Simple transport and cryopreservation of cold-stored mouse          embryos

Mukunoki, Ayumi; Tanaka, Takeo; Nakao, Satohiro; Tamura, Kana; Horikoshi, Yuka; Nakagata, Naomi

doi:10.1538/expanim.20-0042

Cited by 4 publications

(5 citation statements)

References 21 publications

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“…The embryos were derived from in vitro fertilization, using 9–12‐week‐old Crl:CD1(ICR) male and B6D2F1/Crl female mice (Jackson Laboratory, Kanagawa, Japan). In vitro fertilization, embryo vitrification, and thawing were performed as described previously 13,14 . Embryos were frozen after the two pronuclei were visible in the bright field and used for experiments after thawing.…”

Section: Methodsmentioning

confidence: 99%

“…In vitro fertilization, embryo vitrification, and thawing were performed as described previously. 13,14 Embryos were frozen after the two pronuclei were visible in the bright field and used for experiments after thawing. Potassium simplex optimized medium (KSOM; ARK Resource Co., Kumamoto, Japan) was used for embryo culture and observation.…”

Section: Mouse Two-pronuclear Stage Embryosmentioning

confidence: 99%

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An approach for live imaging of first cleavage in mouse embryos using fluorescent chemical probes for DNA, microtubules, and microfilaments

Okabe,

Shirasawa,

Ono

et al. 2023

Reprod Medicine & Biology

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PurposeDynamic morphological changes in the chromosome and cytoskeleton occur in mammals and humans during early embryonic development, and abnormalities such as embryonic chromosomal aneuploidy occur when development does not proceed normally. Visualization of the intracellular organelles and cytoskeleton allows elucidation of the development of early mammalian embryos. The behavior of the DNA and cytoskeleton in early mammalian embryos has conventionally been observed by injecting target molecule mRNAs, incorporating a fluorescent substance‐expressing gene, into embryos. In this study, we visualized the chronological behavior of male and female chromosome condensation in mouse embryos, beginning in the two‐pronuclear zygote, through the first division to the two‐cell stage, using fluorescent chemical probes to visualize the behavior of DNA, microtubules, and microfilaments.MethodMouse two‐pronuclear stage embryo were immersed in medium containing fluorescent chemical probes to visualize DNA, microtubules, and microfilaments. Observation was performed with a confocal microscope.ResultsThis method allowed us to observe how chromosome segregation errors in first somatic cell divisions in mouse embryos and enabled dynamic analysis of a phenomenon called lagging chromosomes.ConclusionsBy applying this method, we can observe any stage of embryonic development, which may provide new insights into embryonic development in other mammals.

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Section: Methodsmentioning

confidence: 99%

Section: Mouse Two-pronuclear Stage Embryosmentioning

confidence: 99%

An approach for live imaging of first cleavage in mouse embryos using fluorescent chemical probes for DNA, microtubules, and microfilaments

Okabe,

Shirasawa,

Ono

et al. 2023

Reprod Medicine & Biology

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show abstract

“…The embryos were derived from in vitro fertilization, using Crl:CD1(ICR) males and B6D2F1/Crl females (Jackson Laboratory, Kanagawa, Japan) aged 9-12 weeks. In vitro fertilization, embryo vitri cation, and thawing were performed as described previously [13,14]. Embryos were frozen after the two pronuclei were visible in the bright-eld and used for experiments after thawing.…”

Section: Mouse 2pn Stage Embryosmentioning

confidence: 99%

Development of Simple live-imaging method for viewing the first cleavage of mammalian embryos by using fluorescent chemical probes for DNA and cytoskeletons

Okabe

Shirasawa

Goto

et al. 2023

Preprint

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Dynamic morphological changes in the chromosomes and cytoskeleton occur in mammals including humans, during early embryonic development, and abnormalities such as embryonic chromosomal aneuploidy occur when development does not proceed normally. In previous reports, the behavior of DNA and cytoskeleton in early mammalian embryos has conventionally been visualized and observed by injecting target molecule mRNA, with a fluorescent substance-expressing gene incorporated, into embryos. However, injecting genetic information into a human embryo to induce the production of unnatural proteins must be carefully considered from an ethical perspective. Therefore, we aimed to develop a simple observation method as a way of gaining knowledge about the first division that can avoid such problems. We visualized the chronological behavior of male and female chromosome condensation in mammalian embryos, beginning in the 2PN zygote, through the first division into the two-cell stage by using fluorescent chemical probes for DNA, microtubules, and microfilaments. This method is simple and does not require genetic manipulation, and its application can be observed at any stage during embryonic development, thereby providing novel insights into embryonic development in many mammals. In particular, it is expected to provide a great deal of cell biological information on the first cleavage of human embryos, which have been reported to exhibit a variety of patterns.

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“…The embryos were derived from in vitro fertilisation, using Crl:CD1(ICR) males and B6D2F1/Crl females (Jackson Laboratory, Kanagawa, Japan) aged 9-12 weeks. In vitro fertilisation, embryo vitri cation, and embryo thawing were performed as previously described [13,14]. Embryos at the pronuclear stage were used for experiments.…”

Section: Embryosmentioning

confidence: 99%

Simple live-imaging method for viewing the first cleavage of mouse embryos that does not require genetic manipulation

Okabe

Shirasawa

Goto

et al. 2022

Preprint

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Time-lapse incubators have become increasingly popular in assisted reproductive technology, allowing for the observation of the developmental process, which may be useful in the selection of human embryos suitable for transplantation. Dynamic morphological changes of chromosomes and the cytoskeleton occur during early embryonic development, including in humans, and abnormalities such as embryonic chromosomal aneuploidy occur when development does not proceed normally. Chromosome and cytoskeletal dynamics are difficult to observe with time-lapse bright field monitoring. However, in recent years, live-cell imaging techniques have been used to analyse these dynamics by injecting fluorescently labelled cytoskeletal proteins or mRNA encoding fluorescein probes. These require complicated procedures and necessitate mechanical invasion of cells. Here, we introduced a fluorescence-labelled probe with cell-membrane permeability that specifically adheres to DNA and to the cytoskeleton as imaged in an incubator-integrated time-lapse confocal laser microscope observation system. This platform enabled us to analyse, in detail, the dynamics of chromosomes, microtubules, and microfilaments from the fertilized pronuclear zygote, through first cleavage, to 2-cell stage embryo. This method is simple and does not require genetic manipulation, and its application can be expected to provide novel insights into embryonic development in many mammals, including humans.

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Simple transport and cryopreservation of cold-stored mouse embryos

Cited by 4 publications

References 21 publications

An approach for live imaging of first cleavage in mouse embryos using fluorescent chemical probes for DNA, microtubules, and microfilaments

An approach for live imaging of first cleavage in mouse embryos using fluorescent chemical probes for DNA, microtubules, and microfilaments

Development of Simple live-imaging method for viewing the first cleavage of mammalian embryos by using fluorescent chemical probes for DNA and cytoskeletons

Simple live-imaging method for viewing the first cleavage of mouse embryos that does not require genetic manipulation

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