48 GLOBAL GENE EXPRESSION PATTERN OF BOS INDICUS AND BOS TAURUS VITRIFIED EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 117
Author(s):  
R. Cancian ◽  
M. Macelai ◽  
G. Tavares ◽  
R. S. Valente ◽  
E. S. Caixeta ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Cellular Response ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Gene Expression Pattern ◽  
Global Gene Expression ◽  
Differentially Expressed ◽  
Canonical Pathways

The cryopreservation of in vitro-produced (IVP) bovine embryos is one of the most challenging areas of the assisted reproductive biotechnologies. The aim of the present study was to evaluate the global gene expression pattern of Bos indicus (Nellore) and Bos taurus (Simmental) IVP embryos after vitrification. Follicular aspiration was performed on Nellore (n = 14) and Simmental (n = 14) cows, and oocytes (n = 840 and 450; respectively) were submitted to in vitro maturation and in vitro fertilization. Presumptive zygotes were denuded and cultured in SOFaa with 0.5% BSA and 2.5% FCS during 7 days under standard culture conditions. Blastocysts (grade 1 and 2) were vitrified, warmed, and cultured for an additional 12 h under the same conditions. Nellore (n = 8) and Simmental (n = 8) IVP blastocysts considered viable after vitrification, with re-expanded blastocoel, were submitted to total RNA extraction (PicoPure, Arcturus, Applied Biosystems®, Foster Dity, CA, USA), DNAse I treatment (Qiagen®, Valencia, CA, USA), and amplification (RiboAmp, Applied Biosystems®). Fragmented cRNA were obtained through 3′IVT Express Kit (Affymetrix®, Santa Clara, CA, USA) to perform the hybridization using GeneChip Bovine Genome Array (Affymetrix®). Microarray data analysis was performed using the FlexArray 1.6.1.1 software. Genes with at least a 1.5-fold change and a P-value of less than 0.05 were considered differentially expressed. Of the 1278 genes differentially expressed between Bos taurus and Bos indicus vitrified embryos, 1108 were annotated, with 1193 genes up-regulated and 85 genes down-regulated in Bos taurus compared with Bos indicus IVP vitrified embryos. Differentially expressed genes were associated with the functional networks of cell cycle, cellular movement and DNA replication, recombination and repair; RNA post-transcriptional modifications; gene expression, protein synthesis; RNA damage and repair; cellular function and maintenance; and cell death and survival. The top 6 canonical pathways generated by Ingenuity Pathway Analysis® with the differentially expressed genes were ELF2 signalling, oxidative phosphorylation, tricarboxylic acid cycle, protein ubiquitination pathway, mTOR signalling, and IGF-1 signalling. In conclusion, Bos taurus IVP embryos seem to trigger different cellular response mechanisms to the vitrification stress in comparison with Bos indicus IVP embryos. Differential response is mainly represented by different expression profiles of genes regulating important canonical pathways involved in cellular response to stress that could be related with the higher post-cryopreservation survival capacity observed in Bos taurus embryos.Research was supported by FAPESP, CNPq, FAPERGS, and LNBio – National Laboratory of Biosciences/MCT.

scholarly journals 82 EFFECT OF HIGH FETAL CALF SERUM CONCENTRATION IN THE GENE EXPRESSION PATTERN OF IN VITRO PRODUCED BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 155
Author(s):  
M. J. Sudano ◽  
D. M. Paschoal ◽  
E. S. Caixeta ◽  
R. R. Maziero ◽  
M. D. Guastali ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Lipid Metabolism ◽  
Expression Pattern ◽  
Expression Profiles ◽  
Rna Extraction ◽  
Gene Expression Pattern ◽  
Differentially Expressed ◽  
Bovine Embryos

