Improving the Quality of Miniature Pig Somatic Cell Nuclear Transfer Blastocysts: Aggregation of SCNT Embryos at the Four‐cell Stage

Terashita, Yukari; Sugimura, Satoshi; Kudo, Yoshihisa; Amano, Ryota; Hiradate, Yuuki; Sato, Eimei

doi:10.1111/j.1439-0531.2010.01614.x

Cited by 26 publications

(26 citation statements)

References 41 publications

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“…We observed fewer putative ICM-specific genes in the cloned embryos than in the normal embryos (Figure 3B). Perhaps cloned embryos contain fewer ICM cells than normal embryos [34, 35]. GO analysis of genes expressed in cloned embryonic putative ICM showed that they participated in pathways involving protein binding and regulation in the cytoplasm, mitochondria and nucleus, presumably because of interactions between the ooplasm and the transferred somatic nucleus.…”

Section: Resultsmentioning

confidence: 99%

Specific gene-regulation networks during the pre-implantation development of the pig embryo as revealed by deep sequencing

et al. 2014

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BackgroundBecause few studies exist to describe the unique molecular network regulation behind pig pre-implantation embryonic development (PED), genetic engineering in the pig embryo is limited. Also, this lack of research has hindered derivation and application of porcine embryonic stem cells and porcine induced pluripotent stem cells (iPSCs).ResultsWe identified and analyzed the genome wide transcriptomes of pig in vivo-derived and somatic cell nuclear transferred (SCNT) as well as mouse in vivo-derived pre-implantation embryos at different stages using mRNA deep sequencing. Comparison of the pig embryonic transcriptomes with those of mouse and human pre-implantation embryos revealed unique gene expression patterns during pig PED. Pig zygotic genome activation was confirmed to occur at the 4-cell stage via genome-wide gene expression analysis. This activation was delayed to the 8-cell stage in SCNT embryos. Specific gene expression analysis of the putative inner cell mass (ICM) and the trophectoderm (TE) revealed that pig and mouse pre-implantation embryos share regulatory networks during the first lineage segregation and primitive endoderm differentiation, but not during ectoderm commitment. Also, fatty acid metabolism appears to be a unique characteristic of pig pre-implantation embryonic development. In addition, the global gene expression patterns in the pig SCNT embryos were different from those in in vivo-derived pig embryos.ConclusionsOur results provide a resource for pluripotent stem cell engineering and for understanding pig development.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-4) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Specific gene-regulation networks during the pre-implantation development of the pig embryo as revealed by deep sequencing

et al. 2014

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“…Furthermore, as we have previously demonstrated [8] , embryo aggregation also enhanced blastocyst diameters on day 7. If the positive effects of embryo aggregation are due to an epigenetic compensation and/or an increase in embryo cell number [10] , [13] , [15] , [25] , then an increase in the number of aggregated zygotes should correlate to an improvement in in vitro embryo development. Some of the reported benefits of embryo aggregation are related to a normalization of gene expression and developmental potential [10] , [11] , [37] , an increase in cell number [10] , [12] , [33] and a consequent improvement in blastocyst development rates [10] , [13] , [33] .…”

Section: Discussionmentioning

confidence: 99%

“…As a means to improve cloning efficiency, the strategy of embryo aggregation has been applied in several species. These include the mouse [10] , [11] , bovine [12] – [14] , pig [15] and horse [8] . These studies have reported benefits of embryo aggregation for in vitro and/or in vivo embryo development.…”

Section: Introductionmentioning

confidence: 99%

The Aggregation of Four Reconstructed Zygotes is the Limit to Improve the Developmental Competence of Cloned Equine Embryos

