Estimating the Survival Probability of Nuclear-Transfer Embryos before Embryo Transfer by a Novel Biopsy: Oct4 and Sox2 Gene Expression Patterns of a Monozygotic Twin Blastocyst Separated at the 2-Cell Stage of Nuclear-Transfer Embryos

2016 ◽  
Vol 33 (1) ◽  
pp. 55-61 ◽  
Author(s):  
Kazuki Ohata ◽  
Yoko Kato
Keyword(s):  
Gene Expression ◽  
Embryo Transfer ◽  
Survival Probability ◽  
Nuclear Transfer ◽  
Expression Patterns ◽  
Monozygotic Twin ◽  
Gene Expression Patterns ◽  
Cell Stage ◽  
Sox2 Gene

68 GENE EXPRESSION COMPARISONS BETWEEN BOVINE IN VIVO AND CLONED EMBRYOS PRODUCED BY THREE DIFFERENT METHODS

2006 ◽  
Vol 18 (2) ◽  
pp. 142
Author(s):  
N. Ruddock ◽  
K. Wilson ◽  
M. Cooney ◽  
R. Tecirlioglu ◽  
V. Hall ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chromatin Remodeling ◽  
Nuclear Transfer ◽  
Expression Patterns ◽  
Experimental Models ◽  
Gene Expression Patterns ◽  
Imprinted Genes ◽  
Mammalian Embryo

Developmental pathways in the mammalian embryo are profoundly influenced by the epigenetic interaction of the environment and the genome. Loss of epigenetic control has been implicated in aberrant gene expression and altered imprinting patterns with consequence to the physiology and viability of the conceptus. Bovine somatic cell nuclear transfer (SCNT) is contingent on in vitro culture, and both SCNT and culture conditions are known to induce changes in embryonic gene expression patterns. Using these experimental models, this study compared gene expression of Day 7 cloned blastocysts created from three different SCNT protocols using the same cell line, with Day 7 in vivo blastocysts to elucidate mechanisms responsible for variations in phenotypic outcomes. SCNT methods included: (1) traditional SCNT by subzonal injection (SI); (2) handmade cloning (HMC); and (3) modified serial nuclear transfer (SNT), developed within the group. Four imprinted genes (Grb10, Ndn, Nnat, and Ube3a), four chromatin remodeling genes (Cbx1, Cbx3, Smarca4, and Smarcb1) and two genes implicated in polycystic liver disease (Prkcsh and Sec63) were analyzed in single blastocysts from each treatment (n = 5). All blastocysts expressed Actin, Oct-4 and Ifn-tau. All genes were sequence verified. Several genes were expressed ubiquitously across all groups, including Ndn, Ube3a, Cbx1, Cbx3, and Smarcb1. Interestingly, Grb10 was not expressed in two HMCs and one SNT blastocyst. Nnat was weakly expressed in one in vivo blastocyst and in the majority of cloned blastocysts in all groups. Prkcsh and Sec63 were expressed in all but one HMC blastocyst. While gene expression patterns were mostly maintained following SCNT, the imprinted genes Nnat and Grb10 showed instances of differential or abnormal expression in SCNT embryos. The chromatin remodeling genes were maintained in all SCNT treatments. Prkcsh and Sec63 were both absent in one HMC blastocyst, with implications for liver dysfunction, a condition previously reported in abnormal cloned offspring. The variable mRNA expression following SCNT provides an insight into genetic and environmental factors controlling implantation, placentation, organ formation, and fetal growth.

171 THE EFFECT OF TRICHOSTATIN A ON THE DEVELOPMENT AND THE GLOBAL GENE EXPRESSION PATTERNS IN MOUSE EMBRYOS DEVELOPING IN VITRO

2008 ◽  
Vol 20 (1) ◽  
pp. 165
Author(s):  
X. S. Cui ◽  
X. Y. Li ◽  
T. Kim ◽  
N.-H. Kim
Keyword(s):  
Gene Expression ◽  
Trichostatin A ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Gene Expression Profiles ◽  
Mouse Embryos ◽  
Differentially Expressed ◽  
Gene Expression Patterns ◽  
Cell Stage