Even though FCS provides energy substrates, amino acids, vitamins, growth factors, and heavy-metal chelators, its supplementation has been associated with several embryo abnormalities such as mitochondrial degeneration, metabolic deviations, excessive lipid accumulation, and decreased embryo survival after cryopreservation. The aim of the present study was to evaluate the effect of high FCS concentration in the gene expression pattern of in vitro-produced bovine embryos. Slaughterhouse ovaries were used to obtain oocytes (N = 360), which were matured and fertilized in vitro (Day 0). Presumptive zygotes were divided in 2 culture media: with low (SOFaa with 0.5% BSA and 2.5% FCS) or high (SOFaa with 0.5% BSA and 10% FCS) FCS concentration. Cleavage was evaluated on Day 3. Embryo development was evaluated after 7 days under standard culture conditions (at 38.5°C in atmosphere of 5% O2, 5% CO2, and 90% N2). The produced blastocysts were placed in PBS solution and washed five times. A single blastocyst was frozen in a minimal volume of PBS and stored at –80°C until RNA extraction. Total RNA extraction was performed using the PicoPure RNA isolation Kit (Applied Biosystems®, Foster City, CA, USA). Extracted RNA was evaluated through 2100-Bioanalyzer (Agilent Technologies®, Palo Alto, CA, USA) and DNAse treated (Qiagen®, Valencia, CA, USA). RiboAmp RNA Amplification Kit (Applied Biosystems®) was used to amplify the RNA (T7 RNA polymerase-catalysed amplification reaction). The aRNA output was evaluated through NanoDrop ND-1000 (NanoDrop Technologies®, Wilmington, DE, USA). A biotin-labelled cRNA and fragmented cRNA were obtained through 3′IVT Express Kit (Affymetrix®, Santa Clara, CA, USA) to perform the hybridization (N = 3 per group) using GeneChip Bovine Genome Array (Affymetrix®). Following hybridization, probe arrays were washed, stained, and scanned. Microarray data analysis was performed in the software FlexArray 1.6.1.1. Genes with a fold change of at least 1.5 and a probability of P < 0.05 were considered differentially expressed. The data from in vitro embryo production were analysed through the PROC GLM (SAS Institute Inc., Cary, NC, USA). Cleavage rate (81.4 ± 1.5 and 85.5 ± 1.4) and blastocyst production (41.8 ± 2.4 and 47.2 ± 2.8) were not different (P > 0.05) between low and high FCS concentrations, respectively. A total of 40 genes were differentially expressed between low and high FCS concentration. A total of 28 genes were annotated, with 37 genes up-regulated and 3 genes down-regulated by high FCS concentration. The associated network functions of gene expression, RNA damage and repair, and post-transcriptional modification; and cell-to-cell signalling and interaction were generated by Ingenuity Pathway Analysis® (Redwood City, CA, USA). Differentially expressed genes involved in carbohydrate metabolism (GAPVD1, MGAT4A), lipid metabolism (ELOVL5), cellular assembly and organisation (EZR, LRP2), and cell death and survival (DRT8) were identified. In conclusion, high FCS supplementation was associated with different expression profiles of genes regulating carbohydrate and lipid metabolism, cellular assembly and organisation, and cell death and survival. The authors acknowledge support from FAPESP and LNBio-CNPEM.

Patients who failed to conceive following an in vitro fertilization cycle can be clustered into different failure causes using gene expression hierarchical analysis†

2020 ◽  
Vol 103 (3) ◽  
pp. 599-607
Author(s):  
Chloé S Fortin ◽  
Scot Hamilton ◽  
Martin Laforest ◽  
Marie-Claude Léveillé ◽  
Marc-André Sirard
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Clustering Analysis ◽  
Gene Expression Pattern ◽  
Hierarchical Analysis ◽  
Stimulation Protocol ◽  
Follicular Cells ◽  
Vitro Fertilization ◽  
Different Response

Abstract The patient’s response to an IVF stimulation protocol is highly variable and thus difficult to predict. When a cycle fails, there are often no apparent or obvious reasons to explain the failure. Having clues on what went wrong during stimulation could serve as a basis to improve and personalize the next protocol. This exploratory study aimed to investigate if it is possible to distinguish different failure causes or different follicular responses in a population of nonpregnant IVF patients. Using qRT-PCR, we analyzed a panel of genes indicative of different failure causes in patients who did not achieve pregnancy following an IVF cycle. For each patient, a pool of follicular cells from all aspirated follicles was used as a sample which gives a global picture of the patient’s ovary and not a specific picture of each follicle. We performed hierarchical clustering analysis to split the patients according to the gene expression pattern. Hierarchical analysis showed that the population of nonpregnant IVF patients could be divided into three clusters. Gene expression was significantly different, and each cluster displayed a particular gene expression pattern. Follicular cells from patients in clusters 1, 2 and 3 displayed respectively a pattern of gene expression related to large incompetent follicles with a higher apoptosis (over matured), to follicles not ready to ovulate (under mature) and to an excess of inflammation with no visible symptoms. This study reinforces the idea that women often have different response to the same protocol and would benefit from more personalized treatments.

