Fate of genetically marked mitochondrial DNA from spermatocytes microinjected into mouse zygotes

Abstract

Cytoplasts from single spermatocytes of NZB/BinJ mice were separated from the nuclei and individually microinjected into B6D2F1 (C57BL/6 × DNBA/2J) hybrid embryos at the pronuclear stage (20 h after hCG injection). Of 363 zygotes injected, 311 (86%) survived and developed. From these experiments, we transferred 222 embryos into 20 pseudopregnant recipients. Eighteen (90%) became pregnant and 82 pups were born (37% of transfers). Mitochondrial DNA (mt DNA) from the NZB/BinJ strain lacks a RsaI restriction site and can thus be distinguished from the host embryo following PCR amplification. We were unable to detect the transferred mtDNA in blastocysts on day 4–5 after injection. Nor could we detect NZB/BinJ mtDNA in placentae, nor in tissues from mice born to host mothers following the transfer of blastocysts that developed from injected zygotes. Rejection of paternal mitochondria by the embryo normally occurs at the 4- to 8-cell stage in mice and is apparently dependent on mutual recognition between the mitochondria and the nuclear genome. We conclude that this mechanism has probably already developed by the time the germ cells have become committed to meiosis.

Ubiquitinated Sperm Mitochondria, Selective Proteolysis, and the Regulation of Mitochondrial Inheritance in Mammalian Embryos1

“…In ROSI, the injected round spermatids should carry mitochondria that are already imprinted with ubiquitin, and the initial studies in mice indeed suggest that their spermatid mitochondria can be recognized and eliminated by egg cytoplasm [65]. Similarly, spermatocyte mitochondria and mtDNA became undetectable when spermatocyte cytoplasts were injected into mouse pronuclear embryos [66]. With regard to cloning, it was suggested that the mismatched mitochondrial gene products may contribute to the high mortality of cloned offspring [67].…”

The Mitochondrial Genome in Embryo Technologies

“…Spermatid-derived mtDNA, however, was detectable by real-time PCR up to the blastocyst stage, but had disappeared by the time of birth (Shitara et al 2000). Primary spermatocyte mitochondria, in contrast, dispersed rapidly throughout the oocyte cytoplasm (Cummins et al 1998a), but 292 S Hiendleder and E Wolf spermatocyte mtDNA could also not be detected in different organs of offspring at a detection level of ‡ 1-5% (Cummins et al 1999). However, these data are at variance with two other studies that detected paternal mtDNA in human embryos derived from IVF and ICSI by a highly sensitive paternal allele-specific nested PCR.…”

“…Primary spermatocyte mitochondria, in contrast, dispersed rapidly throughout the oocyte cytoplasm (Cummins et al. 1998a), but spermatocyte mtDNA could also not be detected in different organs of offspring at a detection level of ≥ 1–5% (Cummins et al. 1999).…”

The Mitochondrion: The Prisoner of a False Origin and The Victim of Wrong Theories

“…Based on the previous points (1)(2)(3)(4)(5) and on studies of the scientists and their wrong assumptions, theories, contradictions, and on known facts I wish to suggest the following 1-Paternal Y sperm has no mitochondria at all (pure maleness-masculinity).…”