103 GENE EXPRESSION PROFILES OF VITRIFIED MOUSE EMBRYOS DETECTED BY MICROARRAYS

Very little is known about the effect of vitrification on gene functions after warming. The goals of our study were to examine the transcript variations and identify genes most affected by the treatment. For this, 8-cell-stage embryos were collected from female ICR mice mated with ICR males. The embryos were washed with CZB-HEPES base medium and suspended briefly in equilibrium medium consisting of 4% ethylene glycol (EG) in base medium at room temperature. Following equilibration, the embryos were vitrified in a 35% EG, 0.4 M trehalose, 5% polyvinylpyrrolidone (PVP) solution by means of a solid-surface vitrification (SSV) technique as described earlier (Dinnyes 2000 Biol. Reprod. 63, 513-518). Then 40 embryos each from the control and the vitrified/warmed groups were cultured in CZB medium for 3 h. Total RNAs were extracted from cultured embryos in each group using TRIzol (Invitrogen, Bio-Science, Ltd., Budapest, Hungary), following the manufacturer's instructions. Two rounds of amplification were employed to produce labeled RNA, using low input RNA amplification kit (Agilent Technologies, Kromat, Ltd., Budapest, Hungary) procedures with modifications. Three micrograms of contrasting RNA samples were hybridized on the Agilent Mouse 22K oligonucleotide slides with subsequent analysis of the results. Moreover, as an independent analysis tool, real time PCR was used with eight designed primers. All of the vitrified embryos were recovered after warming with no morphological signs of cryodamage and used for analysis. The two rounds of amplification yielded 15-16 �g of cRNA. The analysis of repeated hybridizations by Rosetta luminator software (Agilent) showed 20 183 genes and expressed sequence tags (ESTs) that passed the selection criteria and were identified as common signatures in all of the slides. Unsupervised analysis of the gene expression data identified a total of 631 differentially expressed (P < 0.01) genes. However, to support the reliability of the results, only those variations above 1.5 fold differences were considered as significant in the final analysis. Therefore, with this stringent criterion 183 genes were differentially expressed (P < 0.01), of which 109 were up-regulated and the remainder down-regulated. Although genes have multiple and overlapping functions, most of the differentially expressed genes were functionally classified into various physiological categories. These include stress response (8), apoptosis related (6), metabolism (51), temperature response (4), and transcription regulation (15). Moreover, the independent analysis with real time PCR and unamplified samples verified the results of microarray. Thus, based on confirmation of the results by an independent analysis and support by the previous studies for some of the genes, it is possible to conclude that the expression patterns reflect the true biological image of embryos after vitrification, with most effects on stress- and cell metabolism-related genes. This work was supported by EU FP6 (MEXT-CT-2003-59582), Wellcome Trust Foundation (Grant No. 070246), and National Office of Research and Technology (NKTH) (#BIO-00017/2002, #BIO-00086/2002).

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171 THE EFFECT OF TRICHOSTATIN A ON THE DEVELOPMENT AND THE GLOBAL GENE EXPRESSION PATTERNS IN MOUSE EMBRYOS DEVELOPING IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab171 ◽

2008 ◽

Vol 20 (1) ◽

pp. 165

Author(s):

X. S. Cui ◽

X. Y. Li ◽

T. Kim ◽

N.-H. Kim

Keyword(s):

Gene Expression ◽

Trichostatin A ◽

Expression Profiles ◽

Expression Patterns ◽

Gene Expression Profiles ◽

Mouse Embryos ◽

Differentially Expressed ◽

Gene Expression Patterns ◽

Cell Stage

Trichostatin A (TSA) is an inhibitor of histone deacetylase and is able to alter gene expression patterns by interfering with the removal of acetyl groups from histones. The aim of this study was to determine the effect of TSA treatment on the development and gene expression patterns of mouse zygotes developing in vitro. The addition of 100 nm TSA to the culture medium did not affect the cleavage of mouse embryos (TSA treatment, 148/150 (99%) v. control, 107/107 (100%)); however, embryos that were treated with TSA arrested at the 2-cell stage (145/148, 98%). We estimated the number of nuclei in control and TSA-treated embryos by propidium iodide staining, taking into account the presence of any cells with two or more nuclei. At 62–63 h post-hCG stimulation, control zygotes had developed to the 4-cell stage and exhibited one nucleus in each blastomere, indicative of normal development. In contrast, we observed tetraploid nuclei in at least one blastomere in 20.8% (11/53) of the embryos that had been treated with TSA. At 28–29 h post-hCG stimulation (metaphase of the 1-cell stage), there was no difference in the mitotic index (as determined by analyzing the microtubule configuration) in the TSA group compared to the control group. At the 2-cell stage, however, we did not observe mitotic spindles and metaphase chromatin in embryos in the TSA treatment group compared to the controls. Interestingly, when embryos were cultured in TSA-free medium from 35 h post-hCG stimulation (S- or early G2-phase of the 2-cell stage) onward, almost all of them (47/50) developed to the blastocyst stage. In contrast, when embryos were cultured in TSA-free medium from 42 h post-hCG stimulation (middle G2-phase of the 2-cell stage) onward, they did not develop to the 4-cell stage. We used Illumina microarray technology to analyze the gene expression profiles in control and TSA-treated late 2-cell-stage embryos. Applied Biosystems Expression System software was used to extract assay signals and assay signal-to-noise ratio values from the microarray images. Our data showed that 897 genes were significantly (P < 0.05; 2-sample t-test) up- or down-regulated by TSA treatment compared to controls. Analysis using the PANTHER classification system (https://panther.appliedbiosystems.com) revealed that the 575 genes that were differentially expressed in the TSA group compared to the control were classified as being associated with putative biological processes or molecular function. Overall, in terms of putative biological processes, more nucleoside, nucleotide, and nucleic acid metabolism, protein metabolism and modification, signal transduction, developmental process, and cell cycle genes were differentially expressed between the TSA and control groups. In terms of putative molecular function, more nucleic acid-binding transcription factor and transferase genes were differentially expressed between the groups. The results collectively suggest that inhibition of histone acetylation in mouse embryos affects gene expression profiles at the time of zygotic genome activation, and this subsequently affects further development.

