scholarly journals The embryonic stress response to in vitro culture: insight from genomic analysis

Reproduction ◽  
2016 ◽  
Vol 152 (6) ◽  
pp. R247-R261 ◽  
Author(s):  
Gael Cagnone ◽  
Marc-André Sirard
Keyword(s):  
Stress Responses ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Genomic Analysis ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Long Term Effects ◽  
The Impact

Recent genomic studies have shed light on the impact of in vitro culture (IVC) on embryonic homeostasis and the differential gene expression profiles associated with lower developmental competence. Consistently, the embryonic stress responses to IVC conditions correlate with transcriptomic changes in pathways related to energetic metabolism, extracellular matrix remodelling and inflammatory signalling. These changes appear to result from a developmental adaptation that enhances a Warburg-like effect known to occur naturally during blastulation. First discovered in cancer cells, the Warburg effect (increased glycolysis under aerobic conditions) is thought to result from mitochondrial dysfunction. In the case of IVC embryos, culture conditions may interfere with mitochondrial maturation and oxidative phosphorylation, forcing cells to rely on glycolysis in order to maintain energetic homeostasis. While beneficial in the short term, such adaptations may lead to epigenetic changes with potential long-term effects on implantation, foetal growth and post-natal health. We conclude that lessening the detrimental effects of IVC on mitochondrial activity would lead to significantly improved embryo quality.

254 MOLECULAR AND SUBCELLULAR CHARACTERIZATION OF OOCYTES SCREENED FOR THEIR DEVELOPMENTAL COMPETENCE BASED ON G6PDH ACTIVITY

2008 ◽  
Vol 20 (1) ◽  
pp. 207
Author(s):  
H. Torner ◽  
N. Ghanem ◽  
C. Ambros ◽  
M. Hoelker ◽  
W. Tomek ◽  
...  
Keyword(s):  
Map Kinase ◽  
Protein Biosynthesis ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Atp Synthesis ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
G6pdh Activity ◽  
Cresyl Blue

Oocyte selection based on glucose-6-phosphate dehydrogenase (G6PDH) activity has been successfully used to differentiate between competent and incompetent bovine oocytes (Alm 2005 Theriogenology 63, 2194–2205). However, the intrinsic molecular and subcellular characteristics of these oocytes have not yet been investigated. Here we aim to compare the developmental, molecular, and subcellular characteristics of oocytes selected using brilliant cresyl blue (BCB) staining based on G6PDH activity. Immature compact cumulus–oocyte complexes (COCs) were stained with 26 µm BCB (B-5388, Sigma-Alderich, Taufenkirchen, Germany) for 90 min. Based on their coloration, oocytes were divided into BCB– (colorless cytoplasm, high G6PDH activity) and BCB+ (colored cytoplasm, low G6PDH activity). The chromatin configuration and the mitochondrial activity of oocytes were determined by fluorescence labelling and photometric measurement (n = 337). The abundance and phosphorylation pattern of protein kinases Akt and MAP kinase were estimated by western blot analysis (n = 500). A bovine cDNA microarray with 2000 clones was used to analyze the gene expression profiles of BCB+ and BCB– oocytes (n = 580). BCB+ oocytes were found to result in a higher blastocyst rate (33.1 � 3.1%) until Day 8 of in vitro culture compared to BCB– ones (12.1 � 1.5%). Moreover, BCB+ oocytes showed higher phosphorylation levels of Akt and MAP kinase compared to the BCB– oocytes. After array data analysis, BCB+ oocytes were found to be enriched with genes regulating transcription (SMARCA5), cell cycle (NASP), and protein biosynthesis (RPS274A and EEF1A1), while the BCB– oocytes had a higher level of genes involved in ATP synthesis (ATP5A1), mitochondrial electron transport (FL405), calcium ion binding (S100A10), and growth factor activity (BMP-15). Independent real-time quantitative PCR validated 90% (9/10) of the genes investigated to be in agreement with the array expression profile. The study has shown evidence of differences in molecular and subcellular organization of oocytes with different G6PDH activity.

scholarly journals Integrated Genomic Analysis of Sézary Syndrome

2011 ◽  
Vol 2011 ◽  
pp. 1-13 ◽  
Author(s):  
Xin Mao ◽  
Tracy Chaplin ◽  
Bryan D. Young
Keyword(s):  
Copy Number ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Genomic Analysis ◽  
Gene Clusters ◽  
Sézary Syndrome ◽  
Sezary Syndrome ◽  
Snp Microarray ◽  
The Impact ◽  
Chromosome Regions

