123 Effect of heat stress on oocyte developmental competence and global gene expression dynamics in Bos taurus crossbred beef cows

It is known that animals under the effect of heat stress present reduced fertility. We aimed to investigate the effect of heat stress on the developmental competence and global gene expression profile of oocytes through the transition from spring to summer under Louisiana conditions. Oocytes were collected from 6 crossbred, non-lactating cows once a month from May to July. Temperature and humidity indexes for May and July were 72.48 and 78.06, respectively. An index above 75 indicates that cows are under heat stress. All cows underwent dominant follicle removal, and then 7 days later, ovum pickup was performed to aspirate germinal vesicle (GV) oocytes. Half of the oocytes were processed for RNA-seq as GV, and half were matured in vitro to metaphase II (MII). Smart-seq protocol was followed to prepare RNA sequencing libraries from a pool of 4 oocytes (GV n=6; MII n=6). Sequencing reads were pre-filtered and aligned to the bovine genome, and gene expression values were calculated as transcripts per million. Genes were deemed differentially expressed between different conditions if they showed a false discovery rate P-value<0.05 using DESEqn 2 package. DAVID (https://david.ncifcrf.gov) and IPA (ingenuity pathway analysis) were used to reveal the gene ontology and pathways, respectively. The RNA sequencing showed that a total of 212 genes were differentially expressed as a result of heat stress at the GV stage, with 94 and 118 genes up- and down-regulated, respectively. Gene ontology analysis indicated significant over-representation of elements involved in steroid biosynthetic process, oxidation reduction, and mitophagy in response to mitochondrial depolarization. Several pathways were influenced by heat stress, including glucocorticoid biosynthesis, apoptosis signalling, and HIPPO signalling. At the MII oocyte stage, only 93 genes (19 up-regulated and 74 down-regulated) were significantly differentially expressed in oocytes between July and May groups. Oocytes retrieved on different collection days, from the same cows under the same treatments, showed no difference on maturation rates, suggesting that the in vitro maturation process equalizes the expression of several genes. The primary biological processes significantly affected in MII oocytes were regulation of MAPK cascade, melanosome organisation, and negative regulation of transcription. In addition, we found that UBE2I, a gene involved in phosphorylation-dependent sumoylation of heat shock factor 1 (HSF1), was significantly up-regulated in July compared with May in MII oocytes. Interestingly, only 5 common genes were significantly affected by heat in both GV and MII oocytes: E2F8, GATAD2B, BHLHE41, FBXO44, and RAB39B. Our findings provide new insight into the molecular mechanisms of detrimental conditions (heat stress) on bovine oocytes, which may help to reveal master regulators controlling oocyte competence.

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Integrative In Silico and In Vitro Transcriptomics Analysis Revealed Gene Expression Changes and Oncogenic Features of Normal Cholangiocytes after Chronic Alcohol Exposure

International Journal of Molecular Sciences ◽

10.3390/ijms20235987 ◽

2019 ◽

Vol 20 (23) ◽

pp. 5987

Author(s):

Suthipong Chujan ◽

Tawit Suriyo ◽

Jutamaad Satayavivad

Keyword(s):

Gene Expression ◽

Rna Polymerase Ii ◽

Molecular Mechanisms ◽

Function Analysis ◽

Alcohol Exposure ◽

Gene Expression Omnibus ◽

Regulation Of Transcription ◽

Chronic Alcohol ◽

Chronic Alcohol Exposure

Cholangiocarcinoma (CCA) is a malignant tumor originating from cholangiocyte. Prolonged alcohol consumption has been suggested as a possible risk factor for CCA, but there is no information about alcohol’s mechanisms in cholangiocyte. This study was designed to investigate global transcriptional alterations through RNA-sequencing by using chronic alcohol exposure (20 mM for 2 months) in normal human cholangiocyte MMNK-1 cells. To observe the association of alcohol induced CCA pathogenesis, we combined differentially expressed genes (DEGs) with computational bioinformatics of CCA by using publicly gene expression omnibus (GEO) datasets. For biological function analysis, Gene ontology (GO) analysis showed biological process and molecular function related to regulation of transcription from RNA polymerase II promoter, while cellular component linked to the nucleoplasm. KEGG pathway presented pathways in cancer that were significantly enriched. From KEGG result, we further examined the oncogenic features resulting in chronic alcohol exposure, enhanced proliferation, and migration through CCND-1 and MMP-2 up-regulation, respectively. Finally, combined DEGs were validated in clinical data including TCGA and immunohistochemistry from HPA database, demonstrating that FOS up-regulation was related to CCA pathogenesis. This study is the first providing more information and molecular mechanisms about global transcriptome alterations and oncogenic enhancement of chronic alcohol exposure in normal cholangiocytes.

