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

Yan Jiang,Justin C St John,Daniel A Mitchell,Richard Kelly,Adam Dickinson,Helena Fulka,Amy Peters,Joanna Mary Bridger

doi:10.1371/journal.pone.0014805

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.

Highlights

Somatic cell nuclear transfer (SCNT), which involves the transfer of an adult or fetal cell into an enucleated oocyte, utilises the cytoplasmic factors already present within the oocyte to reprogramme the somatic cell [1]
In order to determine the efficiency of the cytoplasm of an oocyte from one species to reprogramme a somatic nucleus from another, we performed SCNT by injecting mouse embryonic fibroblasts (MEFs) into enucleated porcine oocytes
Reconstructed embryos were transferred from porcine zygote medium (PZM) into Human Tubal fluid (HTF) (n = 147) or KSMO (n = 132) at the 2/4-cell stage; or were maintained in PZM (n = 226)

Summary

Introduction

Somatic cell nuclear transfer (SCNT), which involves the transfer of an adult or fetal cell into an enucleated oocyte, utilises the cytoplasmic factors already present within the oocyte to reprogramme the somatic cell [1]. Following incubation of the somatic cell in the recipient oocyte and subsequent activation, the resultant embryos can be cultured to the blastocyst stage, the final stage of preimplantation development. At this stage, cells can be isolated from the inner cell mass (ICM) and cultured in vitro as potential ‘personalised’ embryonic stem cells (ESCs; [2]). The expanding colonies of pluripotent ESCs have the potential to develop into any cell type of the body Such approaches have led to the generation of murine models of haematopoiesis [3], regenerative strategies for Parkinson9s disease [4] and non-human primate ESC lines [5]. A number of reports have highlighted, amongst other factors, the failure of many iSCNT embryos to initiate and progress further than embryonic genome activation (EGA) [12] most likely through unsuccessful reprogramming and initiation of embryonic transcription [13]

Methods

Results

Conclusion