54 EXPRESSION PATTERN OF DNMT1 AND DNMT3a GENES DURING INTERGENERIC SOMATIC CELL NUCLEAR TRANSFER EMBRYO DEVELOPMENT

2013 ◽  
Vol 25 (1) ◽  
pp. 174
Author(s):  
M. Morovic ◽  
F. Strejcek ◽  
O. Ostrup ◽  
A. Lucas-Hahn ◽  
B. Petersen ◽  
...  
Keyword(s):  
Expression Pattern ◽  
Nuclear Transfer ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Expression Patterns ◽  
Preimplantation Embryos ◽  
Specific Primers ◽  
Cell Stage ◽  
Nuclear Transfer Embryo ◽  
Species Specific

One of the most-discussed reasons for developmental incompetence of embryos constructed by the cloning procedure is inadequate reprogramming of the transferred nucleus to a state equivalent to that of an early embryonic nucleus. Previous studies have shown species-dependent expression patterns of DNA methyltransferase (DNMT) genes in mammalian oocytes and preimplantation embryos, and also a correlation between incomplete DNA methylation and the lack of NT success in mammals. In the present study, the expression pattern of DNMT1 and DNTM3a genes at the 2-cell and 4-cell stages of bovine versus porcine intergeneric nuclear transfer (iSCNT) embryos was observed by reverse-transcriptase (RT) PCR. All pools were done in triplicate and contained 10 iSCNT embryos. The species-specific primers for DNMT1 and DNMT3a genes were designed for determination of de novo synthesis of epigenetic enzymes. As positive controls, porcine and bovine parthenogenetic embryos were used. Gene transcription for bovine DNMT1 (bDNMT1) and DNMT3a (bDNMT3a) was not observed in 2- and 4-cell stage embryos generated by bovine fibroblast transfer into the porcine ooplasm; however, using primers for pig DNMT1 (pDNMT1) and DNMT3a (pDNMT3a), positive results were obtained. In the 2- and 4-cell-stage embryos constructed using porcine fibroblast and bovine ooplasm, only the bovine-specific primers showed positive signals. Based on the different timing of major genome activation during embryonic development in bovine and porcine embryos, the strong influence of ooplasm on introduced fibroblast was expected. Despite the mRNA presence of DNMT1 and DNMT3a enzymes of oocyte origin, de novo transcription of somatic DNMT1 and DNMT3a genes was not detected and iSCNT embryos did not develop beyond the 4-cell stage. These results strongly suggest species-specific and maternally driven regulation of epigenetic reprogramming during early embryogenesis. This work was supported by VEGA 1/0077/11.

scholarly journals The Influence of Interspecies Somatic Cell Nuclear Transfer on Epigenetic Enzymes Transcription in Early Embryos

2016 ◽  
Vol 39 (2) ◽  
pp. 209-217 ◽  
Author(s):  
Martin Morovic ◽  
Matej Murin ◽  
Frantisek Strejcek ◽  
Michal Benc ◽  
Dusan Paál ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Coming Out ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Dna Methyltransferase ◽  
Dna Methyltransferases ◽  
Preimplantation Embryos ◽  
Mammalian Development ◽  
Cell Stage ◽  
Early Embryos

AbstractOne of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription in oocytes and early embryos of several species including bovine and porcine zygotes is species-dependent process and the incomplete DNA methylation correlates with the nuclear transfer failure rate in mammals. In this study the transcription of DNA methyltransferase 1 and 3a (DNMT1, DNMT3a) genes in early embryonic stages of interspecies (bovine, porcine) nuclear transfer embryos (iSCNT) by RT-PCR were analyzed. Coming out from the diverse timing of embryonic genome activation (EGA) in porcine and bovine preimplantation embryos, the intense effect of ooplasm on transferred somatic cell nucleus was expected. In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly influenced by the ooplasmic environment.