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193 DIFFERENTIAL GENE EXPRESSION IN CUMULUS CELLS OF DEVELOPMENTALLY COMPETENT V. CHALLENGED BOVINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab193 ◽

2009 ◽

Vol 21 (1) ◽

pp. 195 ◽

Cited By ~ 1

Author(s):

R. R. Payton ◽

L. A. Rispoli ◽

J. L. Edwards

Keyword(s):

Gene Expression ◽

Gene Ontology ◽

Heat Stress ◽

Empirical Bayes ◽

Rna Extraction ◽

Cumulus Cells ◽

Developmental Competence ◽

Blastocyst Development ◽

Oocyte Competence ◽

Extracellular Region

It is well established that exposure of cumulus–oocyte complexes (COC) to heat stress during the first 12 h of maturation reduces blastocyst development by 42 to 65%. Previous research supports the notion that some of the effects of heat stress on oocyte competence may be cumulus-mediated. To determine the extent to which this may occur, COC were matured at 38.5°C for 24 h (control) or 41°C for the first 12 h of maturation followed by 38.5°C for remaining 12 h (heat stress). A subset of COC underwent IVF with Percoll-prepared sperm and then was cultured in KSOM containing 0.5% BSA to assess developmental competence. Remaining oocytes were denuded. Cumulus cells, kept separate by treatment, were stored in lysis buffer at –80°C until RNA extraction. Total RNA from cumulus was amplified prior to hybridization to bovine Affymetrix GeneChips (Affymetrix Inc., Santa Clara, CA, USA; n = 8 pools per treatment collected on 8 different occasions; n = 16 chips). Following pre-processing using the MAS5.0 algorithm, microarray data were subjected to linear modeling and empirical Bayes analyses (Bioconductor, Limma package). False discovery rate was controlled using the Benjamini and Hochberg method, and differentially expressed genes were selected by an adjusted P-value (P < 0.05). Functional annotation of selected genes was performed using NetAffx (Affymetrix Inc.) and Database for Annotation, Visualization and Integrated Discovery (DAVID; NIAID, NIH, Bethesda, MD, USA). Heat stress of COC reduced blastocyst development (27.2 v. 16.1% for control v. heat stress, respectively; SEM = 1.6; P < 0.002). Approximately 66 and 65% of 24 000 possible genes were called present (i.e. expressed) in RNA from cumulus of competent (control) v. challenged (heat-stressed) oocytes, respectively. In cumulus from developmentally challenged COC, increased abundance of 42 genes (36 currently annotated) was noted. Use of DAVID demonstrated enrichment of genes important for electron transport and energy generation (NOS2A, MAOB, CYP11A1, HSD11B1L, LTB4DH). Further examination of gene ontology identified genes associated with mitochondrial function (SLC25A10, MAOB, CYP11A1), cell signaling (similar to JAK-3, FSHR, CYP11A1, WNT2B), cytoskeleton (ACTA1), antioxidant activity (GSTA1), and extracellular region (FMOD). In contrast, cumulus from developmentally competent COC had increased expression of 22 genes (20 currently annotated), of which 15% were related to protein binding (CAV1, MMP9, TGFB2) according to DAVID. Further analysis using gene ontology revealed genes associated with extracellular matrix formation (MMP9, MMP19, PCOLCE2) and neural tissue (METRNL). In summary, alterations in cumulus gene expression were associated with differences in developmental competence of oocytes. Additional research is necessary to examine the extent to which identified genes account for functional differences in oocyte competence. This research was supported in part by National Research Initiative Competitive Grant no. 2004-35203-14772 from the USDA Cooperative State Research, Education, and Extension Service.