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Gene expression profiling of skeletal muscles

10.1101/2021.02.17.431599 ◽

2021 ◽

Author(s):

Sarah I. Alto ◽

Chih-Ning Chang ◽

Kevin Brown ◽

Chrissa Kioussi ◽

Theresa M. Filtz

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Differentially Expressed Genes ◽

Tibialis Anterior ◽

Skeletal Muscles ◽

Expression Profiles ◽

Expression Patterns ◽

Gene Expression Profiles ◽

Differentially Expressed ◽

Analysis Tool

AbstractSoleus and tibialis anterior are two well-characterized skeletal muscles commonly utilized in skeletal muscle-related studies. Next-generation sequencing provides an opportunity for an in-depth biocomputational analysis to identify the gene expression patterns between soleus and tibialis anterior and analyze those genes’ functions based on past literature. This study acquired the gene expression profiles from soleus and tibialis anterior murine skeletal muscle biopsies via RNA-sequencing. Read counts were processed through edgeR’s differential gene expression analysis. Differentially expressed genes were filtered down using a false discovery rate less than 0.05c, a fold-change value larger than twenty, and an association with overrepresented pathways based on the Reactome pathway over-representation analysis tool. Most of the differentially expressed genes associated with soleus encoded for components of lipid metabolism and unique contractile elements. Differentially expressed genes associated with tibialis anterior encoded mostly for glucose and glycogen metabolic pathways’ regulatory enzymes and calcium-sensitive contractile components. These gene expression distinctions partly explain the genetic basis for muscle specialization and may help to explain skeletal muscle susceptibility to disease and drugs and refine tissue engineering approaches.

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189 TRANSCRIPTIONAL PROFILING OF HISTONE-MODIFYING GENES DURING BOVINE PRE-IMPLANTATION EMBRYO DEVELOPMENT IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab189 ◽

2009 ◽

Vol 21 (1) ◽

pp. 193

Author(s):