scholarly journals 25 MATERNAL-TO-EMBRYONIC TRANSITION FOLLOWING NUCLEAR TRANSFER OR PARTHENOGENETIC ACTIVATION

2006 ◽  
Vol 18 (2) ◽  
pp. 121
Author(s):  
T. Brevini ◽  
S. Antonini ◽  
F. Cillo ◽  
I. Lagutina ◽  
S. Colleoni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Nuclear Transfer ◽  
De Novo ◽  
Gene Expression Pattern ◽  
Mrna Levels ◽  
Cell Stage ◽  
Specific Expression ◽  
Parthenogenetic Activation

The successful development of embryos generated by somatic cell nuclear transfer (SCNT) requires the ooplasm to reprogram the nucleus. This establishes the gene expression pattern necessary for full development by mechanisms that are currently being clarified. The ooplasm action on somatic nuclei shows many common aspects to the process that leads to the creation of a functional embryonic genome from the differentiated sperm and egg genomes. In order to investigate this aspect we studied a critical phase of early embryonic development: the maternal to embryonic transition (MET). We compared the pattern and level of gene expression between bovine embryos derived from in vitro fertilization (IVF), from nuclear transfer of adult fibroblasts (NT), or from parthenogenetic activation (PG). The study was performed in cattle because MET, in this species, occurs over four cell cycles, making it easier to detect even small deviations. Oocytes, matured for 22 h and fertilized in vitro or after cumulus removal, were enucleated and fused to fibroblast cells. Nuclear transfer and Met II oocytes were activated at 24-26 h of maturation with ionomycin (5 �M) for 5 min and 6DMAP (2 mM) for 4 h and then cultured in mSOFaa. Embryos were harvested at the required time for analysis at the 2-, 4-, 8-, and 16-cell; morula; and blastocyst stages and stored snap-frozen in a minimal volume of medium in groups of 5-10 embryos. Semiquantitative RT-PCR was used to study the expression of Nanog, Oct-4, Zar-1, and Par-3, because these genes are directly involved in early embryo development and have a specific expression pattern during MET. Data were analyzed with one-way ANOVA followed by Student-Newman-Keuls All Pairwise Multiple Comparison. No difference in pre-implantation development was observed among the three groups. The Nanog expression pattern was unchanged in all three groups, becoming detectable from the 8-16-cell stage onward. Oct-4 mRNA was detected at all stages in every group, but only in NT embryos did a significant increase occur at the 16-cell stage, suggesting the onset of an anticipated embryonic transcription. the Zar-1 expression pattern, with the characteristic de-novo transcription peak at the 4-cell stage, was observed in both IVF and NT embryos but not in PG embryos. In this group, Zar-1 mRNA levels were significantly higher at the 2- and 4-cell stage than in all of the following stages. The Par-3 gene showed the biggest differences among groups: IVF embryos expressed this gene from the 8-cell stage onward, whereas NT embryos showed high levels of Par-3 mRNA already at the 2-cell stage. Surprisingly, PG embryos showed no detectable Par-3 levels at any stages. The results indicate that, although in vitro development was not affected, gene-specific expression differences during MET occurred among groups. Relating the specific functions exerted by each of these genes in early development to the changes observed following the different manipulations provides useful data toward a better understanding of the role of these genes and of the mechanisms of nuclear reprogramming. This work was supported by FIRB RBNE01HPMX, FIRST 2004, and ESF-EuroStells.

30 INHIBITION OF DNA METHYLTRANSFERASE 1 EXPRESSION IN BOVINE FIBROBLAST CELLS FOR NUCLEAR TRANSFER

2008 ◽  
Vol 20 (1) ◽  
pp. 95
Author(s):  
A. M. Giraldo ◽  
J. W. Lynn ◽  
M. N. Purpera ◽  
R. A. Godke ◽  
K. R. Bondioli
Keyword(s):  
Nuclear Transfer ◽  
Dna Methyltransferase ◽  
Transfection Efficiency ◽  
Expression Patterns ◽  
Preimplantation Embryos ◽  
Dna Methyltransferase 1 ◽  
Aberrant Expression ◽  
Transfection Reagent ◽  
Fetal Fibroblasts ◽  
Methyltransferase 1