Sézary syndrome (SS) is a rare variant of primary cutaneous T-cell lymphoma. Little is known about the underlying pathogenesis of S. To address this issue, we used Affymetrix 10K SNP microarray to analyse 13 DNA samples isolated from 8 SS patients and qPCR with ABI TaqMan SNP genotyping assays for the validation of the SNP microarray results. In addition, we tested the impact of SNP loss of heterozygosity (LOH) identified in SS cases on the gene expression profiles of SS cases detected with Affymetrix GeneChip U133A. The results showed: (1) frequent SNP copy number change and LOH involving 1, 2p, 3, 4q, 5q, 6, 7p, 8, 9, 10, 11, 12q, 13, 14, 16q, 17, and 20, (2) reduced SNP copy number at FAT gene (4q35) in 75% of SS cases, and (3) the separation of all SS cases from normal control samples by SNP LOH gene clusters at chromosome regions of 9q31q34, 10p11q26, and 13q11q12. These findings provide some intriguing information for our current understanding of the molecular pathogenesis of this tumour and suggest the possibility of presence of functional SNP LOH in SS tumour cells.

scholarly journals Effect ofEx VivoCulture Conditions on Immunosuppression by Human Mesenchymal Stem Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Myoung Woo Lee ◽  
Dae Seong Kim ◽  
Somi Ryu ◽  
In Keun Jang ◽  
Hye Jin Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ex Vivo ◽  
Expression Profiles ◽  
Human Mesenchymal Stem Cells ◽  
Gene Expression Profiles ◽  
Culture Conditions ◽  
Seeding Density ◽  
Initial Seeding ◽  
Density Treatment

A microarray analysis was performed to investigate whetherex vivoculture conditions affect the characteristics of MSCs. Gene expression profiles were mainly influenced by the level of cell confluence rather than initial seeding density. The analysis showed that 276 genes were upregulated and 230 genes downregulated in MSCs harvested at~90% versus~50% confluence (P<0.05,FC>2). The genes that were highly expressed in MSCs largely corresponded to chemotaxis, inflammation, and immune responses, indicating direct or indirect involvement in immunomodulatory functions. Specifically, PTGES and ULBP1 were up-regulated in MSCs harvested at high density. Treatment of MSCs withPTGESorULBP1siRNA reversed their inhibition of T-cell proliferationin vitro. The culture conditions such as cell confluence at harvest seem to be important for gene expression profile of MSCs; therefore, the results of this study may provide useful guidelines for the harvest of MSCs that can appropriately suppress the immune response.

scholarly journals Breed-specific factors influence embryonic lipid composition: comparison between Jersey and Holstein

2016 ◽  
Vol 28 (8) ◽  
pp. 1185 ◽  
Author(s):  
Luis Baldoceda ◽  
Isabelle Gilbert ◽  
Dominic Gagné ◽  
Christian Vigneault ◽  
Patrick Blondin ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Lipid Composition ◽  
Lipid Droplets ◽  
Cell Damage ◽  
Poor Performance ◽  
Culture Conditions ◽  
Mitochondrial Activity ◽  
The Impact

Some embryos exhibit better survival potential to cryopreservation than others. The cause of such a phenotype is still unclear and may be due to cell damage during cryopreservation, resulting from overaccumulation and composition of lipids. In cattle embryos, in vitro culture conditions have been shown to impact the number of lipid droplets within blastomeres. Thus far, the impact of breed on embryonic lipid content has not been studied. In the present study were compared the colour, lipid droplet abundance, lipid composition, mitochondrial activity and gene expression of in vivo-collected Jersey breed embryos, which are known to display poor performance post-freezing, with those of in vivo Holstein embryos, which have good cryotolerance. Even when housed and fed under the same conditions, Jersey embryos were found to be darker and contain more lipid droplets than Holstein embryos, and this was correlated with lower mitochondrial activity. Differential expression of genes associated with lipid metabolism and differences in lipid composition were found. These results show genetic background can impact embryonic lipid metabolism and storage.

