Functional study of distinct domains of dux in improving mouse SCNT embryonic development

2021 ◽  
Author(s):  
Xingwei Huang ◽  
Xinglin Hu ◽  
Qi Jiang ◽  
Qianzi Cao ◽  
Yanshuang Wu ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Critical Factor ◽  
Preimplantation Development ◽  
Functional Study ◽  
Intermediate Region ◽  
Cell Stage ◽  
Terminal Domain ◽  
Reprogramming Efficiency ◽  
And Function

Abstract 2-cell-like (2C-like) embryonic stem cells (ESCs) are a small group of ESCs that spontaneously express zygotic genomic activation (ZGA) genes and repeats, such as Zscan4 and MERVL, and are specifically expressed in 2-cell-stage mouse embryos. Although numerous types of treatment and agents elevate the transition of ESCs to 2C-like ESCs, Dux serves as a critical factor in this transition by increasing the expression of Zscan4 and MERVL directly. However, the loss of Dux did not impair the birth of mice, suggesting that Dux may not be the primary transitioning factor in fertilized embryos. It has been reported that for 2-cell embryos derived from somatic cell nuclear transfer (SCNT) and whose expression of ZGA genes and repeats was aberrant, Dux improved the reprogramming efficiency by correcting aberrant H3K9ac modification via its C-terminal domain. We confirmed that overexpression of full-length Dux mRNA in SCNT embryos improved the efficiency of preimplantation development (62.16% vs. 41.26% with respect to controls) and also increased the expression of Zscan4 and MERVL. Furthermore, we found that the N-terminal double homeodomains of Dux were indispensable for Dux localization and function. The intermediate region was essential for MERVL and Zscan4 activation, and the C-terminal domain was important for elevating level of H3K27ac. Mutant Dux mRNA containing N-terminal double homeodomains with the intermediate region or the C-terminal domain also improved the preimplantation development of SCNT embryos. This is the first report focusing on distinguishing functional domains of Dux in embryos derived from SCNT.

47 DIFFERENTIAL DEVELOPMENTAL ABILITY OF EMBRYOS CLONED FROM TISSUE-SPECIFIC STEM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 142
Author(s):  
K. Inoue ◽  
N. Ogonuki ◽  
H. Miki ◽  
S. Noda ◽  
S. Inoue ◽  
...  
Keyword(s):  
Stem Cells ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Donor Cell ◽  
Fibroblast Cell ◽  
High Rate ◽  
Full Potential ◽  
Cell Nuclei ◽  
Cell Stage

Although cloning animals by somatic cell nuclear transfer is generally an inefficient process, use of appropriate donor cell types may improve the cloning outcome significantly. Among the donor cells tested so far, mouse embryonic stem cells have given the best efficiency in terms of the development of reconstructed embryos into offspring. In this study, we examined whether 2 in vitro-produced pluripotent stem cells—neural stem cells (NSCs) and mesenchymal stem cells (MSCs)—could be better nuclear donors than other differentiated cells. Embryos were reconstructed by transfer of nuclei from NSCs or MSCs with full potential for differentiation in vitro. Most (76%) of the 2-cell NCS embryos developed to the 4-cell stage; 43% implanted and 1.6% developed to term after transfer to pseudopregnant recipients. These rates were very similar to those of embryos cloned from fibroblast cell nuclei. Interestingly, in the patterns of zygotic gene expression, NSC embryos were more similar to in vitro-fertilized embryos than fibroblast cloned embryos. By contrast, embryos reconstructed using MSC nuclei showed lower developmental ability and no implantation was obtained after embryo transfer. Chromosomal analysis of the donor MSCs revealed very high frequencies of monosomy and trisomy, which might have caused the very poor post-implantation development of embryos following nuclear transfer. Thus, in vitro-produced pluripotent cells can serve as donors of nuclei for cloning mice, but may be prone to chromosomal aberrations leading to a high rate of cloned embryo death.