The aberrant expression of DNA methyltransferase 1 (DNMT1) in cloned embryos has been implicated as a possible factor in the improper donor genome reprogramming during nuclear transfer (NT). DNMT1 is responsible for maintaining DNA methylation and the subsequent differentiation status of somatic cells. NT utilizing cell fusion introduces the somatic form of DNMT1 (DNMT1s), which is not normally present in preimplantation embryos and could perpetuate the somatic-like methylation patterns observed in early cloned embryos. The objective of this study was to decrease the level of DNMT1s in bovine fibroblasts using siRNA, prior to their use as donor cells. Fetal fibroblasts were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in 5% CO2 at 37�C. The transfection efficiency of different ratios of siRNA concentrations and transfection reagent (µg:µL; 1:3, 1:6, and 1:9) as well as siRNA concentrations (0.5, 1.0, and 1.5 µg) were determined using fluorescein isothiocyanate (FITC)-labeled control siRNA and fluorescence analyzed by flow cytometry. A DNMT1s-specific siRNA was used to transfect cells at 50 and 80% of confluence. A non-silencing siRNA was used as a negative control. The expression patterns of DNMT1s were characterized by Q-PCR using the δδCT method. ANOVA was used to detect differences in transfection efficiency and gene expression. The combination of 1.0 or 1.5 µg siRNA and a 1:6 siRNA to transfection reagent ratio produced the highest transient transfection rates without affecting cell viability. Cells treated at 50% confluence with 1.5 µg of DNMT1s-specific siRNA at a 1:6 ratio showed 80% less DNMT1s mRNA than cells treated with non-silencing siRNA. At 8 h post-transfection (PT) these cells displayed vacuole-like structures within the cytoplasm, stopped dividing, and died approximately 24 h PT. Cells treated at 80% confluence with 1.0 µg DNMT1s-specific siRNA at a 1:6 ratio resulted in 57, 66, 24, 50, 22, 62, 64, and 56% less DNMT1s than control cells at 4, 6, 8, 10, 12, 24, 48, and 72 h PT, respectively, but without the cytotoxic effects observed when cells at 50% of confluence were treated under the same transfection conditions. These data indicate that optimization of cell density, siRNA concentration, and the ratio between siRNA concentration and transfection reagent are required to decrease the levels of DNMT1s without causing deleterious effects to the cells. However, levels of DNMT1s mRNA can be effectively reduced using siRNA; protein analysis is required to determine if reduction of the transcript results in lower levels of the protein. Subsequent use of these cells for NT will provide insight as to how the presence of this enzyme affects reprogramming in early cloned embryos. This study was supported by Louisiana State University Board of Regents.

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.

Differential and overlapping expression patterns of X-dll3 and Pax-6 genes suggest distinct roles in olfactory system development of the African clawed frog Xenopus laevis

2001 ◽  
Vol 204 (12) ◽  
pp. 2049-2061 ◽  
Author(s):  
Marie-Dominique Franco ◽  
Michael P. Pape ◽  
Jennifer J. Swiergiel ◽  
Gail D. Burd
Keyword(s):  
Xenopus Laevis ◽  
Expression Pattern ◽  
Olfactory Epithelium ◽  
Olfactory System ◽  
De Novo ◽  
System Development ◽  
Expression Patterns ◽  
Basal Cells ◽  
Olfactory Placode ◽  
Water Borne

SUMMARY In Xenopus laevis, the formation of the adult olfactory epithelium involves embryonic, larval and metamorphic phases. The olfactory epithelium in the principal cavity (PC) develops during embryogenesis from the olfactory placode and is thought to respond to water-borne odorants throughout larval life. During metamorphosis, the PC undergoes major transformations and is exposed to air-borne odorants. Also during metamorphosis, the middle cavity (MC) develops de novo. The olfactory epithelium in the MC has the same characteristics as that in the larval PC and is thought to respond to water-borne odorants. Using in situ hybridization, we analyzed the expression pattern of the homeobox genes X-dll3 and Pax-6 within the developing olfactory system. Early in development, X-dll3 is expressed in both the neuronal and non-neuronal ectoderm of the sense plate and in all cell layers of the olfactory placode and larval PC. Expression becomes restricted to the neurons and basal cells of the PC by mid-metamorphosis. During metamorphosis, X-dll3 is also expressed throughout the developing MC epithelium and becomes restricted to neurons and basal cells at metamorphic climax. This expression pattern suggests that X-dll3 is first involved in the patterning and genesis of all cells forming the olfactory tissue and is then involved in neurogenesis or neuronal maturation in putative water- and air-sensing epithelia. In contrast, Pax-6 expression is restricted to the olfactory placode, larval PC and metamorphic MC, suggesting that Pax-6 is specifically involved in the formation of water-sensing epithelium. The expression patterns suggest that X-dll3 and Pax-6 are both involved in establishing the olfactory placode during embryonic development, but subtle differences in cellular and temporal expression patterns suggest that these genes have distinct functions.

