Transmission of mitochondrial DNA in mammalian clones

Cloning by nuclear transfer is, typically, performed by fusing a donor cell with an enucleated oocyte (recipient cytoplast). Different types of donor cells have been successfully used for nuclear transfer: embryonic, fetal or adult somatic cells. Assuming recognition and proper processing of foreign mitochondria by the cytoplasmic machinery of the oocyte, the cloned progeny should be heteroplasmic harboring mitochondrial DNAs (mtDNAs) from both the donor and recipient cytoplasms. The objective of this project is to analyse the composition and transmission of mtDNA in mammalian clones produced by nuclear transfer. Investigating the issue of mtDNA inheritance during cloning contributes to the understanding of mitochondrial biogenesis, the evaluation of cloning associated genetic mechanisms and is important for evaluating the safety and efficacy of cloning, The results of the analyses would also allow an evaluation of the potential usefulness of mammalian nuclear transfer for human mitochondrial disease research. In detail, within this project we will investigate, the influence of the donor cell type on the mtDNA transmission pattern, whether there is a relationship between the extend of heteroplasmy and the occurrence of developmental anomalities and the issue of species-specificity of mitochondrial heteroplasmy, i.e. whether the generation of heteroplasmic mtDNA clones is restricted to cattle or can under certain conditions also be generated in other species like sheep, pig, rabbit or mice.

Apart from their role in cellular energy supply, mitochondria have a broad range of functions in inter- and intracellular signalling via steroidogenesis, reactive oxygen species, nitric oxide and calcium. Mitochondria are also crucial to three signalling pathways that lead to programmed cell death and, thus, affect embryonic and fetal development (reviewed in (Hiendleder et al., 2005)). This suggests that inadequate or pertubed mitochondria may adversely affect somatic cell nuclear transfer (NT) success. The goals of this project were to: (i) analyse the mtDNA transmission in cross-subspecies NT in cattle, (ii) to analyse mtDNA transmission in NT-derived sheep fetuses and lambs, and (iii) to proof that heteroplasmy can be generated by transfer of donor ooplasm between Bos indicus and Bos taurus. (i) We investigated the mitochondrial DNA (mtDNA) composition in one of the largest adult somatic mammalian clones (n = 20) reported so far. The healthy cloned cattle were derived from NT of an identical nuclear genetic background into enucleated oocytes with either Bos indicus or Bos taurus mtDNA and harbored coexisting mtDNAs of these closely related subspecies. Heteroplasmy (0.6% to 2.8%) was found in four out of eleven cross- subspecies cloned cattle. Intrasubspecific B. indicus heteroplasmy of around 1% (but up to 7.3% and 12.7% in muscle and follicular cells of one animal) was detected in seven out of the nine B. indicus intrasubspecific cloned cattle (Steinborn et al., 2002). (ii) We also investigated the donor mtDNA transmission in cloned ovine fetuses (n = 7) and lambs (n = 5) derived from NT of somatic donor cells at early passages. We report the first cases (n = 3 fetuses, n = 3 lambs) of recipient cytoplast/donor mtDNA heteroplasmy in NT-derived ovine clones. In three cloned fetuses we quantitated low (cells isolated from two fetuses: 0.2% and 0.9%) or high (tissues of a Day-49 fetus: 6.8% to 46.5%) levels of heteroplasmy. In three of the five cloned lambs we found at least two tissue types with heteroplasmies ranging from 0.2% to 0.9%. Sequence analysis of the 13 mitochondrially encoded protein subunits failed to reveal any amino acid substitution between recipient cytoplast and donor cell corresponding to either homoplasmy or heteroplasmy. Remarkably, the most variable mtDNA haplotype possessed the recipient cytoplast leading to the highly heteroplasmic cloned fetus (four amino acid changes compared to the donor. (iii) Ooplasm transfer between B. indicus and B. taurus yielded heteroplasmies between 8 to 15%.