28 EFFECT OF TREATMENT OF OVINE OOCYTES WITH CAFFEINE ON GENE EXPRESSION IN NUCLEAR TRANSFER EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 122 ◽  
Author(s):  
I. Choi ◽  
J.-H. Lee ◽  
K. Campbell
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Early Development ◽  
Nuclear Transfer ◽  
Expression Patterns ◽  
Gene Expression Pattern ◽  
Blastocyst Stage ◽  
Group Iv ◽  
Expression Levels ◽  
Number Of Genes

The efficiency of animal production by somatic cell nuclear transfer (SCNT) remains low and this has been linked to incomplete epigenetic reprogramming of the donor somatic cell nucleus. Previous studies have reported that embryos produced by SCNT exhibit abnormal expression patterns for a number of genes, including IL6, FGF4, FGFr2, Hsp, IF-tau, DNMT, and Mash2 (Daniels et al. 2000 Biol. Reprod. 63, 1034-1040; Wrenzycki et al. 2001 Biol. Reprod. 65, 309-317). Recently, we demonstrated that treatment of telophase I or metaphase II ovine oocytes with 10 mM caffeine for 6 h increased the activities of both MPF and MAPK kinases. In NT embryos produced using caffeine treated oocytes as recipient cytoplasts, no increase in the frequency of development to the blastocyst stage was observed; however, blastocyst stage embryos had an increased cell number (Lee and Campbell 2005 Reprod. Fertil. Dev. 16, 125). The objective of the present study was to examine the effects of caffeine treatment on the expression levels of a number of genes involved in early development. Target genes were categorized into seven groups based on their function: (1) transcription factors (Oct-4, Sox-2), (2) growth factors (IGF-1, IGF-1r, IGF-2r, FGF-2, and FGF-4), (3) stress adaptation (Hsp70.1 and Hsp27), (4) metabolism (Glut-1, Glut-3, and Glut-4), (5) compaction/cavitation (DcII), (6) trophoblastic function (IF-tau), and (7) nuclear reprogramming factors (Hist4h4, H2A.Z, and Lmna). To determine the transcript levels semiquantitatively, different PCR cycle parameters were used (35 and 40 cycles). Expression levels were compared in blastocyst-stage embryos obtained from five groups produced as previously described (Lee and Campbell 2005; Maalouf et al. 2005 Reproduction 32, 49): (I) IVF embryos, (II) caffeine-treated IVF embryos, (III) parthenotes, (IV) SCNT embryos, and (V) caffeine treated SCNT embryos. No differences in overall expression patterns were observed among groups I, II, and III. In group IV non-treated SCNT-derived embryos, an aberrant gene expression pattern was found with respect to Oct-4 and genes regulated by Oct-4: H2A.Z, IF-tau, and FGF-4, Oct-4, H2A.Z, and FGF-4 were down-regulated and IF-tau was up-regulated. In contrast, the expression patterns of group V caffeine-treated SCNT embryos resembled those of control groups I, II, and III. In comparison to gene expression in group IV embryos, Oct-4, FGF-4, and H2A.Z were up-regulated but IF-tau was down-regulated. Previous studies have demonstrated that FGF-4 and H2A.Z play an important role in early development and implantation, whereas expression of IF-tau is related to embryo quality (Feldman et al. 1995 Science 267, 246-249; Fasst et al. 2001 Curr. Biol. 11, 1183-1187; Wrenzycki et al. 2001). Our results demonstrate that treatment of oocytes with caffeine prior to embryo reconstruction can alter the expression patterns of developmentally regulated genes in SCNT embryos to more closely resemble those of IVF controls.

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.

56CARDIOMYOCYTES FOR THE STUDY OF DEDIFFERENTIATION IN BOVINE NUCLEAR TRANSFER

2004 ◽  
Vol 16 (2) ◽  
pp. 150
Author(s):  
A. Lucas-Hahn ◽  
M. Schwarzer ◽  
E. Lemme ◽  
L. Schindler ◽  
H. Niemann
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Nuclear Transfer ◽  
Expression Patterns ◽  
Gene Expression Pattern ◽  
Fusion Rate ◽  
Marker Genes ◽  
Cardiac Marker ◽  
Reconstructed Embryos

