Chromosome transfer in mature oocytes

Division of Reproductive and Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, Oregon, USA.
Fertility and sterility (Impact Factor: 4.59). 05/2012; 97(5):e16. DOI: 10.1016/j.fertnstert.2012.03.048
Source: PubMed


To demonstrate step-by-step micromanipulation procedures required for transfer of spindle-chromosomal complexes between mature oocytes.
Video presentation of reproductive biology study.
In vitro fertilization and embryo manipulation laboratory.
Rhesus (Macaca mulatta) primates.
Transplantation of the genetic material between mammalian oocytes offers many opportunities to study various aspects of nuclear-cytoplasmic interactions during oogenesis, fertilization and embryo development. We demonstrate the feasibility of isolation and transfer of chromosomes between mature metaphase II (MII) primate oocyte. After fertilization, manipulated oocytes were capable of producing healthy offspring or embryonic stem cells.
In this video, we show micromanipulation procedures required for isolation and transfer of spindle-chromosomal complexes between rhesus MII oocytes. In brief, the spindle is visualized using a polarized microscope and extracted into a membrane enclosed karyoplast. Karyoplasts are then reintroduced into an enucleated recipient oocyte (cytoplast, derived from an 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 from another.
This video demonstrates the protocol developed for primate oocytes that successfully allowed of isolation and transfer of chromosomes between mature metaphase II (MII) 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. Video is available at

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Available from: Shoukhrat Mitalipov, Mar 26, 2015
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    • "Early attempts using this technology were largely unsuccessful, with poor rates of fertilization and poor embryo development. However, recent methodological advances have allowed proof of principle experiments to be conducted in primates (Tachibana et al., 2009, 2010). In this novel study by Tachibana et al. (2009), reconstructed MII oocytes were capable of normal fertilization, normal embryo development and four live and healthy offspring were produced. "
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    ABSTRACT: Mitochondrial DNA (mtDNA) mutations are a relatively common cause of progressive disorders that can be severe or even life-threatening. There is currently no cure for these disorders; therefore recent research has been focused on attempting to prevent the transmission of these maternally inherited mutations. Here we highlight the challenges of understanding the transmission of mtDNA diseases, discuss current genetic management options and explore the use of germ-line reconstruction technologies to prevent mtDNA diseases. In particular we discuss their potential, indications, limitations and possible safety concerns. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email:
    Preview · Article · Nov 2014 · Molecular Human Reproduction
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    • "A cytoplast was then placed under the zona pellucida of a second oocyte containing another half cytoplasm with a spindle (karyoplast) to induce fusion. Reconstructed oocytes were next fertilized by ICSI, placed into 4-well dishes (Nalge Nunc) containing embryo culture medium and cultured at 37°C in 6% CO 2 , 5% O 2 , and 89% N 2 (Tachibana et al., 2010). Complete mitochondrial gene replacement by the ST procedure and embryo transfers were carried out as we previously described (Tachibana et al., 2010; Tachibana et al., 2009). "
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    ABSTRACT: Exposure of cells to electric fields is a commonly used technique for parthenogenesis, cloning and tetraploid embryo production. However, little is known about possible detrimental effects of electric fields on embryos and their development. The aim of this study was to investigate the effects of electric fields on early preimplantation development in mice and rats. Mouse and rat metaphase II (MII) and pre-activated oocytes, zygotes and 2-cell stage embryos were treated with electric fields with increasing voltage. Cleavage rate, morula and blastocyst formation were evaluated by in vitro cultivation. The effects of electric fields on embryos were investigated by measurement of reactive oxygen species (ROS) content and microtubule and microfilament distributions using fluorescence staining. Pre-activated oocytes at the pronuclear stage and zygotes are more resistant to electric exposure than freshly isolated oocytes at MII stage in both studied species. Rat zygotes treated with electric fields of increasing voltage showed higher cleavage rates compared with the mouse and some of them developed beyond 4-cell stage in vitro. Embryos blocked at the 2-cell stage after in vitro cultivation of zygotes exposed to electric fields demonstrated increased level of ROS but normal distributions of microtubules and microfilaments. In both species, embryos at the 2-cell stage were more resistant to electric fields because they formed tetraploid embryos after electric field-induced blastomere fusion and these embryos could develop in vitro until the blastocyst stage. There are stage-dependent and species-specific differences in sensitivity to electric fields in mouse and rats.
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