Interspecies bacterial interactions stabilize transcriptional responses of ancestral wild types and biofilm-optimised variants

2020 ◽  
Author(s):  
Mette Burmølle ◽  
Nanna Mee Coops Olsen ◽  
Samuel Jacquiod ◽  
Henriette Lyng Røder
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Niche Partitioning ◽  
Gene Expression Profiles ◽  
Biotic Interactions ◽  
Culture Conditions ◽  
Differentially Expressed ◽  
Natural Environments ◽  
Transcriptional Responses ◽  
The Impact

&lt;p&gt;Most bacteria in natural environments live in multispecies biofilms, featuring high diversity and chemical heterogeneity. The cell-to-cell proximity found in these biofilms results in biotic interactions and niche-partitioning, facilitating co-existence of species that may otherwise out-compete each other. Additionally, due to the fast generation time of microbes and ceaseless biotic interactions, biofilms accelerate adaptation through the emergence of more fit genetic variants, most probably in response to niche-partitioning and local constraints. We have previously isolated and characterized biofilm-optimised (wrinkled) variants of &lt;em&gt;Xanthomonas retroflexus&lt;/em&gt;. These variants emerged in biofilm co-cultures with &lt;em&gt;Paenibacillus amylolyticus&lt;/em&gt; and reinforced the original interspecific mutualistic interaction, due to altered c-di-GMP regulation and spatial organisation.&lt;/p&gt; &lt;p&gt;The aim of the present study was to examine the impact on gene expression profiles of either co-cultivation of the wild type (WT) or the wrinkled variant &lt;em&gt;X. retroflexus&lt;/em&gt; with &lt;em&gt;P. amylolyticus&lt;/em&gt; or its supernatant. We hypothesised that the gene expression of the two &lt;em&gt;X. retroflexus&lt;/em&gt; strains would differ significantly and that these differences would be even more pronounced when co-cultured with &lt;em&gt;P. amylolyticus&lt;/em&gt; or its supernatant.&lt;/p&gt; &lt;p&gt;Mono- and dual species biofilms were grown in 24-well plates for 24 h. The liquid culture was removed, and the remaining biofilm from the sides of the wells and the air-liquid interface was sampled and processed for mRNA sequencing. After sequencing, &lt;em&gt;X. retroflexus&amp;#160;&lt;/em&gt;reads were mapped against its concatenated genome and genes of which expression differed by fold changes of log2 &lt;-1 and &gt;1 were considered differentially expressed.&lt;/p&gt; &lt;p&gt;Unexpectedly, most marked differences in gene expression were observed when comparing mono-cultures of the WT and the wrinkled &lt;em&gt;X. retroflexus&lt;/em&gt;, as approximately 500 genes were differentially expressed in these biofilms. Of these, 30 genes were predicted to encode biofilm-associated functions. When exposed to either live &lt;em&gt;P. amylolyticus&lt;/em&gt; or its supernatant, expression profiles of the WT and the wrinkled variant were more similar, with the living partner &lt;em&gt;P. amylolyticus&lt;/em&gt; being the key factor of this stabilization. Specifically, the stabilisation was caused by opposite regulation of specific genes in the wrinkled &lt;em&gt;X. retroflexus&amp;#160;&lt;/em&gt;variant compared to the WT in mono- vs. co-culture conditions.&lt;/p&gt; &lt;p&gt;In conclusion, our data indicates that differences in gene expression of &lt;em&gt;X. retroflexus&lt;/em&gt; WT and the biofilm-optimised variant were neutralised by co-cultivation with &lt;em&gt;P. amylolyticus&lt;/em&gt;. To our knowledge, such comparative analyses of ancestral and biofilm-optimised variants have not previously been presented, despite being instrumental in elucidating evolutionary trajectories of such variants in complex environments.&lt;/p&gt;

scholarly journals Rescue Potential of Supportive Embryo Culture Conditions on Bovine Embryos Derived from Metabolically Compromised Oocytes

2020 ◽  
Vol 21 (21) ◽  
pp. 8206
Author(s):  
Anouk Smits ◽  
Jo L. M. R. Leroy ◽  
Peter E. J. Bols ◽  
Jessie De Bie ◽  
Waleed F. A. Marei
Keyword(s):  
Embryo Culture ◽  
Embryo Quality ◽  
Negative Impact ◽  
Expression Patterns ◽  
Cellular Metabolism ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
The Impact

Elevated non-esterified fatty acid (NEFA), predominantly palmitic acid (PA), concentrations in blood and follicular fluid are a common feature in maternal metabolic disorders such as obesity. This has a direct negative impact on oocyte developmental competence and the resulting blastocyst quality. We use NEFA-exposure during bovine oocyte in vitro maturation (IVM) as a model to mimic oocyte maturation under maternal metabolic stress conditions. However, the impact of supportive embryo culture conditions on these metabolically compromised zygotes are not known yet. We investigated if the addition of anti-apoptotic, antioxidative and mitogenic factors (namely, Insulin-Transferrin-Selenium (ITS) or serum) to embryo culture media would rescue development and important embryo quality parameters (cell proliferation, apoptosis, cellular metabolism and gene expression patterns) of bovine embryos derived from high PA- or high NEFA-exposed oocytes when compared to controls (exposed to basal NEFA concentrations). ITS supplementation during in vitro culture of PA-exposed oocytes supported the development of lower quality embryos during earlier development. However, surviving blastocysts were of inferior quality. In contrast, addition of serum to the culture medium did not improve developmental competence of PA-exposed oocytes. Furthermore, surviving embryos displayed higher apoptotic cell indices and an aberrant cellular metabolism. We conclude that some supportive embryo culture supplements like ITS and serum may increase IVF success rates of metabolically compromised oocytes but this may increase the risk of reduced embryo quality and may thus have other long-term consequences.