scholarly journals Formation of nucleoli in interspecies nuclear transfer embryos derived from bovine, porcine, and rabbit oocytes and nuclear donor cells of various species

Reproduction ◽  
2011 ◽  
Vol 141 (4) ◽  
pp. 453-465 ◽  
Author(s):  
Irina Lagutina ◽  
Valeri Zakhartchenko ◽  
Helena Fulka ◽  
Silvia Colleoni ◽  
Eckhard Wolf ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
De Novo ◽  
Mammalian Species ◽  
Housekeeping Genes ◽  
Cell Stage ◽  
Whole Process ◽  
Embryonic Genome ◽  
Donor Nucleus ◽  
Donor Cells ◽  
Genome Activation

The most successful development of interspecies somatic cell nuclear transfer (iSCNT) embryos has been achieved in closely related species. The analyses of embryonic gene activity in iSCNT embryos of different species combinations have revealed the existence of significant aberrations in expression of housekeeping genes and genes dependent on the major embryonic genome activation (EGA). However, there are many studies with successful blastocyst (BL) development of iSCNT embryos derived from donor cells and oocytes of animal species with distant taxonomical relations (inter-family/inter-class) that should indicate proper EGA at least in terms of RNA polymerase I activation, nucleoli formation, and activation of genes engaged in morula and BL formation. We investigated the ability of bovine, porcine, and rabbit oocytes to activate embryonic nucleoli formation in the nuclei of somatic cells of different mammalian species. In iSCNT embryos, nucleoli precursor bodies originate from the oocyte, while most proteins engaged in the formation of mature nucleoli should be transcribed from genes de novo in the donor nucleus at the time of EGA. Thus, the success of nucleoli formation depends on species compatibility of many components of this complex process. We demonstrate that the time and cell stage of nucleoli formation are under the control of recipient ooplasm. Oocytes of the studied species possess different abilities to support nucleoli formation. Formation of nucleoli, which is a complex but small part of the whole process of EGA, is essential but not absolutely sufficient for the development of iSCNT embryos to the morula and BL stages.

180 IDENTIFICATION AND QUANTIFICATION OF DIFFERENTIALLY REPRESENTED TRANSCRIPTS IN PREIMPLANTATION BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 169 ◽  
Author(s):  
C. E. McHughes ◽  
G. K. Springer ◽  
L. D. Spate ◽  
R. Li ◽  
R. J. Woods ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Nuclear Transfer ◽  
Developmental Stages ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Production Techniques

Identification of transcripts that are present at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. To that end, this project had two goals. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, including metaphase II-stage oocytes (MPII), and 2-cell-stage (2-cell), precompact morula (PCM), and in vitro-produced blastocyst-stage (IVTBL) embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo- (IVVBL), in vitro-, and nuclear transfer-produced (NTBL) blastocysts. It was our hypothesis that the identification of differentially represented transcripts from these stages would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine EST project (http://genome.rnet.missouri.edu/Bovine/), which focused on female reproductive tissues and embryos, were compared using Fisher's exact test weighted by number of transcripts per tissue by gene (SAS PROC FREQ; SAS Institute, Inc., Cary, NC, USA). Of the 3144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P < 0.01) in at least one pairwise comparison (Table 1). Some transcripts found to increase in representation from the MPII to the 2-cell stage include protein kinases, PRKACA and CKS1, as well as the metabolism-related gene, PTTG1. These same transcripts were also found to decrease in representation from the 2-cell to the PCM stage. RPL15 (translation) and FTH1 (immune function) were both more highly represented in the PCM than in the 2-cell stage. From PCM to IVTBL, we saw an increase in RPS11, another translation-related transcript. When comparing blastocyst-stage embryos from different production techniques, several transcripts involved in energy production (e.g., COX7B and COX8A) were found to be more highly represented in the NTBL than in the IVTBL. COX8A was also more highly represented in the IVVBL than in the IVTBL. By investigating these differentially represented transcripts, we will be able to better understand the developmental implications of embryo manipulation. We may also be able to better develop reproductive technologies that lead to in vitro- and nuclear transfer-derived embryos which more closely follow a normal program of development. Table 1. Differentially represented transcripts between developmental stages