Trichostatin A (TSA) is an inhibitor of histone deacetylase and is able to alter gene expression patterns by interfering with the removal of acetyl groups from histones. The aim of this study was to determine the effect of TSA treatment on the development and gene expression patterns of mouse zygotes developing in vitro. The addition of 100 nm TSA to the culture medium did not affect the cleavage of mouse embryos (TSA treatment, 148/150 (99%) v. control, 107/107 (100%)); however, embryos that were treated with TSA arrested at the 2-cell stage (145/148, 98%). We estimated the number of nuclei in control and TSA-treated embryos by propidium iodide staining, taking into account the presence of any cells with two or more nuclei. At 62–63 h post-hCG stimulation, control zygotes had developed to the 4-cell stage and exhibited one nucleus in each blastomere, indicative of normal development. In contrast, we observed tetraploid nuclei in at least one blastomere in 20.8% (11/53) of the embryos that had been treated with TSA. At 28–29 h post-hCG stimulation (metaphase of the 1-cell stage), there was no difference in the mitotic index (as determined by analyzing the microtubule configuration) in the TSA group compared to the control group. At the 2-cell stage, however, we did not observe mitotic spindles and metaphase chromatin in embryos in the TSA treatment group compared to the controls. Interestingly, when embryos were cultured in TSA-free medium from 35 h post-hCG stimulation (S- or early G2-phase of the 2-cell stage) onward, almost all of them (47/50) developed to the blastocyst stage. In contrast, when embryos were cultured in TSA-free medium from 42 h post-hCG stimulation (middle G2-phase of the 2-cell stage) onward, they did not develop to the 4-cell stage. We used Illumina microarray technology to analyze the gene expression profiles in control and TSA-treated late 2-cell-stage embryos. Applied Biosystems Expression System software was used to extract assay signals and assay signal-to-noise ratio values from the microarray images. Our data showed that 897 genes were significantly (P < 0.05; 2-sample t-test) up- or down-regulated by TSA treatment compared to controls. Analysis using the PANTHER classification system (https://panther.appliedbiosystems.com) revealed that the 575 genes that were differentially expressed in the TSA group compared to the control were classified as being associated with putative biological processes or molecular function. Overall, in terms of putative biological processes, more nucleoside, nucleotide, and nucleic acid metabolism, protein metabolism and modification, signal transduction, developmental process, and cell cycle genes were differentially expressed between the TSA and control groups. In terms of putative molecular function, more nucleic acid-binding transcription factor and transferase genes were differentially expressed between the groups. The results collectively suggest that inhibition of histone acetylation in mouse embryos affects gene expression profiles at the time of zygotic genome activation, and this subsequently affects further development.

scholarly journals 231EXPRESSION PATTERN OF CERTAIN DEVELOPMENTALLY IMPORTANT GENES IN BOVINE NUCLEAR TRANSFER EMBRYOS PRODUCED USING CELL LINES OF DIFFERENT EFFICIENCY

2004 ◽  
Vol 16 (2) ◽  
pp. 236 ◽  
Author(s):  
Z. Beyhan ◽  
N.L. First
Keyword(s):  
Gene Expression ◽  
Statistical Analysis ◽  
Cell Line ◽  
Cell Lines ◽  
Live Birth ◽  
Nuclear Transfer ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Gene Expression Patterns ◽  
Donor Cells

Developmental abnormalities associated with the cloning process suggest that reprogramming of donor nuclei into an embryonic state may not be fully completed in most of the cloned animals. One of the areas of interest in this respect is the analysis of gene expression patterns in nuclear transfer embryos to dissect the processes that failed and to develop means to overcome the limitations imposed by these factors. In this study, we investigated the expression patterns of histone deacetylase-1,-2,-3 (HDAC-1,-2,-3), DNA methyltransferase-3A (DNMT3A) and octamer binding protein-4 gene (POU5F1) in donor cells with different cloning efficiencies (low: no-pregnancy, medium: pregnancy but no live birth and high: live birth) and nuclear transfer embryos derived from these cell lines using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay with SYBR green chemistry. Employing standard protocols, we produced nuclear transfer embryos from three different cell lines categorized as having varying efficiencies in supporting development to term. Embryos were collected at morula, blastocyst and hatched blastocyst stages and total RNA was extracted from pools of 4–5 embryos using Absolutely RNA nanoprep kit (Stratagene, La Jolla, CA, USA). Relative level of expression at these stages was analyzed using ΔΔCT method with HH2A as the reference gene and in vitro-fertilized embryos as the control samples. Statistical analysis was performed on ranked expression data employing SAS statistical analysis software procedure ANOVA. Same set of genes were also analyzed on donor cells using standard curve method. All genes investigated were affected by nuclear transfer and followed somewhat altered expression patterns. In general, expression of HDAC genes was elevated especially at the compact morula stage but became comparable to control embryos at the hatched blastocyst stage. DNMT3A expression in NT embryos was lower than in IVF embryos at all stages. POU5F1 transcript levels were also reduced in nuclear transfer embryos at the compact morula and blastocyst stages. The difference, however, disappeared at the hatched blastocyst stage. There was a cell line effect on the expression patterns of all genes investigated. Cell lines efficient in producing offspring tended to resemble control embryos in gene expression patterns compared to inefficient cell lines. These results agree with several studies reporting altered gene expression patterns for certain genes in cloned embryos. Our data also suggest that cell line differences in developmental competency observed in cloning experiments might be related to physiological differences in transcriptional regulation and nuclear remodeling, DNA methylation, and lineage differentiation in embryos cloned from these cell lines.