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3 IN VITRO MATURATION ALTERS GENE EXPRESSION IN MOUSE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab3 ◽

2008 ◽

Vol 20 (1) ◽

pp. 82

Author(s):

M. Paczkowski ◽

C. Bidwell ◽

D. Spurlock ◽

J. Waddell ◽

R. L. Krisher

Keyword(s):

Gene Expression ◽

Cell Death ◽

Dna Methyltransferase ◽

In Vitro Maturation ◽

Fold Increase ◽

Developmental Competence ◽

Differentially Expressed ◽

Dna Methyltransferase 1

The in vitro culture environment significantly impacts nuclear maturation, fertilization, embryonic development, and epigenetic competence; however, our knowledge of the effects of in vitro maturation on oocyte developmental competence, and specifically cytoplasmic maturation, is limited. The objective of this experiment was to identify alterations in the transcriptome of oocytes matured in vitro compared to those matured in vivo that correlate to developmental competence. Immature oocytes were collected from Day 26 and 7-8-week-old B6D2F1 mice 48 h post-pregnant mare serum gonadotropin (PMSG) administration and matured for 16 h in Gmat supplemented with 0.5 mm citric acid, 0.5 mm cysteamine, 100 ng mL–1 epidermal growth factor (EGF), 0.05% insulin-transferrin-selenium (ITS; v/v), 0.01% recombumin (v/v) and 2 mg mL–1 fetuin. In vivo-matured oocytes from females of the same ages were collected from the oviducts 62 h post-PMSG and 14 h post-hCG and mating to vasectomized males. In vivo- and in vitro-matured oocytes were identified visually by the presence of the first polar body. Mature oocytes were pooled into three groups of 150 oocytes per treatment and lysed; poly A+ RNA was extracted. Samples were processed through two cycles of linear amplification and hybridized to the GeneChip� Mouse Genome 430 2.0 Array (Affymetrix, Inc., Santa Clara, CA, USA), with three arrays per treatment. Microarray data were sorted and filtered to include genes that were classified as having two present calls per treatment. The data were then normalized to the chip median and analyzed using a one-way analysis of variance; the level of significance was calculated at P < 0.01. In total, 2.17% (482/22170) and 1.61% (358/22170) of genes were differentially expressed between in vitro- and in vivo-matured oocytes in Day 26 and 7–8-week-old mice, respectively. However, 72.82% (351/482) and 67.87% (243/358) of differentially expressed genes had increased abundance in the in vitro- and in vivo-matured oocytes, respectively. Transcripts involved in gene expression, cellular growth and proliferation, and cellular development were increased in in vivo-matured oocytes from both age groups compared to those matured in vitro. Cell death was one of the higher ranking functional groups increased in the 7–8-week-old in vitro-matured oocytes compared to the 7–8-week-old in vivo-matured oocytes. Specific genes altered by in vitro maturation conditions in Day 26 oocytes were DNA methyltransferase 1 (>7-fold increase in vivo), caspase 8 (>4-fold increase in vivo), and eukaryotic translation initiation factor 1B (>4-fold increase in vivo). DNA methyltransferase 1 and ubiquitin-conjugating enzyme E2T were significantly increased in in vivo-matured 7–8-week-old oocytes (>3-fold and >5-fold, respectively). These results indicate that gene expression is altered in oocytes matured in vitro compared to those matured in vivo. Based on the functional annotations of genes differentially expressed, dysregulation of gene expression in the oocyte resulting in altered DNA methylation and an up-regulation in cell death pathways are potential developmental mechanisms influenced by in vitro culture conditions that correlate to reduced embryonic developmental potential.

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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 ◽

10.1182/blood.v104.11.2178.2178 ◽

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.

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

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab48 ◽

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.

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Elevated expression of NEU1 sialidase in idiopathic pulmonary fibrosis provokes pulmonary collagen deposition, lymphocytosis, and fibrosis

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00346.2015 ◽

2016 ◽

Vol 310 (10) ◽

pp. L940-L954 ◽

Cited By ~ 15

Author(s):

Irina G. Luzina ◽

Virginia Lockatell ◽

Sang W. Hyun ◽

Pavel Kopach ◽

Phillip H. Kang ◽

...