65 BLASTOCYSTS DERIVED FROM ADULT FIBROBLASTS OF RHESUS MONKEY (MACACA MULATTA) USING INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER

2010 ◽  
Vol 22 (1) ◽  
pp. 191
Author(s):  
D. K. Kwon ◽  
J. T. Kang ◽  
S. J. Park ◽  
M. N. L. Gomez ◽  
S. J. Kim ◽  
...  
Keyword(s):  
Rhesus Monkey ◽  
Somatic Cell ◽  
Macaca Mulatta ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Developmental Rate ◽  
Cell Stage ◽  
Nuclear Number

Interspecies somatic cell nuclear transfer (iSCNT) has alternatively chosen in primate SCNT because of the difficulty in collecting enough oocytes for research. The purpose of this experiment is to produce iSCNT-derived blastocysts using enucleated cow (Bos taurus) metaphase II oocytes and adult rhesus monkey (Macaca mulatta) fibroblasts. Ear skin tissueofrhesus monkey (male, 6 years old) was collected by biopsy and fibroblasts were isolated. Immature COCs from cow ovaries were collected and matured in vitro in TCM-199. Squish enucleation was done in the presence of bisbenzimide and cytochalasin B. After enucleation, a single rhesus monkey somatic cell was injected into the perivitelline space of an enucleated oocyte through the slit in the zona pellucida made during enucleation. Subsequently, the rhesus monkey somatic cell and cow oocyte membranes were electrically fused. The nonactivated interspecies cloned couplets were cultured for 2 h to allow reprogramming to occur. Then, couplets were activated using a 2-step protocol consisting of treatment with 5 μM ionomycin for 4 to 5 min and subsequently with 2mM 6-DMAP for 4 h. Activated iSCNT embryos were cultured for 10 days inmodified SOF with various conditions (at 37 to39°C, 5 to 5.5% CO2 and 5 to 20% O2) to examine the effects ofIVC conditions. As a results, most embryos were arrested at the 8- to 16-cell stage and only 3 blastocysts were derived from rhesus monkey iSCNT. The blastocyst developmental rate was 0.26% generated from the total IVC activated interspecies embryos (n = 1153). Among the 3 blastocysts, 2 of them were used for counting nuclear number using bisbenzimide staining. The nuclear number of the 2 iSCNT-derived blastocysts was 51 and 24, respectively. The other iSCNT-derived blastocyst was used for analyzing mitochondrial (mt)DNAto confirm that it contained both cow and rhesus monkey mtDNA. As a result, mtDNA from both rhesus monkey and cow were detected inPCR analysis. The band intensity was more dominant for cow mtDNA than for rhesus monkey mtDNA. Although the blastocyst developmental rate is extremely low, it is confirmed that two phylogenetically distant species including primate could develop in vitro until the blastocyst stage by iSCNT. The in vitro developmental system of this rhesus monkey iSCNT-derived blastocysts provides a platform for further improvement of developmental rate and quality of rhesus monkey iSCNT-derived blastocysts. It also provides an opportunity to establish rhesus monkey iSCNT-derived embryonic stem cell lines for study of rhesus monkey nucleus and cow mitochondria interaction mechanisms during early developmental stages. This study was financially supported by the Korean MEST, through the BK21 program for Veterinary Science, and SNU foundation (Benefactor; RNL Bio).

57 GENOME ACTIVATION IN MOUSE EMBRYOS OF DIFFERENT ORIGIN

2008 ◽  
Vol 20 (1) ◽  
pp. 109
Author(s):  
O. Svarcova ◽  
A. Dinnyes ◽  
Z. Polgar ◽  
S. Bodo ◽  
M. Adorjan ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Mouse Embryos ◽  
Ultrastructural Changes ◽  
Rrna Synthesis ◽  
Cell Stage ◽  
Stage Of Development ◽  
Nucleolar Proteins ◽  
Genome Activation ◽  
Different Origin

