Distribution of introduced human mitochondrial DNA in early stage mouse embryos
- Authors: Kustova M.E.1, Sokolova V.A.1, Kidgotko O.V.1, Bass M.G.1, Zakharova F.M.1,2, Vasilyev V.B.1,2
-
Affiliations:
- Institute of Experimental Medicine
- Saint Petersburg State University
- Issue: Vol 20, No 2 (2020)
- Pages: 69-78
- Section: Original research
- Published: 02.09.2020
- URL: https://journals.eco-vector.com/MAJ/article/view/34657
- DOI: https://doi.org/10.17816/MAJ34657
- ID: 34657
Cite item
Abstract
Objective. The aim of study was the analysis of human mitochondrial DNA (mtDNA) distribution among murine blastomeres in the embryos developing after an injection of human mitochondria suspension at the stage of one or two cells is presented.
Material and methods. Mice CBA/C57Black from Rappolovo aged three weeks were used. Zygotes were obtained upon hormonal stimulation of animals and mated with males. 3–10 pL of mitochondrial suspension from HepG2 cells was injected into a zygote or one blastomere of a two-cell embryo. Zygotes or two-cell embryos cultured in M3 medium drops covered with mineral oil in Petri dishes. Upon reaching the two-, four- or eight-cell stage the cultured embryos were separated into blastomeres. The latter were lysed and the total DNA was isolated. Human mtDNA was detected by PCR using species-specific primers.
Results. The development of 2848 mouse embryos was monitored. In 520 embryos that achieved the stage of 2, 4, 8 in proper time the presence of human mtDNA was assayed in each blastomere. Along with murine mtDNA all embryos contained human mitochondrial genome, which is an evidence of artificially modelled heteroplasmy. Not every blastomere of transmitochondrial embryos contained foreign (human) mtDNA. Mathematical elaboration evidenced an uneven distribution of human mtDNA in cytoplasm within the time elapsed between the injection of human mitochondria and the subsequent splitting of the embryo.
Conclusion. The results obtained confirm our previous notion of the presence of 10–11 segregation units of human mtDNA in the total amount of mitochondria (about 5 ∙ 102) injected into an embryo.
Keywords
Full Text
About the authors
Maria E. Kustova
Institute of Experimental Medicine
Email: kusmasha@yandex.ru
ORCID iD: 0000-0002-4149-2895
SPIN-code: 7151-4480
PhD in Biological Sciences, senior researcher of the Department of Molecular Genetics
Russian Federation, Saint PetersburgVasilina A. Sokolova
Institute of Experimental Medicine
Email: iva-li@mail.ru
ORCID iD: 0000-0002-9204-2448
SPIN-code: 6348-6616
PhD in Biological Sciences, senior researcher of the Department of Molecular Genetics
Russian Federation, Saint PetersburgOksana V. Kidgotko
Institute of Experimental Medicine
Email: oks-kidgotko@yandex.ru
ORCID iD: 0000-0002-2182-7782
SPIN-code: 6348-6616
PhD in Biological Sciences, researcher of the Department of Molecular Genetics
Russian Federation, Saint PetersburgMikhail G. Bass
Institute of Experimental Medicine
Email: mgb3@yandex.ru
researcher of the Department of Molecular Genetics
Russian Federation, Saint PetersburgFaina M. Zakharova
Institute of Experimental Medicine; Saint Petersburg State University
Email: fzakharova@mail.ru
ORCID iD: 0000-0002-9558-3979
SPIN-code: 9699-5744
PhD in Biological Sciences, researcher of the Department of Molecular Genetics; Senior lecturer, Department of Embryologyat
Russian Federation, Saint PetersburgVadim B. Vasilyev
Institute of Experimental Medicine; Saint Petersburg State University
Author for correspondence.
Email: vadim@biokemis.ru
ORCID iD: 0000-0002-9707-262X
SPIN-code: 6699-6350
Doctor of Medical Sciences, Head of the Department of Molecular Genetics; Professor of Chair of Fundamental Problems of Medicine and Medical Technology
Russian Federation, Saint PetersburgReferences
- Vasilyev V.B. Geneticheskie osnovy mitokhondrial’nykh bolezney. Saint Petersburg: Nestor-Istoriya; 2006. (In Russ.)
- Sokolova VA, Kustova ME, Arbuzova NI, et al. Obtaining mice that carry human mitochondrial DNA transmitted to the progeny. Mol Reprod Dev. 2004;68(3):299-307. https://doi.org/10.1002/mrd.20075.
- Bass MG, Sokolova VA, Kustova ME, et al. Analysis of efficiency of obtaining transmitochondrial mice by microinjections of human mitochondria into mouse zygote. Biochim Biopys Acta. 2006;1757:679-685.
