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.

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 40 CHARACTERIZATION OF EARLY G1 CELLS AS NUCLEAR DONORS FOR SOMATIC CELL CLONING IN CATTLE

2005 ◽  
Vol 17 (2) ◽  
pp. 170
Author(s):  
A. Kasamatsu ◽  
K. Saeki ◽  
T. Tamari ◽  
K. Shirouzu ◽  
S. Taniguchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Somatic Cell ◽  
Cell Cloning ◽  
Proliferating Cells ◽  
Blastocyst Development ◽  
Ki 67 ◽  
Quiescent Cells ◽  
Reconstructed Embryos ◽  
G1 Cells

In somatic cell cloning, the cell cycle phase of the donor cells has critical impact on nuclear reprogramming and chromosomal normality of the reconstructed embryos. Recently, enhanced development to full term was obtained with embryos reconstructed with bovine fibroblasts soon after cell division (early G1 cells, Kasinathan P et al. 2001 Nat. Biotech. 19, 1176–1178; Urakawa M et al. 2004 Theriogenology 62, 714–728). In this study, to investigate the detailed cell cycle characteristics and gene expression of the early G1 cells as nuclear donors, we examined the cell proliferating and nuclear activity by detecting PCNA and Ki-67 in the cells, and the gene expression in the cells transfected with the luciferase gene. Bovine fibroblasts were transfected with chicken β-actin/firefly luciferase fusion gene (β-act/luc+), and stably transfected; cloned cells were used for cell analysis. We compared cell cycle characteristics for quiescent cells (0.4% serum for 7 days), cell doublets (early G1 cells) prepared by the “shake-off” method, and proliferating (30 to 40% confluency) cells. The presence of PCNA and Ki-67 and the incorporation of BrdU in the cells were determined by immunohistochemical analysis. The LUC+ signal (luminescence) in the cells was detected with an imaging photon counter for 10 consecutive min. Embryos reconstructed with these cells were cultured for 168 h for examination of blastocyst development. Experiments were repeated three times, and the data were analyzed with Fisher's PLSD test following ANOVA. Incorporation of BrdU was observed only in proliferating cells (24% of the cells). Neither PCNA nor Ki-67 signals were detected in the quiescent cells. PCNA was detected but Ki-67 was not detected in early G1 cells. Both PCNA and Ki-67 were detected in the proliferating cells. A strong LUC+ signal (6354 ± 673 pixels/cell) was detected in the proliferating cells, and weak signals were detected in the early G1 (2044 ± 303 pixels/cell, P < 0.05) and quiescent cells (617 ± 59 pixels/cell, P < 0.05). The rate of blastocyst development with early G1 cells was higher (45/133, 32%) than that with starved and proliferating cells (47/233, 21%, and 41/258, 14%, respectively, P < 0.05). These results indicate that early G1 cells were actively proliferating cells because of the positive PCNA signals, but their nuclei were silent because of the absence of Ki-67 signals and the weak LUC+ signals. These characteristics of the early G1 cells might enhance the development of the reconstructed embryos. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan MEXT, and by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.

scholarly journals 62 RELATION OF INTENSITY OF GENE EXPRESSION IN BOVINE RECONSTRUCTED EMBRYOS TO SUBSEQUENT DEVELOPMENT

2005 ◽  
Vol 17 (2) ◽  
pp. 181
Author(s):  
K. Saeki ◽  
T. Tamari ◽  
A. Kasamatsu ◽  
K. Shirouzu ◽  
S. Taniguchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Somatic Cells ◽  
Calcium Ionophore ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Critical Event ◽  
Serum Starvation ◽  
Cell Stage ◽  
Sports Science ◽  
Strong Luminescence