124. DNA METHYLATION IN THE 2-CELL MOUSE EMBRYO IS THE RESULT OF DNMT ACTIVITY DURING DEVELOPMENT FROM THE ZYGOTE TO THE 2-CELL STAGE

2009 ◽  
Vol 21 (9) ◽  
pp. 43
Author(s):  
Y. Li ◽  
H. D. Morgan ◽  
L. Ganeshan ◽  
C. O'Neill
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Cytosine Methylation ◽  
De Novo ◽  
Culture Conditions ◽  
Early Embryo ◽  
Cleavage Stage ◽  
Cell Stage ◽  
Stage Of Development ◽  
First Time

In an accompanying abstract we show for the first time that global demethylation of both paternally- and maternally-derived genomes occurs prior to syngamy. It is commonly considered that new methylation of the genome does not commence until late in the preimplantation stage. Yet embryos during cleavage stage are known to show DNA methylation. This creates a paradox, if global demethylation occurs by the time of syngamy yet remethylation does not occur until the blastocysts stage, how can cleavage stage embryos possess methylated DNA. We examined this paradox. We examined DNA methylation in 2-cell embryos by confocal microscopy of anti-methylcytosine immunofluorescence and propidium iodide co-staining of whole mounts. We confirmed that DNA in late zygotes was substantially demethylated in both the male and female pronuclei. By the 2-cell stage, embryos collected direct from the oviduct showed high levels of cytosine methylation. We assessed whether this accumulation of cytosine methylation during the early 2-cell stage was a consequence of DNA methyltransferase (DNMT) activity. This was achieved by treating late stage zygotes with the DNMT inhibitor RG108 (5 μM) for the period of development spanning pronuclear stage 5 to early 2-cell stage. The embryos that developed in the presence of the DNA methyltransferase inhibitor showed significantly less methylcytosine staining than the embryos in the untreated culture conditions (P<0.001). Treatment of embryos during this period with RG108 significantly reduced their capacity to develop to normal blastocysts, indicating that this early DNA re-methylation reaction was important for the normal development of the embryo. Our results show for the first time that de novo methylation of the genome occurs as early as the 2-cell stage of development and that this is mediated by a RG108-sensitive DNMT activity. The results substantially change our understanding of epigenetic reprogramming in the early embryo.

Persistence of human mitochondrial DNA throughout the development to the blastocyst of mouse zygotes microinjected with human mitochondria

Zygote ◽  
1999 ◽  
Vol 7 (4) ◽  
pp. 279-283 ◽  
Author(s):  
V.B. Vasilyev ◽  
V.A. Sokolova ◽  
A.V. Sorokin ◽  
M.G. Bass ◽  
N.I. Arbuzova ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Hepg2 Cell Line ◽  
Chain Reaction ◽  
Specific Primers ◽  
Human Mitochondrial Dna ◽  
Mouse Zygotes ◽  
Polymerase Chain ◽  
Species Specific

The conditions for transfer of human mitochondria into fertilised mouse ova were elaborated. Species-specific primers were designed to discriminate human mitochondrial DNA (mtDNA) and the endogenous mtDNA in the preimplantation embryos. Human mitochondria isolated from the HepG2 cell line were microinjected into murine zygotes, and the latter cultured for 96 h to the blastocyst stage. The polymerase chain reaction allowed the detection of human mtDNA at every stage of embryo cleavage. In some cases a clear disparity in distribution of human mtDNA among blastomeres was evident.

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