Assessment of chromosomal integrity using a novel live-cell imaging technique in mouse embryos produced by intracytoplasmic sperm injection

Yamagata, Kazuya; Suetsugu, Rinako; Wakayama, Teruhiko

doi:10.1093/humrep/dep236

Cited by 52 publications

(56 citation statements)

References 27 publications

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“…Finally, we are wondering whether our reconstitution system might have a potential for clinical applications. For instance, it is known that failures in intracytoplasmic sperm injection, an assisted reproductive technology, are often associated with abnormal chromosome segregation in mouse blastomeres 45 . Pretreatment of sperm chromatin with defined purified factors in vitro, as demonstrated in the current study, could help increase the success rate of intracytoplasmic sperm injection.…”

Section: Discussionmentioning

confidence: 99%

Reconstitution of mitotic chromatids with a minimum set of purified factors

2015

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The assembly of mitotic chromosomes, each composed of a pair of rod-shaped chromatids, is an essential prerequisite for accurate transmission of the genome during cell division. It remains poorly understood, however, how this fundamental process might be achieved and regulated in the cell. Here we report an in vitro system in which mitotic chromatids can be reconstituted by mixing a simple substrate with only six purified factors: core histones, three histone chaperones (nucleoplasmin, Nap1 and FACT), topoisomerase II (topo II) and condensin I. We find that octameric nucleosomes containing the embryonic variant H2A.X-F are highly susceptible to FACT and function as the most productive substrate for subsequent actions of topo II and condensin I. Cdk1 phosphorylation of condensin I is the sole mitosis-specific modification required for chromatid reconstitution. This experimental system will enhance our understanding of the mechanisms of action of individual factors and their cooperation during this process.

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Section: Discussionmentioning

confidence: 99%

Reconstitution of mitotic chromatids with a minimum set of purified factors

2015

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“…S2). Most embryos with ACS arrest their development after implantation, as revealed by live-cell imaging analysis for embryos derived from SCNT or round spermatid injection [66,67]. Nevertheless, embryos cloned from Purkinje cells of both sexes showed better global gene expression profiles and relatively normal Xist expression levels, indicating that the genome of Purkinje cells can potentially support embryonic development after SCNT.…”

Section: Discussionmentioning

confidence: 99%

Generation of Cloned Mice from Adult Neurons by Direct Nuclear Transfer1

Mizutani

Oikawa

Kassai

et al. 2015

Biology of Reproduction

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Whereas cloning mammals by direct somatic cell nuclear transfer has been successful using a wide range of donor cell types, neurons from adult brain remain "unclonable" for unknown reasons. Here, using a combination of two epigenetic approaches, we examined whether neurons from adult mice could be cloned. First, we used a specific antibody to discover cell types with reduced amounts of a repressive histone mark-dimethylated histone H3 lysine 9 (H3K9me2)-and identified CA1 pyramidal cells in the hippocampus and Purkinje cells in the cerebellum as candidates. Second, reconstructed embryos were treated with trichostatin A (TSA), a potent histone deacetylase inhibitor. Using CA1 cells, cloned offspring were obtained at high rates, reaching 10.2% and 4.6% (of embryos transferred) for male and female donors, respectively. Cerebellar Purkinje cell nuclei were too large to maintain their genetic integrity during nuclear transfer, leading to developmental arrest of embryos. However, gene expression analysis using cloned blastocysts corroborated a high rate of genomic reprogrammability of CA1 pyramidal and Purkinje cells. Neurons from the hippocampal dentate gyrus and cerebral cortex, which had higher amounts of H3K9me2, could also be used for producing cloned offspring, but the efficiencies were low. A more thorough analysis revealed that TSA treatment was essential for cloning adult neuronal cells. This study demonstrates, to our knowledge for the first time, that adult neurons can be cloned by nuclear transfer. Furthermore, our data imply that reduced amounts of H3K9me2 and increased histone acetylation appear to act synergistically to improve the development of cloned embryos.

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“…These had been mated with vasectomized ICR males. Six to 10 embryos were transferred into each uterus [25]–[27]. Offspring were obtained at 18.5 days post copulation through Cesarean section or by natural birth, and we recorded the body and placental weights and sex.…”

Section: Methodsmentioning

confidence: 99%

Offspring from Mouse Embryos Developed Using a Simple Incubator-Free Culture System with a Deoxidizing Agent

Itoi¹,

Tokoro²,

Terashita

et al. 2012

PLoS ONE

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To culture preimplantation embryos in vitro, water-jacketed CO2 incubators are used widely for maintaining an optimal culture environment in terms of gas phase, temperature and humidity. We investigated the possibility of mouse embryo culture in a plastic bag kept at 37°C. Zygotes derived from in vitro fertilization or collected from naturally mated B6D2F1 female mice were put in a drop of medium on a plastic culture dish and then placed in a commercially available plastic bag. When these were placed in an oven under air at 37°C for 96 h, the rate of blastocyst development and the cell numbers of embryos decreased. However, when the concentration of O2 was reduced to 5% using a deoxidizing agent and a small oxygen meter, most zygotes developed into blastocysts. These blastocysts were judged normal according to their cell number, Oct3/4 and Cdx2 gene expression levels, the apoptosis rate and the potential for full-term development after embryo transfer to pseudopregnant recipients. Furthermore, using this system, normal offspring were obtained simply by keeping the bag on a warming plate. This culture method was applied successfully to both hybrid and inbred strains. In addition, because the developing embryos could be observed through the transparent wall of the bag, it was possible to capture time-lapse images of live embryos until the blastocyst stage without needing an expensive microscope-based incubation chamber. These results suggest that mouse zygotes are more resilient to their environment than generally believed. This method might prove useful in economical culture systems or for the international shipment of embryos.

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Assessment of chromosomal integrity using a novel live-cell imaging technique in mouse embryos produced by intracytoplasmic sperm injection

Abstract: ACS during first mitosis appears to be a major cause of early pregnancy losses in ICSI-generated mouse embryos. Moreover, this novel imaging technology could be applicable as a method for the assessment of embryo quality.

Cited by 52 publications

References 27 publications

Reconstitution of mitotic chromatids with a minimum set of purified factors

Reconstitution of mitotic chromatids with a minimum set of purified factors

Generation of Cloned Mice from Adult Neurons by Direct Nuclear Transfer1

Offspring from Mouse Embryos Developed Using a Simple Incubator-Free Culture System with a Deoxidizing Agent

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