Global Gene Expression Analysis Demonstrates that Transforming Growth Factor β1 (TGF-β1) Signals Exclusively through Receptor Complexes Involving TGF-β Receptor I and Identifies Numerous Targets of TGF-β Signaling.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2178-2178
Author(s):  
Goran Karlsson ◽  
Yingchun Liu ◽  
Marie-José Goumans ◽  
Jonas Larsson ◽  
Ju-Seog Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Target Genes ◽  
Global Gene Expression ◽  
Differentially Expressed ◽  
Global Gene Expression Analysis ◽  
Β Receptor ◽  
Tgf Β1

Abstract In the hematopoietic system, TGF-β1 is one of the most potent extrinsic regulators, affecting both early progenitors and committed cells. At the top of the hematopoietic hierarchy, TGF-β1 maintains hematopoietic stem cells (HSCs) in quiescence in vitro through transcriptional regulation of genes encoding proteins important in the cell cycle. We have shown that TGF-β receptor I (TβRI) −/− HSCs exhibit increased proliferative capacity in vitro and that TβRII−/− mice develop a multifocal autoimmune disease, mainly mediated by T-cells (Larsson et al, 2003, Levéen et al 2002). The mechanisms of TGF-β signaling in hematopoietic cells are poorly understood and many target genes of TGF-β signaling remain elusive. In this study we have used global gene expression analysis to investigate whether all TGF-β signaling is mediated by TβRI and II. Furthermore, we asked what target genes are affected upon TGF-β stimulation in normal and TGF-β signaling deficient murine embryonic fibroblasts (MEFs). MEFs were grown with and without TGF-β1 stimulation and proliferation, transcriptional responses and expression analysis were performed. We demonstrate through Western Blot analysis, luciferase reporter assays and cell expansion experiments how these cells lack functional TβRI. Additionally, transcriptional assays show that no other Smad activity is triggered by TGF-β1 stimulation. Furthermore, we demonstrate through quantitative RT-PCR that the inhibitor of differentiation family of genes, known targets of TGF-β signaling, are not affected by TGF-β1 in TβRI−/− MEFs, while wt cells downregulate these genes 4–8.5 fold in response to stimulation. In order to completely exclude alternative receptors outside the TGF-β superfamily and signaling pathways activated through TβRII alone, we performed global gene expression profiling on TGF-β1 stimulated TβRI−/− MEFs with unstimulated TβRI deficient cells as reference. Very few (0.05 %) of the more than 37,000 spots on the microarray had a >2 fold differential expression in the two experiments conducted. Similar experiments performed on wt cells resulted in differential expression of between 2.6–3.9 % of the genes printed. From this data we conclude that no signaling affecting gene expression occur in the absence of TβRI in these cells. Additionally we present transcriptional profiles of MEF cell lines that either are normal or are TβRI deficient. By means of cDNA microarray technology, we have identified genes that were differentially expressed when TβRI deficient fibroblasts were compared to wt cells stimulated with TGF-β1. Our results create a data base of 461 significantly differentially expressed (p<0.01) target genes of TGF-β signaling. These include genes potentially responsible for the growth arrest induced by TGF-β1, like Gadd45g, Gas5, Id1, Id2 and Id3. However, the most significantly enriched number of differentially expressed genes are involved in protein folding and chaperone activities (Hspa9a, Hsp105, Hspe1, Hsp60, Cct2, Cct3, Cct8, Tcp1 and Dnaja1. Studies to identify TGF-β signaling responsive genes in HSCs are in progress.

scholarly journals Multitarget Effects of Danqi Pill on Global Gene Expression Changes in Myocardial Ischemia

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Qiyan Wang ◽  
Hui Meng ◽  
Qian Zhang ◽  
Tianjiao Shi ◽  
Xuefeng Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Myocardial Ischemia ◽  
Differentially Expressed Genes ◽  
Sham Group ◽  
Gene Expression Pattern ◽  
Enrichment Analysis ◽  
Global Gene Expression ◽  
Differentially Expressed ◽  
Group Model ◽  
Model Group