Major genome activation is a key event in early embryonic development occurring at the late 2-cell stage in the mouse. Concomitantly occurring molecular and ultrastructural changes in the nucleolus, where the ribosomal RNA genes are transcribed and their transcripts processed, enable the use of this organelle as a sensitive marker of genome activation in embryos produced by different techniques. The aim of this study was to evaluate and compare the genome activation in mouse embryos of different origin using the nucleolus as a marker. Early and late 2-cell- and late 4-cell-stage embryos, prepared by in vitro fertilization (IVF), parthenogenetic activation (PG), and somatic cell nuclear transfer of mouse embryonic fibroblast (MEF), and mouse HM1 embryonic stem cells (HM1) were processed for autoradiography following 3H-uridine incubation and transmission electron microscopy (5 embryos per group) and for immunofluorescence for detection of nucleolar proteins involved in rRNA synthesis (upstream binding factor; UBF) and processing (nucleophosmin; B23) (10–21 embryos per group). Early 2-cell embryos in all groups showed transcriptional activity in the nucleoplasm, but not over nucleolar precursor bodies (NPBs). UBF was localized diffusely in the cytoplasm. B23 was, likewise, localized in the cytoplasm and, in 30% of embryos, in the nucleoplasm. Late 2-cell IVF and PG embryos displayed transcriptional labelling over nucleoplasm and NPBs, which, ultrastructurally, were in the process of transformation into fibrillo-granular nucleoli presenting fibrillar centers, a dense fibrillar component, and a granular component. MEF and HM1 embryos displayed transcriptional labelling over nucleoplasm, but not over NPBs, and the transformation into functional nucleoli was never observed at this stage of development. UBF and B23 were in all groups localized in the nucleoplasm and, in 40–50% of cases, distinctly in the developing nucleoli. At the late 4-cell stage, all embryos presented transcriptional labelling over nucleoplasm and NPBs, which were at different levels of transformation into fibrillo-granular nucleoli. UBF and B23 were distinctly localized in these developing nucleoli. However, whereas fully transformed reticulated fibrillo-granular nucleoli without remnants of NPBs were found in IVF and PG embryos, despite the distinct localization of nucleolar proteins, the nucleoli in MEF and HM1 embryos were not reticulated and still displayed remnants of NPBs. Conclusively, embryos reconstructed by nuclear transfer, independent of cell origin, displayed well-timed extranucleolar genomic activation, but delayed transformation of NPBs into reticulated fibrillo-granular nucleoli. Moreover, the proper nucleolar activation noted in PG embryos activated in the same manner as MEF and HM1 embryos demonstrate that somatic and embryonic stem cell factors exert an influence on nucleolar activation and may cause reduced embryo viability. This work was supported by the Specific Targeted Project (MED-RAT; contract LSHG-CT-2006-518240) and Marie Curie ResearchTraining Networks (CLONET; contract 035468-2).

Blastocysts derived from adult fibroblasts of a rhesus monkey (Macaca mulatta) using interspecies somatic cell nuclear transfer

Zygote ◽  
2011 ◽  
Vol 19 (3) ◽  
pp. 199-204 ◽  
Author(s):  
Dae Kee Kwon ◽  
Jung Taek Kang ◽  
Sol Ji Park ◽  
Ma Ninia Limas Gomez ◽  
Su Jin Kim ◽  
...  
Keyword(s):  
Rhesus Monkey ◽  
Somatic Cell ◽  
Macaca Mulatta ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Development Rate ◽  
Attractive Alternative ◽  
Cell Stage