Nuclear transfer facilitates the study of the dedifferentiation process of differentiated somatic cells. Cardiomyocytes are a good model of terminally differentiated cells showing a unique gene expression pattern of cardiac marker genes. The purpose of this study was to test bovine cardiomyocytes as donor cells in nuclear transfer. Cardiomyocytes were isolated from fetal heart muscle (3–5 months of gestation), which were obtained at the abbatoir and immediately perfused with cold Custodiol (Dr. Franz Köhler Chemie, Germany) to reduce metabolism and protect the cells against ischaemia. Subsequently, hearts were perfused with collagenase in Krebs-Henseleit buffer (KHB) to dissociate the tissue and isolate single elongated, contractile cells. For nuclear transfer and fusion the cardiomyocytes were rounded up by exposure to increasing calcium concentrations (2.5–200μM) in the culture medium before the cells were incubated in suspension for 46–48 hours in MEM medium plus 10% FCS. Nuclear transfer was performed as described earlier (Lucas-Hahn et al., 2002, Theriogenology 57, 433). As a control, adult female fibroblasts were employed. Fusion rate, cleavage (day 3 of in vitro culture) and development up to the morula/blastocyst (day 7 of in vitro culture) were recorded and statistically analysed with Student’s t-test. A total of 243 nuclear transfer complexes with cardiomyocytes and 127 with fibroblasts were produced. Fusion rates for cardiomyocyte complexes were significantly (P<0.001) lower (28.8%) compared to fibroblasts (84.3%). Cleavage rates were 48.1% for cardiomyocytes and 62.8% for the fibroblast-derived embryos. The developmental capacity to the morula/blastocyst was significantly (P<0.01) reduced for cardiomyocyte (9.4%) compared with the fibroblast-derived (32.4%) reconstructed embryos. Most of the Day 7 embryos were frozen for investigation of gene expression patterns of cardiac marker genes. Staining with Hoechst 33342 for counting total cell numbers revealed that 87.3±20.9 blastocysts were derived from fibroblasts and 100 blastocysts from cardiomyocytes. These results indicate that nuclear transfer with terminally differentiated cardiomyocytes is possible, although with reduced rates. Studies are underway to analyze the gene expression of cardiac marker genes in reconstructed embryos to gain insight into dedifferentiation after nuclear transfer using cardiomyocytes as a model. This study was supported by Deutsche Forschungsgemeinschaft (DFG; Ni 256/16-1)

200 REPROGRAMMING OF Oct-4 FOLLOWING EQUINE SOMATIC CELL NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 198
Author(s):  
T. Xiang ◽  
S. Walker ◽  
K. Gregg ◽  
W. Zhou ◽  
V. Farrar ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Expression Patterns ◽  
Somatic Cells ◽  
Blastocyst Stage ◽  
Rt Pcr ◽  
Tissue Samples ◽  
Donor Cells

Oct-4, a POU domain-containing transcription factor encoded by Pou5f1, is selectively expressed in pre-implantation embryos and pluripotent stem cells, but not in somatic cells. Because of such a unique expression feature, Oct-4 can serve as a useful reprogramming indicator in somatic cell nuclear transfer (SCNT). Compared with data of Oct-4 expression in mouse and bovine cloned embryos, little is known about this gene in equine nuclear transfer. In the present study, we investigated Oct-4 expression in donor cells, oocytes, and SCNT embryos to evaluate reprogramming of equine somatic cells following nuclear transfer. Horse ovaries were obtained from a local slaughterhouse and the oocytes collected from the ovaries were matured in vitro in an M199-based medium (Galli et al. 2003 Nature 424, 635) for 24 h. Donor cells were derived from biopsy tissue samples of adult horses and cultured for 1 to 5 passages. Standard nuclear transfer procedures (Zhou et al. 2008 Mol. Reprod. Dev. 75, 744–758) were performed to produce cloned embryos derived from equine adult somatic cells. Cloned blastocysts were obtained after 7 days of in vitro culture of reconstructed embryos. Total RNA were extracted using Absolutely RNA Miniprep/Nanoprep kits (Stratagen, La Jolla, CA) from oocytes (n = 200), donor cells, and embryos (n = 5). DNase I treatment was included in the procedure to prevent DNA contamination. Semiquantitative RT-PCR was performed with optimized cycling parameters to analyze Oct-4, GDF9, and β-actin in equine donor cells, oocytes, and cloned blastocysts. The RT-PCR products were sequenced to verify identity of the genes tested. The relative expression abundance was calculated by normalizing the band intensity of Oct-4 to that of β-actin in each analysis. No transcript of Oct-4 was detected in equine somatic cells used as donor nuclei, consistent with its expression patterns in other animal species, whereas Oct-4 was abundantly expressed in equine SCNT blastocysts derived from the same donor cell line. Oct-4 transcripts were also detected in equine oocytes and whether any maternally inherited Oct-4 mRNA persisted up to the blastocyst stage was unclear in this study. We selected GDF9 to address this question; GDF9 was abundantly detected in equine oocytes, consistent with its expression pattern in mouse and bovine, but not detected in donor cells and cloned blastocysts, suggesting that the GDF9 mRNA from the oocyte was degraded at least by the blastocyst stage. The results from this study imply occurrence of Oct-4 reprogramming in equine SCNT blastocysts, and future analysis for more developmentally important genes is needed to better understand reprogramming at molecular levels in this species.