- Howell N. Human Mitochondrial Diseases: Answering Questions and Questioning Answers. In: International Review of Cytology. Vol. 186. Elsevier; 1998. P. 49-116. https://doi.org/10.1016/s0074-7696(08)61051-7
- Nagy A, Gertsenstein M, Vintersten K, Behringer R. Manipulating the mouse embryo. 2003. New York: Cold Spring Harbor Laboratory Press; 2003.
- Vasilyev VB, Sokolova VA, Sorokin AV, et al. Persistence of human mitochondrial DNA throughout the development to the blastocyst of mouse zygotes microinjected with human mitochondria. Zygote. 1999;7(4):279-283. https://doi.org/10.1017/s0967199499000672.
- Anderson S, Bankier AT, Barrell BG, et al. Sequence and organization of the human mitochondrial genome. Nature. 1981;290(5806):457-465. https://doi.org/10.1038/ 290457a0.
- Bibb MJ, Van Etten RA, Wright CT, et al. Sequence and gene organization of mouse mitochondrial DNA. Cell. 1981;26(2):167-180. https://doi.org/10.1016/0092-8674(81) 90300-7.
- Glantz SA. Primer of Biostatistics. New York – St. Louis – San Francisco – Auckland: McGraw Hill, Inc; 1994.
- Poulton J, Marchington DR. Prospects for DNA-based prenatal diagnosis of mitochondrial disorders. Prenat Diagn. 1996;16(13):1247-1256. https://doi.org/10.1002/(sici)1097- 0223(199612)16:13<1247::aid-pd99>3.0.co;2-p.
- Matthews PM, Brown RM, Morten K, et al. Intracellular heteroplasmy for disease-associated point mutations in mtDNA: implications for disease expression and evidence for mitotic segregation of heteroplasmic units of mtDNA. Hum Genet. 1995;96(3):261-268. https://doi.org/10.1007/bf00210404.
- Tourte M, Besse C, Mounolou JC. Cytochemical evidence of an organized microtubular cytoskeleton in Xenopus laevis oocytes: involvement in the segregation of mitochondrial populations. Mol Reprod Dev. 1991;30(4):353-359. https://doi.org/10.1002/mrd.1080300410.
- Penman S. Rethinking cell structure. Proc Natl Acad Sci U S A. 1995;92(12):5251-5257. https://doi.org/10.1073/pnas.92.12.5251.
- Hermann GJ, King EJ, Shaw JM. The yeast gene, MDM20, is necessary for mitochondrial inheritance and organization of the actin cytoskeleton. J Cell Biol. 1997;137(1):141-153. https://doi.org/10.1083/jcb.137.1.141.
- Nogawa T, Sung WK, Jagiello GM, Bowne W. A quantitative analysis of mitochondria during fetal mouse oogenesis. J Morphol. 1988;195(2):225-234. https://doi.org/10.1002/jmor.1051950208.
- Smith LC, Alcivar AA. Cytoplasmic inheritance and its effects on developments and performance. J Reprod Fertil Suppl. 1993;48:31-43.
- Howell N. Mutational analysis of the human mitochondrial genome branches into the realm of bacterial genetics. Am J Hum Genet. 1996;59(4):749-755.
- Thundathil J, Filion F, Smith LC. Molecular control of mitochondrial function in preimplantation mouse embryos. Mol Reprod Dev. 2005;71(4):405-413. https://doi.org/10.1002/mrd.20260.
- Ashley MV, Laipis PJ, Hauswirth WW. Rapid segregation of heteroplasmic bovine mitochondria. Nucleic Acids Res. 1989;17(18):7325-7331. https://doi.org/10.1093/nar/ 17.18.7325.
- van der Bliek AM. Functional diversity in the dynamin family. Trends Cell Biol. 1999;9(3):96-102. https://doi.org/10.1016/s0962-8924(98)01490-1.
- Hinshaw JE. Dynamin and its role in membrane fission. Annu Rev Cell Dev Biol. 2000;16:483-519. https://doi.org/10.1146/annurev.cellbio.16.1.483.
- Olichon A, Emorine LJ, Descoins E, et al. The human dynamin-related protein OPA1 is anchored to the mitochondrial inner membrane facing the inter-membrane space. FEBS Lett. 2002;523(1-3):171-176. https://doi.org/10.1016/s0014-5793(02)02985-x.
- Borovkov AA. Teoriya veroyatnostey. Moscow: Nauka; 1986. (In Russ.)
- Kidgotko OV, Kustova MY, Sokolova VA, et al. Transmission of human mitochondrial DNA along the paternal lineage in transmitochondrial mice. Mitochondrion. 2013;13(4): 330-336. https://doi.org/10.1016/j.mito.2013.03.004.