SummaryIn non-human primates, it is difficult to collect sufficient numbers of oocytes for producing identical embryos by somatic cell nuclear transfer (SCNT). Because of this factor, inter-species SCNT (iSCNT) using heterospecific oocytes is an attractive alternative approach. The objective of this study was to produce iSCNT-derived blastocysts using enucleated cow (Bos taurus) metaphase II oocytes and adult rhesus monkey (Macaca mulatta) fibroblasts. Ear skin tissue from a 6-year-old male rhesus monkey was collected by biopsy and fibroblasts were isolated. Immature cumulus–oocyte complexes from cow ovaries were collected and matured in vitro in Medium 199. The enucleated oocytes were reconstructed with rhesus monkey fibroblasts and iSCNT embryos were cultured in modified synthetic oviduct fluid in an atmosphere of 5–5.5% CO2 under various conditions (37–39 °C and 5–20% O2) to examine the effects of in vitro culture conditions. Most embryos were arrested at the 8- or 16-cell stage and only three blastocysts were derived in this way using iSCNT from a total of 1153 cultured activated embryos (0.26% production rate). Two of the three blastocysts were used for counting nuclear numbers using bisbenzimide staining, which were 51 and 24. The other iSCNT-derived blastocyst was used to analyse mitochondrial DNA (mtDNA) by PCR, and both rhesus monkey and cow mtDNA were detected. Although the development rate was extremely low, this study established that iSCNT using two phylogenetically distant species, including a primate, could produce blastocysts. With improvements in the development rate, it may be possible to produce rhesus monkey iSCNT-derived embryonic stem cell lines for studies on primate nucleus and cow mitochondria interaction mechanisms.

183 Generation of porcine embryonic stem cell lines derived from nuclear transfer embryos reconstructed with induced pluripotent stem cells

2019 ◽  
Vol 31 (1) ◽  
pp. 216
Author(s):  
S. Haraguchi ◽  
T. Q. Dang-Nguyen ◽  
D. Wells ◽  
D. Fuchimoto ◽  
T. Fukuda ◽  
...  
Keyword(s):  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Feeder Cells ◽  
Es Cell ◽  
Cell Stage ◽  
Reprogramming Factors ◽  
Induced Pluripotent

To establish a porcine embryonic stem (ES) cell line that not only maintains self-renewing capacity but also exhibits pluripotency [Haraguchi et al. 2012 J. Reprod. Dev. 58, 707-716], 6 synthetic porcine RNAs (Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28) were chemically transfected into outgrowth cultured cells derived from the inner cell mass of in vitro-produced porcine embryos. Subsequently, cells grew as compact, dome-shaped colonies displaying alkaline phosphatase activity and were cultured for more than 20 passages. Although 13 candidate cell lines were generated (13/43, 30%), none formed teratomas after injection of the cells into SCID (sever combined immunodeficiency) mice. We also observed that when transfection of the exogeneous RNAs was discontinued, the cells no longer maintained a stem cell morphology and began to differentiate (13/13, 100%). This suggests that continuous expression of exogenous reprogramming factors is necessary to maintain induced pluripotency in the pig. Next, we used cloned embryos reconstructed with porcine induced pluripotent stem cells (piPSC), which were created using a recombinant lentivirus expression vector carrying 6 mouse reprogramming factor genes (the same as above) and green fluorescent protein (GFP) (Fukuda et al. 2017 J. Cell Biochem. 118, 537-553]. The piPSC were dispersed to a single cell suspension and electrically fused to cytoplasts prepared following enucleation of in vitro-matured zona-free metaphase II-arrested oocytes. A second cytoplast was then fused to the first reconstruct (double cytoplast nuclear transfer). Reconstructs were electrically activated and cultured in microwells with porcine zygote medium-3 (PZM3). After 5 days, reconstructed embryos developed to GFP-positive blastocysts (10/93, 11%) and 4- to 8-cell stages (25/93, 27%). The blastocysts (10) and 4- to 8-cell-stage embryos (25) were transferred onto mouse embryonic fibroblast feeder cells for outgrowth culture in FCS-based ES cell medium supplemented with 2% polyvinylpyrrolidone. After 24h, the medium was changed to piPSC medium containing CHIR99021, PD0325901, thiazovivin, and GF-109203x. Embryos attached to the feeder cells began to outgrow (8/10 of blastocysts and 6/25 of 4- to 8-cell-stage embryos). To date, 3 ES-like cell lines have been established from blastomeres of embryos (3/25, 12%) but not from blastocysts (0/10, 0%). They show GFP fluorescence and have been maintained continuously in culture for more than 20 passages without any overt changes in morphology. These results suggest that the constant expression of reprogramming factors and the use of combinations of specific small molecule inhibitors largely contribute to the establishment of pluripotent cells in the pig. Further characterisation of the cells is ongoing, including methylation status of the X chromosome and the capacity for in vivo differentiation.