89 ZYGOTICALLY ACTIVATED GENES ARE SUPPRESSED IN MOUSE NUCLEAR-TRANSFERRED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 162
Author(s):  
T. Suzuki ◽  
N. Minami ◽  
H. Imai
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Early Stage ◽  
Mammalian Species ◽  
Expression Patterns ◽  
Gene Activation ◽  
Blastocyst Stage ◽  
Success Rates ◽  
Cell Nuclei ◽  
Cell Stage

Mammalian oocytes have the ability to confer totipotency to terminally differentiated somatic cell nuclei. Viable cloned animals have been produced by somatic cell nuclear transfer (NT) into oocytes in many mammalian species including mouse. However, the success rates of the production were quite low in all species. Many studies have measured differences in gene expression between NT and fertilized embryos in relatively advanced stages of development such as pre- and post-natal stages or the blastocyst stage. In the mouse, major zygotic gene activation (ZGA) occurs at the 2-cell stage after fertilization and leads to the transition of gene regulation from maternal control to embryonic control. Suppression of the ZGA by a transcription inhibitor was shown to decrease the viability of embryos, and causes developmental arrest at the 2-cell stage. An abnormal ZGA may therefore affect the viability of NT embryos and cause further abnormalities in later embryonic development. In the present study, we compared gene expression patterns using differential display RT-PCR (DDRT-PCR) between the NT and IVF embryos at the 2-cell stage to detect some abnormalities affecting later development of NT embryos. The developmental rate of NT embryos to blastocysts (32.9%) was significantly lower than that of IVF (92.7%) or PA (92.8%). In addition, the cell numbers of NT embryos at the blastocyst stage (39.5 � 2.6; n = 19) were less than those of IVF (66.8 � 2.1; n = 30) or PA embryos (48.2 � 2.1; n = 30). Using these embryos, we first identified 4 genes that were differentially expressed between NT and IVF embryos at the 2-cell stage. Among the identified genes, Inpp5b and Chst12 were up-regulated, and MuERV-L and Dnaja2 were down-regulated in the NT embryos compared with IVF embryos. Further analysis showed that the expression of zygotically activated genes such as Interferon-γ, Dub-1, Spz1, DD2106, and DD2111 were not properly activated in NT embryos, suggesting that the cellular process involved in the control of the zygotic genome activation is not appropriately regulated. These results indicate that abnormal gene expression has already occurred at the early stage of pre-implantation development as a failure of nuclear reprogramming.

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

Bovine pretransfer endometrium and embryo transcriptome fingerprints as predictors of pregnancy success after embryo transfer

2010 ◽  
Vol 42 (2) ◽  
pp. 201-218 ◽  
Author(s):  
Dessie Salilew-Wondim ◽  
Michael Hölker ◽  
Franca Rings ◽  
Nasser Ghanem ◽  
Mehmet Ulas-Cinar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Estrous Cycle ◽  
Embryo Transfer ◽  
Expression Pattern ◽  
Transcriptome Analysis ◽  
Expression Patterns ◽  
Gene Expression Pattern ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Quantitative Alteration

Aberrant gene expression in the uterine endometrium and embryo has been the major causes of pregnancy failure in cattle. However, selecting cows having adequate endometrial receptivity and embryos of better developmental competence based on the gene expression pattern has been a greater challenge. To investigate whether pretransfer endometrial and embryo gene expression pattern has a direct relation with upcoming pregnancy success, we performed a global endometrial and embryo transcriptome analysis using endometrial and embryo biopsy technology and the pregnancy outcome information. For this, endometrial samples were collected from Simmental heifers at day 7 and 14 of the estrous cycle, one cycle prior to embryo transfer. In the next cycle, blastocyst stage embryos were transferred to recipients at day 7 of the estrous cycle after taking 30–40% of the blastocyst as a biopsy for transcriptome analysis. The results revealed that at day 7 of the estrous cycle, the endometrial gene expression pattern of heifers whose pregnancy resulting in calf delivery was significantly different compared with those resulting in no pregnancy. These differences were accompanied by qualitative and quantitative alteration of major biological process and molecular pathways. However, the transcriptome difference was minimal between the two groups of animals at day 14 of the estrous cycle. Similarly, the transcriptome analysis between embryos biopsies that resulted in calf delivery and those resulted in no pregnancy revealed a total of 70 differentially expressed genes. Among these, the transcript levels of 32 genes including SPAG17, PF6, UBE2D3P, DFNB31, AMD1, DTNBP1, and ARL8B were higher in embryo biopsies resulting in calf delivery. Therefore, the present study highlights the potential of pretransfer endometrial and embryo gene expression patterns as predictors of pregnancy success in cattle.