scholarly journals Bovine blastocysts with developmental competence to term share similar expression of developmentally important genes although derived from different culture environments

Reproduction ◽  
2011 ◽  
Vol 142 (4) ◽  
pp. 551-564 ◽  
Author(s):  
N Ghanem ◽  
D Salilew-Wondim ◽  
A Gad ◽  
D Tesfaye ◽  
C Phatsara ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Developmental Competence ◽  
Bovine Embryo ◽  
Placental Development ◽  
Similar Expression ◽  
Maternal Interaction

This study was conducted to investigate the gene expression profile of in vivo-derived bovine embryo biopsies based on pregnancy outcomes after transferring to recipients. For this, biopsies of 30–40% embryos were taken from grade I blastocysts (International Embryo Transfer Society Manual) and the remaining 60–70% of the intact embryos were transferred to recipients. Frozen biopsies were pooled into three distinct groups based on the pregnancy outcome after transferring the corresponding parts, namely those resulting in no pregnancy (NP), pregnancy loss (PL), and calf delivery (CD). Array analysis revealed a total of 41 and 43 genes to be differentially expressed between biopsies derived from blastocysts resulting in NP versus CD and PL versus CD respectively. Genes regulating placental development and embryo maternal interaction (PLAC8) were found to be upregulated in embryo biopsies that ended up with CD. Embryo biopsies that failed to induce pregnancy were enriched with mitochondrial transcripts (Fl405) and stress-related genes (HSPD1). Overall, gene expression profiles of blastocysts resulting in NP and CD shared similar expression profiles with respect to genes playing significant roles in preimplantation development of embryo. Finally, comparing the transcript signatures of in vivo- and in vitro-derived embryos with developmental competence to term revealed a similarity in the relative abundance of 18 genes. Therefore, we were able to present a genetic signature associated with term developmental competence independent of the environmental origin of the transferred blastocysts.

scholarly journals Effect of in vitro growth on mouse oocyte competency, mitochondria and transcriptome

Reproduction ◽  
2021 ◽  
Author(s):  
Tomoya Takashima ◽  
Tsubasa Fujimaru ◽  
Yayoi Obata
Keyword(s):  
Expression Profiles ◽  
Mammalian Species ◽  
Gene Expression Profiles ◽  
Developmental Competence ◽  
Ceramide Level ◽  
Expression Of Genes ◽  
Mitochondrial Dna Copy Number ◽  
O2 Concentration

In vitro generation of fertile oocytes has been reported in several mammalian species. However, oocyte integrity is compromised by in vitro culture. Here, we aimed to understand the factors affecting oocyte competency by evaluating mitochondrial function and transcriptome as well as lipid metabolism in in vivo-derived oocytes and in vitro grown and matured (IVGM) oocytes under atmospheric (20%) and physiological (7%) O2 concentration. We used single-cell RNA-sequencing as well as Gene Ontology and KEGG analyses to identify the molecular pathways affecting developmental competence of oocytes. Oocytes grown under 20% O2 conditions showed significant decrease in mitochondrial membrane potential, upregulation of ceramide synthesis pathway-associated genes, and high ceramide accumulation compared with oocytes grown under 7% O2 conditions and in vivo-grown oocytes. This suggests that excess ceramide level causes mitochondrial dysfunction and poor developmental ability of the oocytes. Mitochondrial DNA copy number was lower in IVGM oocytes irrespective of O2 concentration in culture, although there was no common abnormality in the expression of genes related to mitochondrial biosynthesis. In contrast, some oocytes produced under 7% O2 conditions showed gene expression profiles similar to those of in vivo-grown oocytes. In these oocytes the expression of transcription factors, including Nobox, was restored. Nobox expression correlated with the expression of genes essential for oocyte development. Thus, Nobox may contribute to the establishment of oocyte competency before and after the growth phase. The comprehensive analysis of IVGM oocytes presented here provides a platform for elucidating the mechanism underlying functional oocyte production in vivo.

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