Analysis of transcription factor expression during oogenesis and preimplantation development in mice

Zygote ◽  
2007 ◽  
Vol 15 (2) ◽  
pp. 117-128 ◽  
Author(s):  
S. Kageyama ◽  
W. Gunji ◽  
M. Nakasato ◽  
Y. Murakami ◽  
M. Nagata ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Germ Cell ◽  
Expression Patterns ◽  
Preimplantation Development ◽  
Global Changes ◽  
Regulation Of Transcription ◽  
Cell Stage ◽  
High Level ◽  
And Function

SummaryThe transition from a differentiated germ cell into a totipotent zygote during oogenesis and preimplantation development is critical to the creation of a new organism. During this period, cell characteristics change dynamically, suggesting that a global alteration of gene expression patterns occurs, which is regulated by global changes in various epigenetic factors. Among these, transcription factors (TFs) are essential in the direct regulation of transcription and also play important roles in determining cell characteristics. However, no comprehensive analysis of TFs from germ cells to embryos had been undertaken. We used mRNA amplification systems and microarrays to conduct a genomewide analysis of TFs at various stages of oogenesis and preimplantation development. The greatest alteration in TFs occurred between the 1- and 2-cell stages, at which time zygotic genome activation (ZGA) occurs. Our analysis of TFs classified by structure and function revealed several specific patterns of change. Basic transcription factors, which are the general components of transcription, increased transiently at the 2-cell stage, while homeodomain (HD) TFs were expressed specifically in the oocyte. TFs containing the Rel homology region (RHR) and Ets domains were expressed at a high level in 2-cell and blastocyst embryos. Thus, the global TF dynamics that occur during oogenesis and preimplantation development seem to regulate the transition from germ-cell-type to embryo-type gene expression.

scholarly journals Expression and function of FGF-4 in peri-implantation development in mouse embryos

Development ◽  
1994 ◽  
Vol 120 (8) ◽  
pp. 2259-2269 ◽  
Author(s):  
D.A. Rappolee ◽  
C. Basilico ◽  
Y. Patel ◽  
Z. Werb
Keyword(s):  
Embryonal Carcinoma ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Carcinoma Cells ◽  
Embryonal Carcinoma Cells ◽  
Cell Stage ◽  
And Function

One of the earliest events in mammalian embryogenesis is the formation of the inner cell mass (ICM) and the subsequent delamination of primitive endoderm. We have found that mRNA for fibroblast growth factor (FGF)-4, but not FGF-3, is expressed in preimplantation mouse blastocysts and that the FGF-4 polypeptide is present in ICM cells. ICM-like embryonal carcinoma cells and embryonic stem cells also express FGF-4. Conversely, differentiated embryonal carcinoma cells in the endoderm lineage express FGF-3, but not FGF-4 mRNA. Although mouse embryos expressed FGF-4 mRNA from the 1-cell stage, embryos cultured from the 2-cell through the blastocyst stage in the presence of recombinant FGF-4 did not respond mitogenically. However, when ICMs that were isolated by immunosurgery were cultured with FGF-4, the number of morphologically distinct, differentiated parietal endoderm cells growing out onto the coverslip increased, without an increase in the number of undifferentiated ICM cells. ICM outgrowths cultured with FGF-4 increased their secretion of 92 × 10(3) M(r) gelatinase and tissue plasminogen activator, a hallmark of migrating cells. Receptors for FGF-4 (FGFR-3 and FGFR-4) are expressed in all cells of the mouse blastocyst. These findings indicate that FGF-4 produced by undifferentiated ICM cells acts in the peri-implantation period of embryogenesis to influence the production and behavior of endoderm cells derived from them.

