Aberrant Allocations of Inner Cell Mass and Trophectoderm Cells in Bovine Nuclear Transfer Blastocysts1

Koo, Deog‐Bon; Kang, Yong‐Kook; Choi, Young-Hee; Park, Jung Sun; Kim, Hana; Oh, Keon Bong; Son, Dong-Soo; Park, Humdai; Lee, Kyung-Kwang; Han, Yong Mahn

doi:10.1095/biolreprod67.2.487

Cited by 181 publications

(126 citation statements)

References 38 publications

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“…A higher total cell number in the embryo correlated with IVF (van Soom et al 1997) and SCNT embryos' developmental potential (Renard et al 2007). Moreover, it has been suggested that aberrant allocation of ICM and TE cells to the blastocyst stage may be responsible for the abnormalities observed after transfer of SCNT embryos (Koo et al 2002). Like other observers, we found that SCNT embryos activated using ionomycin/ DMAP presented a significantly higher ratio of ICM:total cells when compared with IVF and in vivo-produced embryos (Koo et al 2002).…”

Section: Discussionsupporting

confidence: 79%

“…Moreover, it has been suggested that aberrant allocation of ICM and TE cells to the blastocyst stage may be responsible for the abnormalities observed after transfer of SCNT embryos (Koo et al 2002). Like other observers, we found that SCNT embryos activated using ionomycin/ DMAP presented a significantly higher ratio of ICM:total cells when compared with IVF and in vivo-produced embryos (Koo et al 2002). On the other hand, we saw no differences between embryos activated using ionomycin/ CHX or PLCZ cRNA injection and IVF controls, which : bars with different superscripts differ significantly (P!0.05).…”

Section: Discussionmentioning

confidence: 99%

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Activation of bovine somatic cell nuclear transfer embryos by PLCZ cRNA injection

Ross¹,

Rodríguez²,

Iager³

et al. 2009

Reproduction

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The production of cloned animals by the transfer of a differentiated somatic cell into an enucleated oocyte circumvents fertilization. During fertilization, the sperm delivers a sperm-specific phospholipase C (PLCZ) that is responsible for triggering Ca 2C oscillations and oocyte activation. During bovine somatic cell nuclear transfer (SCNT), oocyte activation is artificially achieved by combined chemical treatments that induce a monotonic rise in intracellular Ca 2C and inhibit either phosphorylation or protein synthesis. In this study, we tested the hypothesis that activation of bovine nuclear transfer embryos by PLCZ improves nuclear reprogramming. Injection of PLCZ cRNA into bovine SCNTunits induced Ca 2C oscillations similar to those observed after fertilization and supported high rates of blastocyst development similar to that seen in embryos produced by IVF. Furthermore, gene expression analysis at the eight-cell and blastocyst stages revealed a similar expression pattern for a number of genes in both groups of embryos. Lastly, levels of trimethylated lysine 27 at histone H3 in blastocysts were higher in bovine nuclear transfer embryos activated using cycloheximide and 6-dimethylaminopurine (DMAP) than in those activated using PLCZ or derived from IVF. These results demonstrate that exogenous PLCZ can be used to activate bovine SCNT-derived embryos and support the hypothesis that a fertilization-like activation response can enhance some aspects of nuclear reprogramming.

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Section: Discussionsupporting

confidence: 79%

Section: Discussionmentioning

confidence: 99%

Activation of bovine somatic cell nuclear transfer embryos by PLCZ cRNA injection

Ross¹,

Rodríguez²,

Iager³

et al. 2009

Reproduction

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“…Data from in vivo-produced bovine embryos indicate that an ICM/TCN proportion between 20 and 40% is within the normal range (Van Soom et al 1997, Koo et al 2002, Rho et al 2007. In this study, a significant percentage of embryos treated with supraphysiological concentrations of IGF1 developed a high ICM/TCN proportion (O40%).…”

Section: Discussionsupporting

confidence: 49%

“…A high ICM/TCN proportion (41.3%) was found in experiments in which cysteine addition to the synthetic oviductal fluid (SOF) medium supplemented with foetal bovine serum increased apoptosis and reduced blastocyst formation and hatching rates (Van Soom et al 2002). Furthermore, high ICM/TCN proportions (42-60%) have been reported in bovine somatic cell nuclear transfer (SCNT) embryos (Koo et al 2002, Amarnath et al 2004, Li et al 2004, Oh et al 2006. The aberrant cell allocation of bovine SCNT embryos has been suggested to be partially responsible for the high embryonic losses occurring during early pregnancy (first trimester) after embryo transfer (Koo et al 2002).…”