Danqi pill (DQP) is a widely prescribed traditional Chinese medicine (TCM) in the treatment of cardiovascular diseases. The objective of this study is to systematically characterize altered gene expression pattern induced by myocardial ischemia (MI) in a rat model and to investigate the effects of DQP on global gene expression. Global mRNA expression was measured. Differentially expressed genes among the sham group, model group, and DQP group were analyzed. The gene ontology enrichment analysis and pathway analysis of differentially expressed genes were carried out. We quantified 10,813 genes. Compared with the sham group, expressions of 339 genes were upregulated and 177 genes were downregulated in the model group. The upregulated genes were enriched in extracellular matrix organization, response to wounding, and defense response pathways. Downregulated genes were enriched in fatty acid metabolism, pyruvate metabolism, PPAR signaling pathways, and so forth. This indicated that energy metabolic disorders occurred in rats with MI. In the DQP group, expressions of genes in the altered pathways were regulated back towards normal levels. DQP reversed expression of 313 of the 516 differentially expressed genes in the model group. This study provides insight into the multitarget mechanism of TCM in the treatment of complex diseases.

94 EFFICIENT BOVINE EMBRYO SPLITTING FOR GENE EXPRESSION AND IN VIVO DEVELOPMENT STUDIES

2014 ◽  
Vol 26 (1) ◽  
pp. 161
Author(s):  
A. Velasquez ◽  
D. Veraguas ◽  
F. O. Castro ◽  
J. F. Cox ◽  
L. l. Rodriguez-Alvarez
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Gene Expression Pattern ◽  
Blastocyst Stage ◽  
Embryo Morphology ◽  
Bovine Embryo ◽  
Gene Markers ◽  
Developmental Potential

It is known that embryos produced in vitro are less competent than their in vivo-derived counterparts. When embryos are produced or manipulated in vitro, their developmental potential decreases significantly, which impinges upon the production of viable offspring. In bovines, embryos that will be transferred to a surrogate mother are selected at the blastocysts stage using noninvasive methods, such as their morphological features. However, many of those embryos are not able to implant or to maintain a normal pregnancy because embryo morphology does not reflect its developmental potential and a correct gene expression pattern that support a normal development. It seems that the ideal method for embryo selection would be based on the screening of gene markers that correlate with successful pregnancy after embryo transfer. In that sense, we have proposed an approach to characterise gene expression pattern of early (Day 7) bovine blastocysts and to correlate this gene expression with further developmental potential in vivo, i.e. upon elongation until Day 17. For that, it was established an efficient method to produce identical and viable hemi-embryos by splitting IVF bovine blastocysts in order to set the expression profile of certain genes in one hemi-embryo at blastocyst stage, while the counterpart embryo elongates in vivo for 10 days. A total of 129 blastocysts were split. Six groups of blastocysts were used for splitting and the results compared: 1) Day-7 early blastocysts (n = 20); 2) Day-7 expanded blastocysts (n = 25); 3) Day-7 hatched blastocysts (n = 17); 4) Day-8 early blastocysts (n = 10); 5) Day-8 expanded blastocysts (n = 12); and 6) Day-8 hatched blastocysts (n = 45). Hemi-embryos derived from day-8 grade I and well expanded blastocysts had the greatest survival rate, in vitro re-expansion (67.7%; P < 0.05) and both hemi-embryos conserved a normal morphology with a total cell number over 80 after 6 h in culture. Also both hemi-embryos at blastocyst stage showed homogeneous expression pattern of the genes OCT4, SOX2, NANOG, CDX2, ACTB, and GAPDH (P < 0.05). Finally, the in vivo survival of hemi-embryos was assessed and compared with nonsplit embryos (control) by transferring to recipient cow and collecting at Day 17 of development. For this, hemi-embryos derived from Day-8 hatched blastocyst were used. From 14 transferred hemi-embryos, 5 (35.7%) were collected, and 9 elongated from 17 controls were recovered (52.9%). Also the elongation rate was significantly lower in hemi-embryos than in control; the length of hemi-embryos had a range between 1 and 5 cm, whereas 60% of the control embryos were longer than 10 cm. Our results provide an initial approach to study the correlation among the gene expression characteristics of early bovine embryos with their further development. However, it seems that embryo splitting hampers their elongation in vivo. It might be possible that the development of split embryos is retarded because of manipulation. This work was partially supported by Fondecyt grant no. 11100082 from the Ministry of Education of Chile.

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