During embryo development, embryonic gene activation (EGA) is the first critical event. We previously showed that EGA is also critical for further development in somatic cell-cloned embryos (Saeki K et al. 2004 Reprod. Fertil. Dev. 16, 157–158 abst). To show this, we reconstructed bovine embryos with bovine somatic cells transfected with chicken β-actin/firefly luciferase fusion gene (β−act/luc+) and showed that only luminescent embryos at 60 hours post-fusion (hpf) developed to the blastocyst stage. In this study, we examined the relation between the intensity of expression of the same reporter gene in embryos reconstructed with bovine β−act/luc+ fibroblasts and their subsequent development to the blastocyst stage. Bovine fibroblasts were transfected with β−act/luc+ as described earlier (Saeki K et al. 2004 Reprod. Fertil. Dev. 16, 157–158 abst). The stably transfected and cloned cells were cultured for several passages. The cells were cultured under serum starvation (0.4% FCS) for 7 days and then used as donor cells. In vitro-matured bovine oocytes derived from slaughterhouse ovaries were enucleated at 20 h post maturation. Enucleated oocytes were electrofused with the cells, and activated with a calcium ionophore and cycloheximide. The LUC+ signal (luminescence) in the embryos was detected in medium containing 500 μg mL−1 luciferin with an imaging photon counter (ARGUS 50, Hamamatsu, Japan) for 30 consecutive min at 60 hpf. The intensity of luminescence in embryos (4- to 8-cell stage) was graded as being strong (>10 × 104 pixels/embryo), intermediate (5 to 10 × 104 pixels/embryo), weak (<5 × 104 pixels/embryo), or absent. The embryos were cultured separately until 168 hpf, and examined for blastocyst development. Experiments were repeated four times, and the data were analyzed with Fisher's PLSD test following ANOVA by Stat View software (Ver. 5.0; abacus Concepts, Berkeley, CA, USA). Of 125 embryos that were reconstructed, 74 (59%) developed to the 4- to 8-cell stage at 60 hpf. The luminescence was strong in 29 (39%) of the embryos, intermediate in 12 (16%), weak in 19 (26%), and absent in 14 (19%). Blastocysts were obtained from a group of embryos that exhibited strong luminescence (10/29, 34%), but none of the embryos from the other groups developed to blastocysts. These results suggest that active gene expression in embryos reconstructed with somatic cells is important for their subsequent development. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Ministry of Education, Culture, Sports, Science, and Technology, and by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.

Sodium butyrate improves the cloned yak embryo viability and corrects gene expression patterns

Zygote ◽  
2013 ◽  
Vol 23 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Xian-rong Xiong ◽  
Dao-liang Lan ◽  
Jian Li ◽  
Yong Wang ◽  
Jin-cheng Zhong
Keyword(s):  
Gene Expression ◽  
Expression Patterns ◽  
Formation Rate ◽  
Cell Number ◽  
Developmental Competence ◽  
Gene Expression Patterns ◽  
Embryo Viability ◽  
Cell Stage ◽  
Expression Levels

SummaryInterspecies somatic cell nuclear transfer (iSCNT), a powerful tool in basic scientific research, has been used widely to increase and preserve the population of endangered species. Yak (Bos grunniens) is one of these species. Development to term of interspecies cloned yak embryos has not been achieved, possibly due to abnormal epigenetic reprogramming. Previous studies have demonstrated that treatment of intraspecies cloned embryos with (NaBu) significantly improves nuclear–cytoplasmic reprogramming and viability in vitro. Therefore, in this study, we evaluated the effect of optimal NaBu concentration and exposure time on preimplantation development of yak iSCNT embryos and on the expression patterns of developmentally important genes. The results showed that 8-cell rate, blastocyst formation rate and total cell number increased significantly compared with their untreated counterparts when yak iSCNT embryos were treated with 5 nM NaBu for 12 h after activation, but that the 2-cell stage embryo rate was not significantly different. The treatment of NaBu also increased significantly the expression levels of Oct-4 and decreased the expression levels of HDAC-2, Dnmt-1 and IGF-1; the expression patterns of these genes were more similar to that of their bovine–yak in vitro fertilization (BY-IVF) counterparts. The results described above indicated that NaBu treatment improved developmental competence in vitro and ‘corrected’ the gene expression patterns of yak iSCNT embryos.

Gene expression patterns in blood predict future severe endpoints in community acquired pneumonia

Pneumologie ◽  
2018 ◽  
Vol 72 (S 01) ◽  
pp. S8-S9
Author(s):  
M Bauer ◽  
H Kirsten ◽  
E Grunow ◽  
P Ahnert ◽  
M Kiehntopf ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Patterns ◽  
Community Acquired Pneumonia ◽  
Gene Expression Patterns
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