If cells harboring these mutant mitochondria are selected for SCNT, several possible fates might await them. As "foreign" (i.e., not oocytic) mitochondria, they might actively be eliminated, much as sperm mitochondria typically are eliminated. They might fail to replicate and thus be eliminated by attrition. They might persist in the embryo, leading to mitochondrial heteroplasmy. Last, they might experience a replicative advantage over endogenous ooplasmic mitochondria, and thus become the predominant mitochondria in the cloned animal.
In one study, a mouse model was employed in which mitochondrial dysfunction was associated with telomere shortening and genomic instability (Liu et al., 2002). Using nuclear transfer to transfer nuclei from cells with defective mitochondria to oocytes with normal mitochondria alleviated these defects. This suggests that mitochon-drial defects related to aging might be eliminated by nuclear transfer. To what extent replacement of "aged"
mitochondria by "young" ooplasmic mitochondria would occur is not clear. A variety of nuclear transfer studies have described diverse results concerning the fate of mitochondria after various micromanipulations involving oocytes.
Oocytes are designed to eliminate the mitochondria that are transmitted via the sperm (Kaneda et al., 1995). These paternal mitochondria carry ubiquitin tags that seem to target them for elimination by the ooplasm (Sutovsky et al., 2000). Interestingly, with interspecies crosses, paternal mitochondria can avoid elimination (Kaneda et al., 1995), indicating that there exists species specificity to the recognition of the sperm ubiquitinated proteins by the ooplasm. Cell type specificity also exists. Injection of Mus spretus spermatid or liver mitochondria into oocytes of Mus domesticus revealed that the liver mitochondria persisted to birth, whereas spermatid mitochondria were gradually eliminated and then were not detected in progeny (Shitara et al., 2000). It is therefore unlikely that donor cell mitochondria would be eliminated in cloned embryos by the same mechanism that eliminates sperm mitochondria, as the donor cell mitochondria would not be expected to carry the appropriate ubiquitinated tags.
If not actively eliminated, then to what degree might donor cell mitochondria in SCNT embryos be eliminated by attrition, persist at a low level, or assume a replicative advantage over endogenous mitochondria? Mitochondrial heteroplasmy was not observed in sheep clones (Evans et al., 1999). In SCNT embryos made with Bos indicus, donor nuclei transferred to Bos taurus oocytes, donor cell mitochondria were nearly undetect-able in cloned fetuses (Hiendler et al., 2003) and calves (Meirelles et al., 2001). However, donor cell mitochondria were not eliminated in monkey-to-rabbit or panda-to-rabbit NT embryos (Chen et al., 2002; Yang et al., 2003). Mitochondrial heteroplasmy was also reported for nuclear transfers made between Holstein and Luxi Yellow cow (Han et al., 2004), and in other studies involving bovine species (Takeda et al., 2003; Steinborn et al., 2002). Embryonic blastomere nuclear transfer in Macaca mullatta produces offspring with mitochondria from both the recipient oocyte and the donor blastomere (St. John and Schatten, 2004). In this case, both maternal and paternal mitochondria derived from the donor embryonic blastomere were detected, indicating that the sperm mitochondria were not eliminated as expected, revealing a possible defect in the process of paternal mitochondrial elimination in cloned constructs. Similarly, with ooplasm transfer approaches in human infertility treatment, mitochondrial heteroplasmy can be detectable in the resulting children (Barritt et al., 2001). Collectively, the results obtained to date indicate that mitochondrial heteroplasmy is likely to accompany the application of SCNT methods in the human.
The persistence of mitochondrial heteroplasmy to birth indicates that the donor cell mitochondria replicate following SCNT. In some cases, the donor cell mitochondria may actually have a replicative advantage. In the panda-to-rabbit NT embryos, donor cell mitochondria, in fact, can become the predominant organelle by fetal life (Chen et al., 2002). In bovine NT studies, donor cell mitochondria can account for as much as 40% of the total (Takeda et al., 2003), again indicating a replicative advantage over recipient mitochondria at some point during development. It is worth noting that, although it is often assumed that donor cell mitochondria might be eliminated due to incompatibilities with recipient cell nuclear genomes, these observations indicate that this need not always be the case. It is thus of interest that mitochondria carrying mutated genomes can be unevenly distributed within adult tissues, consistent with a clonal expansion (both intracellular and cellular) during aging (Khrapko et al., 2003). The ability of such mutated mitochondria from aging cells to expand within adult tissues might also result in their expansion, and possibly a replicative advantage, in SCNT embryos.
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