Keyword(s):

Gene Expression ◽

Endothelial Cells ◽

Idiopathic Pulmonary Fibrosis ◽

Pulmonary Fibrosis ◽

Molecular Mechanisms ◽

Transforming Growth Factor ◽

Recombinant Adenovirus ◽

Global Gene Expression

Idiopathic pulmonary fibrosis (IPF) poses challenges to understanding its underlying cellular and molecular mechanisms and the development of better therapies. Previous studies suggest a pathophysiological role for neuraminidase 1 (NEU1), an enzyme that removes terminal sialic acid from glycoproteins. We observed increased NEU1 expression in epithelial and endothelial cells, as well as fibroblasts, in the lungs of patients with IPF compared with healthy control lungs. Recombinant adenovirus-mediated gene delivery of NEU1 to cultured primary human cells elicited profound changes in cellular phenotypes. Small airway epithelial cell migration was impaired in wounding assays, whereas, in pulmonary microvascular endothelial cells, NEU1 overexpression strongly impacted global gene expression, increased T cell adhesion to endothelial monolayers, and disrupted endothelial capillary-like tube formation. NEU1 overexpression in fibroblasts provoked increased levels of collagen types I and III, substantial changes in global gene expression, and accelerated degradation of matrix metalloproteinase-14. Intratracheal instillation of NEU1 encoding, but not control adenovirus, induced lymphocyte accumulation in bronchoalveolar lavage samples and lung tissues and elevations of pulmonary transforming growth factor-β and collagen. The lymphocytes were predominantly T cells, with CD8+ cells exceeding CD4+ cells by nearly twofold. These combined data indicate that elevated NEU1 expression alters functional activities of distinct lung cell types in vitro and recapitulates lymphocytic infiltration and collagen accumulation in vivo, consistent with mechanisms implicated in lung fibrosis.

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Construction and analysis of mRNA, miRNA, lncRNA, and TF regulatory networks reveal the key genes in prostate cancer

10.1101/323543 ◽

2018 ◽

Author(s):

Su-Liang Li ◽

Yun Ye ◽

Sheng-Yu Wang

Keyword(s):

Gene Expression ◽

Prostate Cancer ◽

Large Scale ◽

Molecular Mechanisms ◽

Scale Effects ◽

Expression Profiles ◽

Differentially Expressed ◽

Regulation Of Transcription ◽

Normal Prostate ◽

Ppi Networks

AbstractPurpose: Prostate cancer (PCa) causes a common male urinary system malignant tumour, and the molecular mechanisms of PCa remain poorly understood. This study aims to investigate the underlying molecular mechanisms of PCa with bioinformatics.Methods: Original gene expression profiles were obtained from the GSE64318 and GSE46602 datasets in the Gene Expression Omnibus (GEO). We conducted differential screens of the expression of genes (DEGs) between two groups using the R software limma package. The interactions between the differentially expressed miRNAs, mRNAs and lncRNAs were predicted and merged with the target genes. Co-expression of the miRNAs, lncRNAs and mRNAs were selected to construct the mRNA-miRNA and-lncRNA interaction networks. Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed for the DEGs. The protein-protein interaction (PPI) networks were constructed, and the transcription factors were annotated. The expression of hub genes in the TCGA datasets was verified to improve the reliability of our analysis.Results: The results demonstrated that 60 miRNAs, 1578 mRNAs and 61 lncRNAs were differentially expressed in PCa. The mRNA-miRNA-lncRNA networks were composed of 5 miRNA nodes, 13 lncRNA nodes, and 45 mRNA nodes. The DEGs were mainly enriched in the nuclei and cytoplasm and were involved in the regulation of transcription, related to sequence-specific DNA binding, and participated in the regulation of the PI3K-Akt signalling pathway. These pathways are related to cancer and focal adhesion signalling pathways. Furthermore, we found that 5 miRNAs, 6 lncRNAs, 6 mRNAs and 2 TFs play important regulatory roles in the interaction network. The expression levels of EGFR, VEGFA, PIK3R1, DLG4, TGFBR1 and KIT were significantly different between PCa and normal prostate tissue.Conclusion: Based on the current study, large-scale effects of interrelated mRNAs, miRNAs, lncRNAs, and TFs were revealed and a model for predicting the mechanism of PCa was provided. This study provides new insight for the exploration of the molecular mechanisms of PCa and valuable clues for further research.

