioscience, Falmouth, England, UK). Lysis events were recorded between 24–48 hpa. Cleavage was recorded at 48 hpa. Development to 16-cells was recorded at 96 hpa. A general linear model was used for statistical analysis and the Tukey post hoc test was applied to the means. Embryos injected with KDM4E mRNA (n = 51), caprine mitochondrial extract (n = 61), or a combination of mRNA and extract (n = 45) had similar (P > 0.05) cleavage rates (91.67%, 90.38%, and 97.73%, respectively), lysis rates (5.88%, 12.86%, and 2.22%, respectively), and rate of development to 16-cells (13.64%, 25.0%, and 11.63%, respectively). The rates were also similar (P > 0.05) when compared to cleavage, lysis, and 16-cell development rates of control embryos (95.38%, 0%, and 19.35%, respectively; n = 3 replicates). Though the lysis rate of embryos injected with mitochondrial extract was not significantly higher than other groups, it may be greater due to the viscosity of the extract. Attempting to decrease the degree of mitochondrial heteroplasmy in iSCNT embryos with donor cell mitochondrial extract supplementation did not support the in vitro development of caprine-bovine embryos past 16 cells. This was also the case when we attempted to overcome the embryonic developmental arrest at the EGA timepoint through the injection of KDM4E mRNA. Uninjected and injected (KDM4E mRNA) iSCNT caprine-bovine embryos were collected for ATAC-seq analysis to assess differences in chromatin accessibility; data analysis is ongoing. Further research is required to increase the overall efficiency of iSCNT so that it can effectively be used as a method of rescuing endangered species.