Treating Cloned Embryos, But Not Donor Cells, with 5-aza-2’-deoxycytidine Enhances the Developmental Competence of Porcine Cloned Embryos

Huan, Yan Jun; Zhu, Jiang; Xie, Bing; Wang, Jian Yu; Liu, Shi Chao; Zhou, Yang; Kong, Qing Ran; He, Hong; Liu, Zhong Hua

doi:10.1262/jrd.2013-026

Cited by 40 publications

(61 citation statements)

References 31 publications

(44 reference statements)

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“…So it was great difficulty, if possible, to determine which genes were responsible for the improvement of embryonic development. It had been reported that regulating epigenetic status can improve the reprogramming efficiency both in SCNT and iPS (Kishigami et al 2006, Huangfu et al 2008, Onder et al 2012, Huan et al 2013, Matoba et al 2014. Therefore, we also examined the iPS efficiency after reducing H3K27me3 level.…”

Section: Discussionmentioning

confidence: 98%

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Histone H3 lysine 27 trimethylation acts as an epigenetic barrier in porcine nuclear reprogramming

et al. 2016

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Aberrant epigenetic reprogramming is the main obstacle to the development of somatic cell nuclear transfer (SCNT) embryos and the generation of induced pluripotent stem (iPS) cells, which results in the low reprogramming efficiencies of SCNT and iPS. Histone H3 lysine 27 trimethylation (H3K27me3), as a repressive epigenetic mark, plays important roles in mammalian development and iPS induction. However, the reprogramming of H3K27me3 in pig remains elusive. In this study, we showed that H3K27me3 levels in porcine early cloned embryos were higher than that in IVF embryos. Then GSK126 and GSK-J4, two small molecule inhibitors of H3K27me3 methylase (EZH2) and demethylases (UTX/JMJD3), were used to regulate the H3K27me3 level. The results showed that H3K27me3 level was reduced in cloned embryos after treatment of PEF with 0.75 mM GSK126 for 48 h, incubation of one-cell reconstructed oocytes with 0.1 mM GSK126 and injection of antibody for EZH2 into oocyte. Meanwhile, the development of the cloned embryos was significantly improved after these treatments. On the contrary, GSK-J4 treatment increased the H3K27me3 level in cloned embryos and decreased the cloned embryonic development. Furthermore, iPS efficiency was both increased after reducing the H3K27me3 level in donor cells and in early reprogramming phase. In summary, our results suggest that H3K27me3 acts as an epigenetic barrier in SCNT and iPS reprogramming, and reduction of H3K27me3 level in donor cells and in early reprogramming phase can enhance both porcine SCNT and iPS efficiency.

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Section: Discussionmentioning

confidence: 98%

“…Similarly, abnormal epigenetics reprogramming also often occur in these areas during iPS induction, and the reprogramming efficiency could be improved by regulating these epigenetic status both in SCNT and iPS (Kishigami et al 2006, Huangfu et al 2008, Onder et al 2012, Huan et al 2013, Matoba et al 2014.…”

Section: Introductionmentioning

confidence: 99%

Histone H3 lysine 27 trimethylation acts as an epigenetic barrier in porcine nuclear reprogramming

et al. 2016

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“…Increased blastocyst rates and OCT4, SOX2, NANOG expression when used in fetal fibroblast cells Huan et al, 2013 Buffalo somatic cells…”

Section: Histone Demethylating Strategiesmentioning

confidence: 99%

Probability, odds and random chance: the difficult task of modulating the epigenetic profile of cloned embryos

et al. 2017

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Since the beginning of modern embryology, scientists have wondered about how a small number of totipotent embryonic cells can become an individual with a wide variety of organs and tissues with distinct functions. Also, the idea of generating a cloned animal using a nucleus from a donor cell is not recent. However, it has taken years of research to achieve this goal, especially regarding mechanisms of cell reprogramming required to return a differentiated cell to totipotency. Cloning by somatic cell nuclear transfer (SCNT) has been a valuable tool to understand epigenetic mechanisms related to cellular reprogramming. However, cloning efficiency is still low, with a low percentage of embryos resulting in healthy animals. The high attrition rate is associated with incomplete or abnormal epigenetic reprogramming, such that many cloned embryos have DNA methylation patterns different than controls, resulting in faulty gene expression and subsequent developmental failures. Attempts to improve genome reprogramming by modulation of oocyte quality and/or somatic cell plasticity, thereby increasing cloning efficiency and preventing detrimental effects on development, have proven ineffective. The recent development of DNA editing techniques may facilitate an improved understanding of cellular reprogramming and the role of DNA methylation in development. These novel tools may lead to new means to modulate epigenetic programming and inheritance, and hold great promise to assist in epigenetic remodeling of the donor nucleus. Such strategies are likely to improve the odds for successful cloning.

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“…Successful cloning using mammalian somatic cell indicates that epigenetic modifications in the differentiated nucleus can be remodeled to a totipotent state [2]. The first living cloned piglet obtained by nuclear transfer of adult granulosa cells was reported in 2000 [4], and subsequently, additional porcine clones have been obtained [5][6][7].…”

Section: Introductionmentioning

confidence: 99%

“…Embryo aggregation consists of placing more than one zona free embryos in the same well; some of the reported benefits in mammals are: improvement in embryo development in pig [6], equine [8,9], feline [10] and mouse [11], increasing the density of cells in the inner cell mass on cattle [12], blastocyst diameter in equine and in bovine [8,13], reduction in apoptosis in bovine and pigs [13,14,15], normalization of gene expression in bovine [15]and in some cases, it has also improved in vivo embryo development and early pregnancy rates in mouse [11], equine [8] and in cattle [13,16,17]. As a single clone may have epigenetic defects [15], embryo aggregation could compensate the reprogramming deficiencies through the interactions between blastomeres [11] or by paracrine pathways [11].…”

Section: Introductionmentioning

confidence: 99%

18 Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality

Buemo

Gambini

Moro

et al. 2016

Reprod. Fertil. Dev.

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In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

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Treating Cloned Embryos, But Not Donor Cells, with 5-aza-2’-deoxycytidine Enhances the Developmental Competence of Porcine Cloned Embryos

Cited by 40 publications

References 31 publications

Histone H3 lysine 27 trimethylation acts as an epigenetic barrier in porcine nuclear reprogramming

Histone H3 lysine 27 trimethylation acts as an epigenetic barrier in porcine nuclear reprogramming

Probability, odds and random chance: the difficult task of modulating the epigenetic profile of cloned embryos

18 Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality

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