Expression and function of transcription factor AP-2γ in early embryonic development of porcine parthenotes

2016 ◽  
Vol 28 (8) ◽  
pp. 1197 ◽  
Author(s):  
Sung-Hyun Lee ◽  
Jung-Woo Kwon ◽  
Inchul Choi ◽  
Nam-Hyung Kim
Keyword(s):  
Transcription Factor ◽  
De Novo ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Preimplantation Development ◽  
Binding Motif ◽  
Cell Stage ◽  
Preimplantation Embryo Development ◽  
Species Specific ◽  
And Function

Transcription factor AP-2γ (TFAP2C) is a member of the transcription factor activating enhancer binding protein (AP) family. In the present study we determined the temporal and spatial expression patterns of TFAP2C in porcine parthenotes during preimplantation development. Porcine TFAP2C transcripts were expressed at all stages of preimplantation development, with highest expression at the 8-cell stage. In contrast with the mouse, TFAP2C protein was not restricted to the trophectoderm and was also detected in the ICM in blastocyst stage porcine parthenotes. In knockdown (KD) experiments, most TFAP2C-depleted embryos were arrested before the compacted 8-cell stage. This developmental failure is attributed to abnormal expression of genes involved in cell adhesion, tight junction biogenesis and cell proliferation. Interestingly, although the conserved region 4 (CR4) of the porcine OCT4 5′ upstream regionlacked the AP2C-binding motif, OCT4 transcript levels were elevated in porcine TFAP2C-KD 8-cell embryos, suggesting TFAP2C may be involved in the regulation of OCT4 in porcine embryos through other mechanisms. In summary, the results suggest that TFAP2C is necessary for the transition from de novo transcript synthesis by activation to compaction and further development, and the different expression patterns of TFAP2C in porcine embryos may reflect species-specific functions during preimplantation embryo development.

The development and expression of pluripotency genes in embryos derived from nuclear transfer and in vitro fertilization

Zygote ◽  
2013 ◽  
Vol 22 (4) ◽  
pp. 540-548 ◽  
Author(s):  
Li-Bing Ma ◽  
Xiao-Ying He ◽  
Feng-Mei Wang ◽  
Jun-Wei Cao ◽  
Teng Cheng
Keyword(s):  
Nuclear Transfer ◽  
Developmental Stages ◽  
Es Cells ◽  
Embryonic Stem ◽  
Developmental Rate ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Pluripotency Genes

SummarySomatic cell nuclear transfer can be used to produce embryonic stem (ES) cells, cloned animals, and can even increase the population size of endangered animals. However, the application of this technique is limited by the low developmental rate of cloned embryos, a situation that may result from abnormal expression of some zygotic genes. In this study, sheep–sheep intra-species cloned embryos, goat–sheep inter-species cloned embryos, or sheep in vitro fertilized embryos were constructed and cultured in vitro and the developmental ability and expression of three pluripotency genes, SSEA-1, Nanog and Oct4, were examined. The results showed firstly that the developmental ability of in vitro fertilized embryos was significantly higher than that of cloned embryos. In addition, the percentage of intra-species cloned embryos that developed to morula or blastocyst stages was also significantly higher than that of the inter-species cloned embryos. Secondly, all three types of embryos expressed SSEA-1 at the 8-cell and morula stages. At the 8-cell stage, a higher percentage of in vitro fertilized embryos expressed SSEA-1 than occurred for cloned embryos. However, at the morula stage, all detected embryos could express SSEA-1. Thirdly, the three types of embryos expressed Oct4 mRNA at the morula and blastocyst stages, and embryos at the blastocyst stage expressed Nanog mRNA. The rate of expression of Oct4 and Nanog mRNA at these developmental stages was higher in in vitro fertilized embryos than in cloned embryos. These results indicated that, during early development, the failure to reactivate some pluripotency genes maybe is a reason for the low cloning efficiency found with cloned embryos.

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