69 RELATION OF SPATIAL GENE EXPRESSION PATTERNS IN BOVINE EMBRYOS RECONSTRUCTED WITH SOMATIC CELLS TO BLASTOCYST DEVELOPMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 142
Author(s):  
K. Saeki ◽  
T. Tamari ◽  
A. Kasamatsu ◽  
D. Iwamoto ◽  
S. Kameyama ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Patterns ◽  
Somatic Cells ◽  
Calcium Ionophore ◽  
Gene Expression Patterns ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Reconstructed Embryos ◽  
M Phase ◽  
G1 Cells

Recently, enhanced development to full term was obtained with embryos reconstructed with bovine early G1 cells rather than with G0 cells (Kasinathan et al. 2001 Nat. Biotechnol. 19, 1176-1178; Urakawa et al. 2004 Theriogenology 62, 714-728). However, the reason why donor somatic cells at the early G1 phase are better for embryo reconstruction is unclear. In this study, we investigated the relation of spatial gene expression patterns at the 4- to 8-cell stage to blastocyst development of embryos reconstructed with early G1 cells. Bovine fibroblasts stably transfected with �-act/luc+/IRES/EGFP were used for embryo reconstruction. M phase cells were prepared as described by Urakawa et al. (2004). Early G1 cells were obtained from cultured M phase cells soon after the M phase cells divided. Quiescent cells (cultured in 0.4% serum for 7 days) were used as G0 cells for a control. The cells were electrofused with enucleated bovine oocytes matured in vitro, and activated with a calcium ionophore and cycloheximide. The reconstructed embryos were cultured until 60 hours post fusion (hpf), and zonae pellucidae of 4- to 8-cell embryos were removed by pronase. To determine gene expression, the LUC+ activity (luminescence) in the embryo blastomeres was detected with an imaging photon counter (Hamamatsu Photonics, Hamamatsu City, Shikuoka Prefecture, Japan) for 10 min. The embryos were categorized as being positive, mosaic, or negative depending on whether all, some or no blastomeres were luminescent, respectively. The embryos were cultured in mSOF medium individually until 168 hpf to assess development to the blastocyst stage. Blastocyst development of reconstructed embryos without detection of luminescence was also examined. Experiments were repeated three times, and the data were analyzed with Fisher's PLSD test following ANOVA. At 60 hpf, 75% (74/99) of embryos reconstructed with early G1 cells and 55% (46/83) of embryos with G0 cells developed to 4- to 8-cell stage embryos. The difference is significant (P < 0.05). The percentages of positive, mosaic, and negative embryos with G1 cells were 49, 35 and 16%, and blastocyst rates were 30, 11, and 0%, respectively. With G0 cells, the percentages were 32, 56, and 12%, and the blastocyst rates were 15, 4, and 0%, respectively. More positive embryos were obtained with early G1 cells than with G0 cells (P < 0.05). Blastocyst rates of the positive embryos with early G1 cells were the same as with G0 cells (P > 0.05). Blastocyst development of positive embryos was higher than that of mosaic and negative embryos in early G1 and G0 groups (P < 0.05). Without detection of luminescence, the blastocyst rates from the reconstructed embryos were 43% (35/81) and 16% (20/125) with early G1 and G0 cells, respectively (P < 0.05). These results suggest that the higher developmental capacity of embryos reconstructed with early G1 cells might be related to the appropriate spatial gene expression at the 4- to 8-cell stage. A part of this study was supported by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.