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Hypoxia Dependent and Independent Dysregulation of the Transcriptome in Sickle Cell Anemia

Blood ◽

10.1182/blood-2019-132078 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2262-2262

Author(s):

Xu Zhang ◽

Jihyun Song ◽

Binal N. Shah ◽

Taif Hassan ◽

Galina Miasnikova ◽

...

Keyword(s):

Gene Expression ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Research Funding ◽

Molecular Mechanisms ◽

Target Genes ◽

Differentially Expressed ◽

Specific Gene ◽

Regulation Of Transcription ◽

Disease Specific

Sickle cell anemia (SCA) is associated with an increased hypoxic response from anemia and vaso-occlusion-impaired tissue perfusion. The effects of hypoxia are mediated by hypoxia transcription factors (HIFs). Chuvash erythrocytosis (CE) is an inherited condition due to homozygosity for the missense mutation in VHL gene (VHLR200W) that impairs interactions of VHL with HIF-α subunits, thereby augmenting transcription of HIF-regulated genes. CE and SCA share increased expression of erythropoietin (EPO) and other HIF target genes. As HIF-regulation of transcription is tissue and differentiation-stage specific, in this study we used reticulocytes, which are easily accessible and purified peripheral blood erythroid cells. We compared the transcriptomes of SCA and CE reticulocytes to differentiate HIF-mediated dysregulation from non-hypoxic dysregulation of SCA transcripts. Our study revealed reticulocyte gene expression changes that are common to both diseases as well as SCA-specific changes. The reticulocytes were purified from 5 HbSS and 5 age- and gender-matched HbAA African-American individuals, and from 17 VHLR200W homozygotes from Chuvashia and 13 wild type Chuvash individuals. Total RNA was depleted of ribosomal RNA and globin transcripts, and reverse transcribed. Strand-specific libraries were constructed for 100 or 125 bp paired-end sequencing to 30-45 million reads using Illumina HiSeq 2500 or 4000 platform. The sequencing data were mapped to human reference genome version GRCh37 using the splice-aware aligner STAR and analyzed using DESeq2. In separate analyses of the two diseases, we identified 1435 genes differentially expressed in SCA among 6965 analyzed genes, 848 increased and 587 decreased in SCA relative to control individuals. We also identified 1498 genes differentially expressed in CE among 8989 analyzed genes, 862 increased and 636 decreased in CE relative to control individuals. Across all analyzed genes, there was a moderate correlation (r=0.30) of expression changes between the two diseases. Among genes differentially expressed, 258 up-regulated and 155 down-regulated genes overlapped between the two diseases, representing a 1.4-fold enrichment. In a combined analysis of the two diseases, we identified 1228 genes among 6924 analyzed genes that shared altered regulation in both diseases. The 693 genes increased in both diseases were enriched (adjusted P < 0.05) in multiple metabolic, inflammatory, and oxidative pathways. The 535 genes decreased in both diseases were enriched in a cell cycle pathway. Among the commonly increased genes, the expression level of ERFE encoding erythroferrone was increased by 9.4-fold in CE and by 4.3-fold in SCA, suggesting markedly altered iron regulation in CE and SCA. This would be expected as both SCA and CE share upregulated erythropoiesis that is associated with augmented erythroferrone. Among the commonly decreased genes, RPL3L encoding Ribosomal Protein L3 Like was decreased by 68% in CE and by 93% in SCA. These common expression changes reflect hypoxic regulation related to chronic anemia in SCA. To assess disease-specific gene expression change, we tested the disease (SCA versus CE) by genotype (mutation versus wildtype) interaction effect. We identified 822 genes that showed disease-specific expression changes among 6924 analyzed genes. Of these disease-specific genes, 304 were increased and 153 decreased in SCA (adjusted P <0.05) but not in CE (nominal P >0.05). For example, SLC16A1, encoding Solute Carrier Family 16 Member 1 and associated with an erythrocyte lactate transporter defect, was increased in SCA by 11-fold whereas PPBP encoding Pro-Platelet Basic Protein was decreased in SCA by 92%; both genes showing no change in CE. The 304 genes with SCA-specific increased expression were enriched in "Thyroid hormone signaling" (5.1 fold) and "Glioma" (7.0-fold) pathways. The 153 genes with SCA-specific decreased expression were enriched in "Ribosome" (12-fold) pathway. Among the disease-specific genes, only 34 increased and 28 decreased in CE but not in SCA. Our study demonstrates high HIF transcriptional activity in both CE and SCA reticulocytes but also reveals hypoxia-independent gene expression changes in SCA reticulocytes. These results suggest that HIF might be a therapeutic target of SCA. These data also shed light on the different molecular mechanisms underlying SCA complications. Disclosures Gordeuk: Pfizer: Research Funding; Modus Therapeutics: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Emmaus: Consultancy, Honoraria; Global Blood Therapeutics: Consultancy, Honoraria, Research Funding; Inctye: Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Ironwood: Research Funding; Imara: Research Funding.