Section: Discussionmentioning

confidence: 99%

Increased apoptosis in bovine blastocysts exposed to high levels of IGF1 is not associated with downregulation of the IGF1 receptor

Velazquez¹,

Hermann²,

Kues³

et al. 2011

Reproduction

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The hypothesis that high concentrations of IGF1 can impair embryo development was investigated in a bovine in vitro model to reflect conditions in polycystic ovary syndrome (PCOS) patients. Embryos were either cultured in the absence or presence of a physiological (100 ng/ml) or supraphysiological (1000 ng/ml) IGF1 concentration. Cell allocation, apoptosis, transcript and protein expression of selected genes involved in apoptosis, glucose metabolism and the IGF system were analysed. Supraphysiological IGF1 concentration did not improve blastocyst formation over controls, but induced higher levels of apoptosis, decreased TP53 protein expression in the trophectoderm and increased the number of cells in the inner cell mass (ICM). The increase in ICM cells corresponded with an increase in IGF1 receptor (IGF1R) protein in the ICM. A small, but significant, percentage of blastocysts displayed a hypertrophic ICM, not observed in controls and virtually absent in embryos treated with physiological concentrations of IGF1. Physiological IGF1 concentrations increased total IGF1R protein expression and upregulated IGFBP3 transcripts leading to an increase in blastocyst formation with no effects on cell number or apoptosis. In conclusion, the results support the hypothesis of detrimental effects of supraphysiological IGF1 concentrations on early pregnancy. However, our results do not support the premise that increased apoptosis associated with high levels of IGF1 is mediated via downregulation of the IGF1R as previously found in preimplantation mouse embryos. This in vitro system with the bovine preimplantation embryo reflects critical features of fertility in PCOS patients and could thus serve as a useful model for in-depth mechanistic studies.

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“…Differential staining of ICM and TE cells of blastocysts (day 8) was performed as described by Koo et al (2002). The zona pellucida of each blastocyst was removed by a brief exposure to acidified PBS (pH 3.0).…”

Section: Blastomere Countingmentioning

confidence: 99%

Recombinant human activin A promotes development of bovine somatic cell nuclear transfer embryos matured in vitro

Shao¹,

Lan²,

Liu³

et al. 2010

CZECH J ANIM SCI

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ABSTRACT:To improve the culture system of bovine somatic cell nuclear transfer (SCNT) embryos, we studied the effects of activin A on developmental competence of bovine SCNT embryos during the early development stage based on the traditional culture method, and analyzed the expression level of the genes related to blastocyst hatching (Na/K-ATPase, Glut-1) and related to activin A signalling pathway (ActRII and Smad2). We generated the bovine SCNT embryo using a Holstein cow oocyte as recipient cytoplasm and a foetal ear fibroblast (Holstein cow, 120 days) as donor cell. The embryos were cultured as follows: experiment 1, the addition of activin A at the concentrations of 0 (control), 20 (M1-20), 40 (M1-40) or 80 ng/ml (M1-80) to the media during the first three days and no addition during the subsequent 5 days; experiment 2, no addition of activin A to the media during the first 3 days and the addition of activin A at the concentrations of 0 (control), 20 (M2-20), 40 (M2-40) or 80 ng/ml (M2-80) during the subsequent 5 days. The results indicated that the blastocyst formation rate and hatching rate, and total blastomere numbers as well as ICM/TE obtained in experiment 1 were not significantly different from the control group (P > 0.05). In contrast, these values obtained in experiment 2 were significantly higher than in the control group (P < 0.05). In addition, the relative abundance (ratio to GAPDH mRNA) of each gene (Glut-1, ActR II and Smad2) was not significantly different among the treatments in the experiment. The expression levels of 4 genes (Na/K-ATPase, Glut-1, ActR II and Smad2) in blastocysts obtained in experiment 2 were higher than those obtained in experiment 1. In conclusion, the present study suggests that the addition of activin A to the culture media from day 4 to day 8 can enhance the developmental competence of bovine SCNT embryos.

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Aberrant Allocations of Inner Cell Mass and Trophectoderm Cells in Bovine Nuclear Transfer Blastocysts1

Cited by 181 publications

References 38 publications

Activation of bovine somatic cell nuclear transfer embryos by PLCZ cRNA injection

Activation of bovine somatic cell nuclear transfer embryos by PLCZ cRNA injection

Increased apoptosis in bovine blastocysts exposed to high levels of IGF1 is not associated with downregulation of the IGF1 receptor

Recombinant human activin A promotes development of bovine somatic cell nuclear transfer embryos matured in vitro

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