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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.

SUBMITTER: Tachibana M

PROVIDER: S-EPMC3244345 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Chromosome transfer in mature oocytes.

Tachibana Masahito M Sparman Michelle M Mitalipov Shoukhrat S

Nature protocols 20100527 6

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 ...[more]

PMID: 20539289

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Project description:OBJECTIVE:To evaluate the effect of cytoplasm transfer from mature oocytes to germinal vesicle(GV)s on promoting the maturation of cytoplasm of GV at the mRNA level. MATERIALS AND METHODS:Sixty six in vitro fertilization (IVF) operations between June 2012 and November 2013 were included in this study. Totally 120 GVs were obtained. Normal GVs were categorized into 3 groups (n = 40) randomly: the first comprised oocytes that did not receive the cytoplasm of mature oocytes; the second group comprised oocytes that did not receive the cytoplasm of mature oocytes but were incubated for 24 h; and the third group comprised oocytes that received 10-15% the cytoplasm of mature oocytes and were then incubated for 24 h. Each group was separately analyzed by quantitative polymerase chain reaction (qPCR) and the expression levels of selected genes were assessed. RESULTS:The expression levels of genes involved in the cytoplasmic maturity, and energy-producing mitochondria were significantly higher in the pooled oocytes of 2(nd) control group than those of the 1(st) control and intervention groups (p < 0.001). The genes involved in the meiosis, spindle check point, DNA repairing and cell cycle checkpoint did not have any expression in the 1(st) and intervention groups; however, these genes were expressed in the 2(nd) group, significantly. In the 2(nd) group, the highest expression level was observed for genes involved in the DNA repairing and cell cycle checkpoint. In the intervention group, none of the genes were expressed except for energy-producing mitochondria gene; even in this case, the expression level of this gene in this group of oocytes was significantly lower than that in other groups (p < 0.001). After 24 h meiosis assumption was significantly higher in the third group than in the second group (95% vs. 68%, p < 0.001). CONCLUSION:The cytoplasm transfer technique is not effective in cytoplasmic maturity of the recipient GV oocytes. In contrast, 24-hr in-vitro culture is associated with increased expression of studied genes in GVs.

Dataset Information

Chromosome transfer in mature oocytes.

Publications

Chromosome transfer in mature oocytes.

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