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80 GLOBAL GENE EXPRESSION PATTERNS OF FRESH AND VITRIFIED IN VITRO-PRODUCED BOVINE BLASTOCYSTS

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab80 ◽

2013 ◽

Vol 25 (1) ◽

pp. 187

Author(s):

M. J. Sudano ◽

E. S. Caixeta ◽

D. M. Paschoal ◽

T. S. Rascado ◽

L. F. Crocomo ◽

...

Keyword(s):

Gene Expression ◽

Messenger Rna ◽

Expression Patterns ◽

Rna Extraction ◽

Global Gene Expression ◽

Differentially Expressed ◽

Microarray Data Analysis ◽

Gene Expression Patterns ◽

Mrna Levels

Over the past decades, there have been great advances in in vitro production (IVP) systems with improved culture methods and new knowledge regarding embryo genetics, physiology, ultrastructure, and morphology. Nevertheless, a major obstacle for dissemination of this technology is the great sensitivity of IVP embryos to cryopreservation. The objective was to study the global gene-expression patterns of fresh and vitrified IVP bovine embryos. Oocytes (N = 1290) were matured and fertilized in vitro (Day 0). Presumptive zygotes were cultured in SOFaa with 0.5% BSA and 2.5% of FCS. Cleavage and blastocyst production was evaluated after 3 and 7 days under standard culture conditions (at 38.5°C in atmosphere of 5% O2, 5% CO2, and 90% N2). On Day 7, half of the blastocysts were vitrified (n = 94), warmed (Sudano et al. 2011 Theriogenology 75, 1211–1220), and returned for 24 h of additional culture (re-expansion and hatching; hatched was evaluated 12 and 24 h after warming, respectively) when their RNA was extracted (vitrified group). The remaining embryos returned to culture until Day 8 when their RNA was extracted (fresh group). Total RNA extraction of a single blastocyst was performed using the PicoPure Kit (Applied Biosystems®, Foster City, CA, USA). The RNA samples were DNAse treated (Qiagen®, Valencia, CA, USA), and mRNA was amplified (RiboAmp Kit®). The aRNA output was evaluated with a NanoDrop (Thermo®, Wilmington, DE, USA) and Bioanalyzer (Agilent®, Santa Clara, CA, USA). Biotin-labelled and fragmented cRNA were obtained with the 3′IVT Kit (Affymetrix®, Santa Clara, CA, USA) to perform hybridization (N = 6–7, respectively, for vitrified and fresh groups) using the GeneChip Bovine Array (Affymetrix®). Microarray data analysis was performed with the FlexArray 1.6.1.1. Genes with a fold change of at least 2 and a probability of P ≤ 0.05 were considered differentially expressed. Real-time PCR was used to validate microarray results (N = 11–15, respectively, for vitrified and fresh groups). As a control, a pool of 200 blastocysts was submitted or not to mRNA amplification followed by the reverse transcription and qPCR of 17 genes. For statistical analyses, PROC GLIMMIX, PROC LOGISTIC, and PROC CORR were used. Cleavage and blastocyst production rates were 86.8 ± 1.0 and 32.5 ± 1.9%, respectively. Re-expansion and hatching/hatched rates were 69.3 and 19.3%, respectively. Messenger RNA abundance of amplified and nonamplified RNA had a high correlation (r = 0.89, P < 0.01). The microarray analysis indicated 383 differentially expressed genes (P ≤ 0.05) between fresh and vitrified blastocysts. Genes involved in apoptosis (PRDX2), heat shock (HSPA5), maternal recognition of pregnancy (IFNT2 and PAG2), and cell differentiation and placenta formation (KRT18) were downregulated in vitrified embryos. According to qPCR analysis, mRNA abundance of IFNT2, PRDX2, and KRT18 was downregulated, whereas HSPA5 mRNA levels were upregulated in vitrified blastocysts. Messenger RNA abundance of PAG2 was not different (P = 0.46) between fresh and vitrified embryos. In conclusion, vitrification alters the expression profile of the genes IFNT2, PRDX2, KRT18, and HSPA5 that can be related with embryo postcryopreservation survival capacity. FAPESP and LNBio-CNPEM are acknowledged.