scholarly journals Aberrant gene expression patterns in placentomes are associated with phenotypically normal and abnormal cattle cloned by somatic cell nuclear transfer

2008 ◽  
Vol 33 (1) ◽  
pp. 65-77 ◽  
Author(s):  
Robin E. Everts ◽  
Pascale Chavatte-Palmer ◽  
Anthony Razzak ◽  
Isabelle Hue ◽  
Cheryl A. Green ◽  
...  
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Expression Patterns ◽  
Pathological Finding ◽  
Culture Methods ◽  
Vitro Fertilization ◽  
Near Term

Transcription profiling of placentomes derived from somatic cell nuclear transfer (SCNT, n = 20), in vitro fertilization (IVF, n = 9), and artificial insemination (AI, n = 9) at or near term development was performed to better understand why SCNT and IVF often result in placental defects, hydrops, and large offspring syndrome (LOS). Multivariate analysis of variance was used to distinguish the effects of SCNT, IVF, and AI on gene expression, taking into account the effects of parturition (term or preterm), sex of fetus, breed of dam, breed of fetus, and pathological finding in the offspring (hydrops, normal, or other abnormalities). Differential expression of 20 physiologically important genes was confirmed with quantitative PCR. The largest effect on placentome gene expression was attributable to whether placentas were collected at term or preterm (i.e., whether the collection was because of disease or to obtain stage-matched controls) followed by placentome source (AI, IVF, or SCNT). Gene expression in SCNT placentomes was dramatically different from AI ( n = 336 genes; 276 >2-fold) and from IVF ( n = 733 genes; 162 >2-fold) placentomes. Functional analysis of differentially expressed genes (DEG) showed that IVF has significant effects on genes associated with cellular metabolism. In contrast, DEG associated with SCNT are involved in multiple pathways, including cell cycle, cell death, and gene expression. Many DEG were shared between the gene lists for IVF and SCNT comparisons, suggesting that common pathways are affected by the embryo culture methods used for IVF and SCNT. However, the many unique gene functions and pathways affected by SCNT suggest that cloned fetuses may be starved and accumulating toxic wastes due to placental insufficiency caused by reprogramming errors. Many of these genes are candidates for hydrops and LOS.

scholarly journals Different Gene Expression Patterns of Hexose Transporter Genes Modulate Fermentation Performance of Four Saccharomyces cerevisiae Strains

Fermentation ◽  
2021 ◽  
Vol 7 (3) ◽  
pp. 164
Author(s):  
Chiara Nadai ◽  
Giulia Crosato ◽  
Alessio Giacomini ◽  
Viviana Corich
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Fermentation Process ◽  
Expression Patterns ◽  
Gene Expression Pattern ◽  
Gene Expression Patterns ◽  
Specific Gene ◽  
Facilitated Diffusion ◽  
Fermentation Performance ◽  
Expression Levels

In Saccharomyces cerevisiae, the fermentation rate and the ability to complete the sugar transformation process depend on the glucose and fructose transporter set-up. Hexose transport mainly occurs via facilitated diffusion carriers and these are encoded by the HXT gene family and GAL2. In addition, FSY1, coding a fructose/H+ symporter, was identified in some wine strains. This little-known transporter could be relevant in the last part of the fermentation process when fructose is the most abundant sugar. In this work, we investigated the gene expression of the hexose transporters during late fermentation phase, by means of qPCR. Four S. cerevisiae strains (P301.9, R31.3, R008, isolated from vineyard, and the commercial EC1118) were considered and the transporter gene expression levels were determined to evaluate how the strain gene expression pattern modulated the late fermentation process. The very low global gene expression and the poor fermentation performance of R008 suggested that the overall expression level is a determinant to obtain the total sugar consumption. Each strain showed a specific gene expression profile that was strongly variable. This led to rethinking the importance of the HXT3 gene that was previously considered to play a major role in sugar transport. In vineyard strains, other transporter genes, such as HXT6/7, HXT8, and FSY1, showed higher expression levels, and the resulting gene expression patterns properly supported the late fermentation process.

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