Gene Expression Patterns in Bovine In vitro-Produced and Nuclear Transfer-Derived Embryos and Their Implications for Early Development

2002 ◽  
Vol 4 (1) ◽  
pp. 29-38 ◽  
Author(s):  
H. Niemann ◽  
C. Wrenzycki ◽  
A. Lucas-Hahn ◽  
T. Brambrink ◽  
W.A. Kues ◽  
...  
Keyword(s):  
Gene Expression ◽  
Early Development ◽  
Nuclear Transfer ◽  
Expression Patterns ◽  
Gene Expression Patterns

scholarly journals 26ABERRANT REPROGRAMMING OF IMPRINTED GENE EXPRESSION IN ENLARGED PLACENTAS OF MICE CLONED FROM ES CELLS TREATED WITH TSA OR 5AZAC

2004 ◽  
Vol 16 (2) ◽  
pp. 135
Author(s):  
S.G. Baqir ◽  
Q. Zhou ◽  
A. Jouneau ◽  
J.-P. Renard ◽  
D.H. Betts ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Histone Acetylation ◽  
Nuclear Transfer ◽  
Es Cells ◽  
Expression Patterns ◽  
Gene Expression Patterns ◽  
Imprinted Genes ◽  
Imprinted Gene ◽  
Donor Cells

The success rate of producing cloned animals is very low, and in many cases is associated with the formation of enlarged placentas. Increasing evidence has pointed towards epigenetic deregulation of imprinted genes due to incomplete or abnormal resetting of DNA methylation and/or histone acetylation patterns during development. It has previously been shown that drugs that alter DNA methylation (5AzaC) and histone acetylation (TSA) over-express imprinted genes in mouse ES cells (Baqir and Smith, 2001, Theriogenology 55, 410). Our objective in this study was to determine whether nuclear transfer is able to reprogram imprinted gene expression patterns in the placenta of mice cloned from ES donor nuclei exposed to 5AzaC and TSA. ES donor cells were treated with either TSA or 5AzaC prior to injection into enucleated oocytes. Total RNA was extracted from placentas of day 14–15 fetus clones, and reversed transcribed; the expression pattern of imprinted genes (Ipl, Mash2, Igf2, H19, Igf2r, p57, Peg1), non-imprinted placental-specific genes (Esx1, Dlx3, Tpbp) and a housekeeping gene (Gapdh) was examined by Real Time PCR. Samples were standardized with an exogenous control (Globin) and expressed as fold changes in relation to placentas of cloned fetus derived from non-treated donor cells. Data were analyzed by ANOVA and mean gene expression values were compared using the Tukey-Kramer test. Our results show that several imprinted genes (Mash2, H19, Ipl) and placenta-specific genes (Esx1 and Dlx1) were properly reprogrammed in non-enlarged (71mg) placentas of fetus clones derived from the TSA and 5AzaC treated ES donor cells. Although Gapdh expression did not differ among normal and enlarged 210mg) placenta groups, the expression level of Igf2 and Mash2 was higher in enlarged placentas from fetus clones produced from TSA-treated ES donor cells (4.6 and 3.5 fold) compared to non-enlarged placentas from non-treated ES cells (1 fold). Conversely, oversized placentas from cloned fetuses derived from TSA-treated donor ES cells under-expressed Peg1, H19 and Ipl (0.5, 0.2 and 0.2 fold, respectively) compared to control placentas (1 fold). In addition, enlarged placentas from the TSA- and 5AzaC-treated group displayed down-regulation of placenta specific genes Esx1 and Dlx3 and up-regulation of Tpbp, suggesting the presence of abnormal distribution of placental layers. These results indicate that while several imprinted and non-imprinted placenta specific genes were correctly expressed in normal size placentas of fetus clones derived from TSA and 5AzaC treated donor ES cells, enlarged placentas displayed aberrant gene expression patterns, suggesting that improper resetting of the epigenetic program after nuclear transfer is directly related to altered DNA methylation and histone acetylation patterns. Funded by NSERC &amp; CIHR.

Sign in / Sign up

Export Citation Format

Share Document