Interspecies Somatic Cell Nuclear Transfer Is Dependent on Compatible Mitochondrial DNA and Reprogramming Factors

Abstract

Interspecies somatic cell nuclear transfer (iSCNT) involves the transfer of a nucleus or cell from one species into the cytoplasm of an enucleated oocyte from another. Once activated, reconstructed oocytes can be cultured in vitro to blastocyst, the final stage of preimplantation development. However, they often arrest during the early stages of preimplantation development; fail to reprogramme the somatic nucleus; and eliminate the accompanying donor cell's mitochondrial DNA (mtDNA) in favour of the recipient oocyte's genetically more divergent population. This last point has consequences for the production of ATP by the electron transfer chain, which is encoded by nuclear and mtDNA. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility on successful development. Initially, we transferred murine fetal fibroblasts into enucleated porcine oocytes, which resulted in extremely low blastocyst rates (0.48%); and failure to replicate nuclear DNA and express Oct-4, the key marker of reprogramming. Using allele specific-PCR, we detected peak levels of murine mtDNA at 0.14±0.055% of total mtDNA at the 2-cell embryo stage and then at ever-decreasing levels to the blastocyst stage (<0.001%). Furthermore, these embryos had an overall mtDNA profile similar to porcine embryos. We then depleted porcine oocytes of their mtDNA using 10 µM 2′,3′-dideoxycytidine and transferred murine somatic cells along with murine embryonic stem cell extract, which expressed key pluripotent genes associated with reprogramming and contained mitochondria, into these oocytes. Blastocyst rates increased significantly (3.38%) compared to embryos generated from non-supplemented oocytes (P<0.01). They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout early preimplantation development. At later stages, these embryos possessed 49.99±2.97% murine mtDNA. They also exhibited an mtDNA profile similar to murine preimplantation embryos. Overall, these data demonstrate that the addition of species compatible mtDNA and reprogramming factors improves developmental outcomes for iSCNT embryos.

On the cellular and developmental lethality of a Xenopus nucleocytoplasmic hybrid

“…Also, it could be that the supplied factors may not localize properly in the recipient cytoplasm. An experiment resembling this one has been recently tried in a murine-to-porcine iSCNT system, whereby mouse ES cell extracts were injected along with a mouse nucleus into mtDNA-depleted recipient porcine oocytes 17 . This significantly improved cybrid development to the blastocyst stage, yet because there was more than one difference between the control and treated iSCNT embryos, it is difficult to conclude whether the improvement came from the mtDNA exchange, and/or from anything else contained in the extracts.…”

Incompatibility of Nucleus and Mitochondria Causes Xenomitochondrial Cybrid Unviable Across Human, Mouse, and Pig Cells

“…Inter-species somatic hybrid cells between mouse and human have always been obtained (37,38), but the maintenance of mitochondrial genomes has been shown to require essentially a complete set of cognate chromosomes. It has been reported that bovine oocyte cytoplasm can support the introduced differentiated nucleus of sheep, pig, monkey, and rat (39), and the recent study of rabbit-human (15) and pig-mouse (14,40) also resulted in early embryonic arrest. As a result, the relationship of nucleus and mitochondria is a huge challenge that can solve the iSCNT problem.…”

The Oocyte Determinants of Early Reprogramming

“…This scenario does not exclude the possibility that the oocyte has also additional factors that operate exclusively for reprogramming. The inefficiency of interspecies SCNT may be used as argument that reprogramming relies on factors that are used for sperm processing (Jiang et al 2011). The oocyte from one species often fails to reprogram the nucleus from another species also due to mitochondrial incompatibilities, which interfere with correct EGA (Jiang et al 2011).…”

“…The inefficiency of interspecies SCNT may be used as argument that reprogramming relies on factors that are used for sperm processing (Jiang et al 2011). The oocyte from one species often fails to reprogram the nucleus from another species also due to mitochondrial incompatibilities, which interfere with correct EGA (Jiang et al 2011). In contrast to the view supported by interspecies SCNT, there are factors that seem to be only required for the development of cloned embryos, such as DJ-1 (Table 1), a factor that is not needed for development in fertilized embryos, suggesting specific molecules to be recruited (Miyamoto et al 2011).…”