Mouse &lt;i&gt;in vivo&lt;/i&gt;-derived late 2-cell embryos have higher developmental competence after high osmolality vitrification and −80°C preservation than IVF or ICSI embryos

Hayashi, Erika; Wakayama, Sayaka; Ito, Daiyu; Hasegawa, Ayumi; Mochida, Keiichi; Ooga, Masatoshi; Ogura, Atsuo; Wakayama, Teruhiko

doi:10.1262/jrd.2021-115

Cited by 8 publications

(9 citation statements)

References 25 publications

(39 reference statements)

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“…To evaluate embryo quality using this system, morulae/blastocysts cultured for 3 days in ETC were transferred to mice. In a control experiment in which 2-cell stage embryos removed immediately after thawing in the ETC, were transferred to the oviduct of the recipient female, we showed a birth rate of 47%, which was comparable to birth rates of our usual frozen-thawed embryos [21]. When embryos collected after 3 days of culture in the ETC were transferred to the uterus, birth rate was 31%, which was slightly lower than the control (Table 4).…”

Section: Plos Onesupporting

confidence: 52%

“…However, the embryo recovery rate obtained by the ETC was lower than that shown in this study (submitted). We tried various improvements right up to launch, such as a good equilibration medium for embryo culture in ETC [20], and identified the most appropriate stage of mouse two-cell embryos for the present project [21]. However, space experiments can be challenging.…”

Section: Discussionmentioning

confidence: 99%

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Development of a new device for manipulating frozen mouse 2-cell embryos on the International Space Station

Wakayama

Soejima²,

Kikuchi³

et al. 2022

PLoS ONE

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Whether mammalian embryos develop normally under microgravity remains to be determined. However, embryos are too small to be handled by inexperienced astronauts who orbit Earth on the International Space Station (ISS). Here we describe the development of a new device that allows astronauts to thaw and culture frozen mouse 2-cell embryos on the ISS without directly contacting the embryos. First, we developed several new devices using a hollow fiber tube that allows thawing embryo without practice and observations of embryonic development. The recovery rate of embryos was over 90%, and its developmental rate to the blastocyst were over 80%. However, the general vitrification method requires liquid nitrogen, which is not available on the ISS. Therefore, we developed another new device, Embryo Thawing and Culturing unit (ETC) employing a high osmolarity vitrification method, which preserves frozen embryos at −80°C for several months. Embryos flushed out of the ETC during thawing and washing were protected using a mesh sheet. Although the recovery rate of embryos after thawing were not high (24%-78%) and embryonic development in ETC could not be observed, thawed embryos formed blastocysts after 4 days of culture (29%-100%) without direct contact. Thus, this ETC could be used for untrained astronauts to thaw and culture frozen embryos on the ISS. In addition, this ETC will be an important advance in fields such as clinical infertility and animal biotechnology when recovery rate of embryos were improved nearly 100%.

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Section: Plos Onesupporting

confidence: 52%

Section: Discussionmentioning

confidence: 99%

Development of a new device for manipulating frozen mouse 2-cell embryos on the International Space Station

Wakayama

Soejima²,

Kikuchi³

et al. 2022

PLoS ONE

Self Cite

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“…The first two cleavage events in an embryo take approximately 20 hours each, and these are significantly longer than successive cleavage steps which take approximately 12 hours each to complete (Mihajlović & Bruce, 2017). The main disadvantage of trying to produce metaphase spreads from IVF-produced two-cell embryos was that many embryos were developmentally arrested before the division into four-cells, and thus did not progress through to metaphase (Hayashi et al, 2022). In general, 10-15% of all human embryos made via IVF arrest at this two-cell stage mainly due to a build-up of reactive oxygen species (ROS) (Betts & Madan, 2008).…”

Section: Ivf Produced Embryosmentioning

confidence: 99%

“…Two-cell embryos produced from timed matings can be flushed at the later stages of the cell cycle (i.e., on the cusp of division to four-cells) making them an ideal sample type for metaphase spread preparation (Bedford, 1971). It has also been shown that late stage two-cell embryos collected via embryo flushing have a higher chance of survival in comparison to early stage two-cell embryos (Hayashi et al, 2022). Early stage two-cell embryos have a higher chance of remaining in interphase (S or G2 phase of the cell cycle) in comparison to dividing late stage two-cell embryos (Hayashi et al, 2022).…”