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263 GENE EXPRESSION PROFILING OF IMMATURE AND IN VITRO-MATURED BOVINE OOCYTES USING AFFYMETRIX GENECHIP TECHNOLOGY

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab263 ◽

2007 ◽

Vol 19 (1) ◽

pp. 248 ◽

Cited By ~ 1

Author(s):

F. Carter ◽

T. Fair ◽

S. Park ◽

M. Wade ◽

A. C. O. Evans ◽

...

Keyword(s):

Gene Expression ◽

Expression Profiles ◽

Bovine Genome ◽

Calf Serum ◽

Developmental Competence ◽

Differentially Expressed ◽

Total Rna ◽

Rna Content ◽

Bovine Oocytes

Previous studies by our group have demonstrated that oocyte maturation is a crucial event in the determination of subsequent developmental competence. The objective of the current study was to characterize changes in gene expression profiles of bovine oocytes during meiotic maturation. To this end, 5 replicate pools of 200 bovine cumulus–oocyte complexes (COCs)were collected from the ovaries of slaughtered heifers. Upon recovery, 100 COCs from each replicatewere immediately denuded, and the oocytes were snap frozen in liquid nitrogen. The remaining 100 COCs were matured in vitro in TCM-199 supplemented with 10% (v/v) fetal calf serum and 10 ngmL-1 epidermal growth factor for 24 h at 39�C under an atmosphere of 5% CO2 in air with maximum humidity. Following maturation, the remaining COCs were denuded and snap frozen. Total RNA was isolated (mean total RNA content 106.08�38.87 ng per 100 oocytes) and subjected to 2 rounds of amplification incorporating biotin-labeled nucleotides during the second in vitro transcription reaction (mean total RNA content 155.15�51.14 �g per 100 oocytes post-amplification). The resulting labeled antisense RNA was hybridized to a GeneChip Bovine Genome Arrays (Affymetrix, Inc., Santa Clara, CA, USA) (10 chips, 5 replicates each of immature and mature oocytes, n=100 oocytes/chip). Expression data were analysed using Genespring software (Agilent Technologies, Palo Alto, CA, USA), and data were normalized to the median. Overall, 54.9�1.3% and 53.3�3.3% of the 24 178 probe sets representing 23 000 transcripts spotted on the arrays were expressed in immature and in vitro-matured oocytes, respectively. Across the 5 array comparisons, 52 genes were consistently exclusively present in immature oocytes, whereas 16 genes were exclusively present in mature oocytes. A further 821 genes were found to be differentially expressed (≥2-fold) between the 2 groups (P <0.05), of which 209 were up-regulated and 612 were down-regulated in the in vitro-matured oocytes compared with their immature counterparts. The differentially expressed transcripts were classified according to their gene ontology (http://benzer.ubic.ca/ermineJ). The existing Affymetrix annotation was updated by blasting the sequences against bovine, human, and murine databases (≥90% homology; increasing molecular function annotation from 14% to 42%). In terms of olecular function, the majority of these genes were associated with protein or nucleic acid binding (>42%), catalytic activity (24%), signal transduction (7%), transporter activity (5%), and structural molecule activity (5%). In conclusion, we have stablished the molecular transcriptome blueprint of immature and in vitro-matured bovine oocytes. Through comparisons with in vivo-matured oocytes, this resource will be invaluable in determining genes that are involved in controlling the developmental competence of oocytes. This research was funded by the Science Foundation Ireland (02/IN1/B78).

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