Since these initial studies in humans, a large compendium of reports in animals have shown that the transfer of cytoplasm or an enriched fraction of mitochondria can reduce in vitro fragmentation of oocytes and increase cleavage rates of recipient embryos compared with noninjected controls. These studies also have demonstrated that this technique can produce healthy embryo and offspring ().
Summary of Previous Cytoplasm and Mitochondrial Transfer Studies.
. Oocytes that have had their mitochondria damaged or have not converted from glycolytic to aerobic respiration have a lower likelihood of development after fertilization [29
]. Early studies demonstrated the feasibility of injection of mitochondria into an oocyte to increase intracellular ATP concentrations [87
]. Mitochondrial injections increase the viability of mouse oocytes destined for apoptotic cell death [88
]. When mitochondria were stained with green fluorescent protein (GFP) prior to transfer to the oocyte, GFP-marked mitochondria were observed until blastocyst stage [89
]. Following infusion of GFP mitochondria, dense clustering of mitochondria occurred around spindles, where ATP was needed.
Injection of mitochondria into mouse oocytes did not negatively affect survival rate or development rate to morula stage compared with buffer-injected controls [90
]. No long-term effects on growth of offspring or phenotypic abnormalities were observed when intra- and interstrain cytoplasmic transfers were compared [91
]. When mitochondrial concentrates were injected into mouse 2 pronuclear stage embryos, there was an increase in progression to blastocyst stage with no untoward effects [92
]. These authors commented on the need to avoid heteroplasmy of mitochondria to maintain appropriate nuclear-mitochondrial communication required for optimal embryo development.
. Transfer of homoplasmic cytoplasm into a high quality MII oocyte did not affect survival of oocyte, fertilization rate or progression to 2 or 8 cell stage, and morula or blastocyst stage of embryo [93
]. On the other hand, transfer of heteroplasmic ooplasm resulted in a decrease in the number of fertilized oocytes reaching the blastocyst stage. The authors conclude that transfer of homogeneous cytoplasm is required for optimal preimplantation embryo development [93
As with other species, oocyte and embryo quality as well as reproductive outcome depend in part on the quality of mitochondria and ATP content in the oocyte [94
]. Treatment of bovine oocytes with ethidium bromide depletes mtDNA content in oocytes, and as a result, they are arrested in pre-implantation development [20
]. Cytoplasmic transfer to these impaired oocytes resulted in the complete rescue of the oocytes, which then are developed into normal calves.
Treatment of poor quality bovine oocytes with mitochondria obtained from the animal's own granulosa cells resulted in dramatic improvements in oocyte quality as well as rates of morula, blastocyst, and hatched blastocysts [100
]. Addition of mitochondria obtained from the same breed improved embryo quality during preimplantation development. There is a segregation of donor mitochondria by the oocyte after cytoplasmic transfer [20
] supporting the hypothesis that mitochondrial homoplasmy is optimal.
. Supplementation of developmentally incompetent oocytes by injection of mitochondria resulted in a doubling of mitochondria number and normal embryo development. As a result fertilization rates were doubled or tripled (from approximately 10–20% to 30–40%) [31