Section: Flushed Embryosmentioning

confidence: 99%

“…It has also been shown that late stage two-cell embryos collected via embryo flushing have a higher chance of survival in comparison to early stage two-cell embryos (Hayashi et al, 2022). Early stage two-cell embryos have a higher chance of remaining in interphase (S or G2 phase of the cell cycle) in comparison to dividing late stage two-cell embryos (Hayashi et al, 2022). This increases the chance of successfully timing the colcemid incubation to produce metaphase spreads for the validation of FISH probes.…”

Section: Flushed Embryosmentioning

confidence: 99%

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Synthesis and Validation of Fluorescence in situ Hybridisation Probes (FISH) for Genotyping Mouse Models of Embryonic Lethal Aneuploidies

Derecourt¹

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Approximately 50% of all embryos produced in fertility clinics have an abnormal number of chromosomes (i.e., aneuploid)(Vera-Rodriguez et al., 2015). Of these, 25% have aneuploidies that are embryonic lethal (O’Connor, 2008) and result in spontaneous abortions in the first trimester(Chen et al., 2017). My research contributes to wider research programme that aimsto develop a non-invasive method for detecting aneuploidies in preimplantation humanembryos. For the overarching research program, several models of embryonic lethal aneuploidies must be developed and validated. One way to produce embryonic lethal aneuploidies is through mouse breeding programmes that make use of Robertsonian translocations. Robertsonian translocations (RobT) are one of the most common forms of chromosomal rearrangements in humans and can cause whole chromosome duplications/deletions(Zhuang et al., 2014). A balanced carrier of these RobT is viable but produces normal gametes, carrier gametes, as well as gametes that will result in aneuploid (trisomy or monosomy) embryos(Beyer & Willats, 2017). As these RobT mouse models are being developed in our laboratory, an accurate method for identifying the trisomic and monosomic (i.e., aneuploid) embryos produced is critical. This project aims to synthesise and validate fluorescence in situ hybridisation (FISH) point probes for mouse chromosomes 4, 10, 11, and 15 using a cost-effective, PCR-based method (Bienko et al., 2013). Herein, I present data from experiments that were conducted to validate FISH probes for mouse chromosome 4, 10, 11, and 15. I also describe a series of experiments to optimise metaphase spread preparation from two different sample types, mouse lymphocytes and cleavage stage embryos. From this study, four FISH probes (specific for chromosome 4, 10, 11, & 15) were synthesised. Based on existing metrics, these were determined to be sufficiently labelled and at a high enough concentration to be evaluated as FISH probes. However, when FISH was performed using each of these probes, no specific signal due to hybridisation was observed. This is despite attempts to identify and address the cause of this lack of binding. Despite this, a positive, pan-centromeric control was successfully validated using the optimised FISH protocol. Also, metaphase spread protocols for two different tissue types were also devised to produce a reliable sample source for FISH validation. Overall, this project has shown that these PCR based point probes are not appropriate for the genotyping of RobT induced aneuploidies and other forms of genotyping should be considered.

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Mouse Cloning Using Outbred Oocyte Donors and Nontoxic Reagents

Wakayama

Terashita

Tanabe³

et al. 2023

Methods in Molecular Biology

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Mouse <i>in vivo</i>-derived late 2-cell embryos have higher developmental competence after high osmolality vitrification and −80°C preservation than IVF or ICSI embryos

Cited by 8 publications

References 25 publications

Development of a new device for manipulating frozen mouse 2-cell embryos on the International Space Station

Development of a new device for manipulating frozen mouse 2-cell embryos on the International Space Station

Synthesis and Validation of Fluorescence in situ Hybridisation Probes (FISH) for Genotyping Mouse Models of Embryonic Lethal Aneuploidies

Mouse Cloning Using Outbred Oocyte Donors and Nontoxic Reagents

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