Intact mouse sperm or mouse sperm tails alone, labelled with MitoTracker Green FM® fluorochrome, were injected into mouse oocytes and the cells cultured in vitro for up to 5 days. The dye stained midpiece mitochondria, the sperm tail coarse fibres and the sperm perforatorium. Intact sperm (or tails injected with separated heads) induced normal embryonic development. The mitochondria could be identified in embryos up to the 4-cell stage, remaining associated with the sperm tail. They largely disappeared by the 8-cell stage, when only a minority of embryos (6/43) could be found with small patches of mitochondria. Axonemal elements could be identified coiled up in single external blastomeres as late as day 5 blastocysts. By contrast, mitochondria as well as tail components could be identified up to 5 days after injection of sperm tails alone into non-activated oocytes and also in embryos that arrested development before the 8-cell stage. We conclude that disappearance of the labelled sperm mitochondria in normally cleaving embryos is not due to fading or inactivation of the fluorochrome marker, but is rather an event specifically tied to cell cycle activities around the second cell division.

Mouse round spermatids labelled with MitoTracker were microinjected into Sr2+-activated mouse oocytes. The labelled mitochondria were tracked up to the morula/blastocyst stage using fluorescence microscopy. The overall incidence of embryos with labelled mitochondria fell from 80% in the 1-cell zygote to 25% in 2-cell, 9% in 4-cell and ~1% in 8-cell or later stages. Thus it appears that almost all round spermatid mitochondria finally disappear from embryos during the 4-cell to 8-cell transition, as happens for mature spermatozoa (Cummins et al.Zygote 1997, 5: 301–8). The spermatid mitochondria remained tightly bound together during this process. In contrast, labelled primary spermatocyte and cumulus mitochondria dispersed rapidly throughout the oocyte cytoplasm within 3 h. We hypothesise that spermatid mitochondria may be bound together by cytoskeletal elements produced in the early haploid spermatid. These elements, together with terminal differentiation of the sperm mitochondria, may be central to the processes by which the embryo ‘recognises’ the sperm mitochondria and inhibits inheritance of paternal mitochondrial DNA. These results suggest that round spermatid injection for infertile men will not pose a significant risk to offspring by transmitting abnormal mitochondrial genomes.