et al. 2014

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Embryo aggregation has been demonstrated to improve cloning efficiency in mammals. However, since no more than three embryos have been used for aggregation, the effect of using a larger number of cloned zygotes is unknown. Therefore, the goal of the present study was to determine whether increased numbers of cloned aggregated zygotes results in improved in vitro and in vivo embryo development in the equine. Zona-free reconstructed embryos (ZFRE's) were cultured in the well of the well system in four different experimental groups: I. 1x, only one ZFRE per microwell; II. 3x, three per microwell; III. 4x, four per microwell; and IV. 5x, five ZFRE's per microwell. Embryo size was measured on day 7, after which blastocysts from each experimental group were either a) maintained in culture from day 8 until day 16 to follow their growth rates, b) fixed to measure DNA fragmentation using the TUNEL assay, or c) transferred to synchronized mares. A higher blastocyst rate was observed on day 7 in the 4x group than in the 5x group. Non-aggregated embryos were smaller on day 8 compared to those aggregated, but from then on the in vitro growth was not different among experimental groups. Apoptotic cells averaged 10% of total cells of day 8 blastocysts, independently of embryo aggregation. Only pregnancies resulting from the aggregation of up to four embryos per microwell went beyond the fifth month of gestation, and two of these pregnancies, derived from experimental groups 3x and 4x, resulted in live cloned foals. In summary, we showed that the in vitro and in vivo development of cloned zona-free embryos improved until the aggregation of four zygotes and declined when five reconstructed zygotes were aggregated.

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“…Furthermore, in a recent report, expression of Cdx2 was corrected by increasing the total cell number of SCNT embryos by embryo aggregation, suggesting that the cell number is a critical parameter in the regulation of extraembryonic linage gene expression in SCNT embryos [28]. If cells within a single SCNT embryo vary in their transcriptional activity or developmental ability because of epigenetic differences, aggregation may increase the number of normal cells with the correct gene expression required for further development [38,39]. Considering the result that no cloned calves were obtained after transfer of 10 nonaggregated SCNT embryos in the present study, the rate of normal cells contained in these SCNT embryos seems to be low.…”

Section: Discussionmentioning

confidence: 99%

Developmental Ability of Somatic Cell Nuclear Transferred Embryos Aggregated at the 8-cell Stage or 16- to 32-cell Stage in Cattle

Akagi

Yamaguchi

Matsukawa

et al. 2011

Journal of Reproduction and Development

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Abstract. Aggregation of somatic cell nuclear transfer (SCNT) embryos in mice is reported to improve full-term development. In the present study, we attempted to improve the development of SCNT embryos by aggregation in cattle. In Experiment 1, to examine the effect of the timing of aggregation on in vitro development of cumulus-cell NT embryos, we aggregated two or three SCNT embryos (2X or 3X embryos) at the 1-cell, 8-cell and 16-to 32-cell stages. Irrespective of the timing of aggregation, 3X embryos developed to the blastocyst stage at a high rate. However, aggregation did not improve the total blastocyst formation rate of the embryos used. The cell numbers of 3X embryos aggregated at the 1-cell stage and 2X embryos tended to be higher than that of single NT embryos (1X embryos). Furthermore, a significant increase in cell number was observed in 3X embryos aggregated at the 8-cell stage and 16-to 32-cell stage. In Experiment 2, we used fibroblast cells as nuclear donors and examined in vitro development of 3X embryos aggregated at the 8-cell stage and 16-to 32-cell stage. As a result, 3X embryos had high blastocyst formation rates and higher cell numbers than 1X embryos, which was consistent with the results of Experiment 1. In Experiment 3, we examined the full-term developmental ability of 3X embryos aggregated at the 8-cell stage and 16-to 32-cell stage. After transfer of fibroblast-derived NT embryos into recipient animals, a significantly higher pregnancy rate was obtained on Day 60 in 3X embryos than in 1X embryos. Two embryos aggregated at 8-cell stage and one embryo aggregated at the 16-to 32-cell stage developed to term, while no pregnancies derived from 1X embryos that lasted to Day 60. However, two of the cloned calves were stillborn. These results suggest that aggregation of the 8-cell stage or 16-to 32-cell stage SCNT embryos may improve the pregnancy rate, but that it cannot reduce the high incidence of fetal loss and stillbirth, which is often observed in bovine SCNT.

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Improving the Quality of Miniature Pig Somatic Cell Nuclear Transfer Blastocysts: Aggregation of SCNT Embryos at the Four‐cell Stage

Cited by 26 publications

References 41 publications

Specific gene-regulation networks during the pre-implantation development of the pig embryo as revealed by deep sequencing

Specific gene-regulation networks during the pre-implantation development of the pig embryo as revealed by deep sequencing

The Aggregation of Four Reconstructed Zygotes is the Limit to Improve the Developmental Competence of Cloned Equine Embryos

Developmental Ability of Somatic Cell Nuclear Transferred Embryos Aggregated at the 8-cell Stage or 16- to 32-cell Stage in Cattle

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