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Chromosome transfer in mature oocytes

Abstract

In this article, we describe detailed protocols for the isolation and transfer of spindle–chromosomal complexes between mature, metaphase II-arrested oocytes. In brief, the spindle–chromosomal complex is visualized using a polarized microscope and extracted into a membrane-enclosed karyoplast. Chromosomes are then reintroduced into an enucleated recipient egg (cytoplast), derived from another female, by karyoplast–cytoplast membrane fusion. Newly reconstructed oocytes consist of nuclear genetic material from one female and cytoplasmic components, including mitochondria and mitochondrial DNA (mtDNA), from another female. This approach yields developmentally competent oocytes suitable for fertilization and producing embryonic stem cells or healthy offspring. The protocol was initially developed for monkey oocytes but can also be used in other species, including mouse and human oocytes. Potential clinical applications include mitochondrial gene replacement therapy to prevent transmission of mtDNA mutations and treatment of infertility caused by cytoplasmic defects in oocytes. Chromosome transfer between the cohorts of oocytes isolated from two females can be completed within 2 h.

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Figure 1: Equipment and pipette setting for chromosome transfer.
Figure 2: Experimental design for serial spindle–chromosomal complex transfer between two cohorts of oocytes.
Figure 3: Schematic diagram of the chromosome transfer procedure.
Figure 4: Experimental examples of good and poor outcomes during chromosome transfer.

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References

  1. Cui, L.B., Huang, X.Y. & Sun, F.Z. Transfer of germinal vesicle to ooplasm of young mice could not rescue ageing-associated chromosome misalignment in meiosis of oocytes from aged mice. Hum. Reprod. 20, 1624–1631 (2005).

    Article  PubMed  Google Scholar 

  2. Liu, H., Wang, C.W., Grifo, J.A., Krey, L.C. & Zhang, J. Reconstruction of mouse oocytes by germinal vesicle transfer: maturity of host oocyte cytoplasm determines meiosis. Hum. Reprod. 14, 2357–2361 (1999).

    Article  CAS  PubMed  Google Scholar 

  3. Roberts, R.M. Prevention of human mitochondrial (mtDNA) disease by nucleus transplantation into an enucleated donor oocyte. Am. J. Med. Genet. 87, 265–266 (1999).

    Article  CAS  PubMed  Google Scholar 

  4. Paulson, R.J. et al. Pregnancy in the sixth decade of life: obstetric outcomes in women of advanced reproductive age. JAMA 288, 2320–2323 (2002).

    Article  PubMed  Google Scholar 

  5. Takeuchi, T., Ergun, B., Huang, T.H., Rosenwaks, Z. & Palermo, G.D. A reliable technique of nuclear transplantation for immature mammalian oocytes. Hum. Reprod. 14, 1312–1317 (1999).

    Article  CAS  PubMed  Google Scholar 

  6. Sato, A. et al. Gene therapy for progeny of mito-mice carrying pathogenic mtDNA by nuclear transplantation. Proc. Natl Acad. Sci. USA 102, 16765–16770 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Meirelles, F.V. & Smith, L.C. Mitochondrial genotype segregation in a mouse heteroplasmic lineage produced by embryonic karyoplast transplantation. Genetics 145, 445–451 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Meirelles, F.V. & Smith, L.C. Mitochondrial genotype segregation during preimplantation development in mouse heteroplasmic embryos. Genetics 148, 877–883 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Wilding, M. et al. Mitochondrial aggregation patterns and activity in human oocytes and preimplantation embryos. Hum. Reprod. 16, 909–917 (2001).

    Article  CAS  PubMed  Google Scholar 

  10. Tachibana, M. et al. Mitochondrial gene replacement in primate offspring and embryonic stem cells. Nature 461, 367–372 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Cohen, J. et al. Ooplasmic transfer in mature human oocytes. Mol. Hum. Reprod. 4, 269–280 (1998).

    Article  CAS  PubMed  Google Scholar 

  12. Tanaka, A. et al. Metaphase II karyoplast transfer from human in-vitro matured oocytes to enucleated mature oocytes. Reprod. Biomed. Online 19, 514–520 (2009).

    Article  PubMed  Google Scholar 

  13. Mitalipov, S.M. & Wolf, D.P. Nuclear transfer in nonhuman primates. Methods Mol. Biol. 348, 151–168 (2006).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by start-up funds from the Oregon National Primate Research Center, the Oregon Stem Cell Center and grants from the National Institutes of Health HD057121, HD059946, HD063276, HD047721, HD047675, RR0000163 and U54 HD18185.

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Contributions

S.M., M.S. and M.T. developed and wrote this protocol.

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Correspondence to Shoukhrat Mitalipov.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Video ~Chromosome transfer in mature oocytes~

Micromanipulation procedures for chromosome transfer involve: 1) MII oocyte positioning for chromosome-spindle detection, laser assisted zona drilling and chromosome-spindle aspiration into a karyoplast; 2) karyoplast exposure to HVJ-E; 3) and karyoplast transfer into a perivitelline space of a cytoplast. (MOV 6059 kb)

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Tachibana, M., Sparman, M. & Mitalipov, S. Chromosome transfer in mature oocytes. Nat Protoc 5, 1138–1147 (2010). https://doi.org/10.1038/nprot.2010.75

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