G. D. Linger ◽

C. L. Bormann ◽

M. D. Peoples ◽

M. C. Golding ◽

C. R. Long

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Real Time ◽

Real Time Pcr ◽

Expression Profiles ◽

Geometric Mean ◽

Cell Stage ◽

Embryonic Genome ◽

Morula Stage

The proper removal of gametic epigenetic marks and coordinated re-establishment of the epigenome is critical to mammalian embryonic development. This global reprogramming of the embryonic genome includes fluctuations in both DNA methylation and histone modifications that are necessary to control chromatin structure and thus gene expression. In the bovine model, epigenetic changes occur from fertilization through blastocyst stages; in particular, and concurrent with the maternal-embryonic transition, de novo DNA methylation begins at the 8-cell stage. In order to understand which factors might be playing key roles in this epigenetic process, we used quantitative real-time PCR to characterize the temporal expression profiles of several genes involved in DNA and/or histone methylation: G9a, SetB1, Suv39h1, Suv420h1, SmyD3, Suz12, and LSH. Bovine ova and embryos were produced via in vitro maturation, fertilization, and culture from multiple pools of ova. Groups of 12–25 bovine ova or embryos, pooled at the 2-, 4 to 7-, mid 8-, late 8-, 12 to 16-cell, morula, and blastocyst stages, were washed twice through 1X PBS and stored in RNA lysis buffer at –80°C until further use. RNA was isolated from each sample using the RNeasy® Mini kit (Qiagen, Valencia, CA, USA), optimized for isolating RNA from single embryos, and treated to remove any contaminating genomic DNA. cDNA was generated with iScript™ reverse transcriptase (Bio-Rad Laboratories, Hercules, CA, USA) and diluted 1:10 with RNase/DNase-free water for further use in real-time PCR. Relative gene expression from each RNA sample was calculated in triplicate using the SYBR Green comparative Ct method (Applied Biosystems, Foster City, CA, USA) adjusted for individual PCR efficiencies (Bustin 2003) and normalized to the geometric mean Ct of 3 endogenous controls (GAPDH, YWHAZ, and SDHA) in order to account for differences in both cell number and amount of total mRNA present in each sample (Goossens et al. 2005). G9a and SetB1, both lysine-specific methyltransferases, were expressed at their highest levels in the metaphase II (MII) oocyte and 2-cell stage, before expression decreased gradually to basal levels by the morula and blastocyst stages. Suv39h1, Suv420h1, and SmyD3, also lysine-specific methyltransferases, all shared a similar pattern of expression: transcript levels were fairly high in the MII oocyte, increased at the 2-cell stage, then gradually dropped off around the 8–16-cell stage to basal levels by the morula stage. Interestingly, Suz12 and LSH both showed low expression from the MII oocyte until the 4 to 7-cell stage, increased dramatically at the 8-cell stage, then decreased again by the morula stage. Suz12 is a member of several Polycomb group complexes (PRCs); LSH associates with PRC-mediated gene silencing as well as DNMT3a and 3b. These data suggest that Suz12 and LSH may be implicated in bovine embryonic genome activation, while the latter genes are active during earlier cleavage events. Ongoing studies will evaluate the role of each of these epigenetic modifiers in bovine pre-implantation embryos by selective silencing via RNA interference.

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Comprehensive Comparison of Amnion Stromal Cells and Chorion Stromal Cells by RNA-Seq

International Journal of Molecular Sciences ◽

10.3390/ijms22041901 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1901

Author(s):

Brielle Jones ◽

Chaoyang Li ◽

Min Sung Park ◽

Anne Lerch ◽

Vimal Jacob ◽

...

Keyword(s):

Gene Expression ◽

Stromal Cells ◽

Expression Profiles ◽

Expression Patterns ◽

Principal Component ◽

Differentially Expressed ◽

Inflammatory Factor ◽

Next Generation Sequencing Technology ◽

Angiogenic Potential ◽

Anti Inflammatory

Mesenchymal stromal cells derived from the fetal placenta, composed of an amnion membrane, chorion membrane, and umbilical cord, have emerged as promising sources for regenerative medicine. Here, we used next-generation sequencing technology to comprehensively compare amniotic stromal cells (ASCs) with chorionic stromal cells (CSCs) at the molecular and signaling levels. Principal component analysis showed a clear dichotomy of gene expression profiles between ASCs and CSCs. Unsupervised hierarchical clustering confirmed that the biological repeats of ASCs and CSCs were able to respectively group together. Supervised analysis identified differentially expressed genes, such as LMO3, HOXA11, and HOXA13, and differentially expressed isoforms, such as CXCL6 and HGF. Gene Ontology (GO) analysis showed that the GO terms of the extracellular matrix, angiogenesis, and cell adhesion were significantly enriched in CSCs. We further explored the factors associated with inflammation and angiogenesis using a multiplex assay. In comparison with ASCs, CSCs secreted higher levels of angiogenic factors, including angiogenin, VEGFA, HGF, and bFGF. The results of a tube formation assay proved that CSCs exhibited a strong angiogenic function. However, ASCs secreted two-fold more of an anti-inflammatory factor, TSG-6, than CSCs. In conclusion, our study demonstrated the differential gene expression patterns between ASCs and CSCs. CSCs have superior angiogenic potential, whereas ASCs exhibit increased anti-inflammatory properties.

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Regional variations in ABC transporter expression along the mouse intestinal tract

Physiological Genomics ◽

10.1152/physiolgenomics.00150.2003 ◽

2004 ◽

Vol 17 (1) ◽

pp. 11-20 ◽

Cited By ~ 40

Author(s):

David M. Mutch ◽

Pascale Anderle ◽

Muriel Fiaux ◽

Robert Mansourian ◽

Karine Vidal ◽

...

Keyword(s):

Gene Expression ◽

Abc Transporter ◽

Expression Profile ◽

Abc Transporters ◽

Intestinal Tract ◽

Expression Profiles ◽

Expression Patterns ◽

Assessment Model ◽

Differentially Expressed ◽

Distal Intestine

The ATP-binding cassette (ABC) family of proteins comprise a group of membrane transporters involved in the transport of a wide variety of compounds, such as xenobiotics, vitamins, lipids, amino acids, and carbohydrates. Determining their regional expression patterns along the intestinal tract will further characterize their transport functions in the gut. The mRNA expression levels of murine ABC transporters in the duodenum, jejunum, ileum, and colon were examined using the Affymetrix MuU74v2 GeneChip set. Eight ABC transporters (Abcb2, Abcb3, Abcb9, Abcc3, Abcc6, Abcd1, Abcg5, and Abcg8) displayed significant differential gene expression along the intestinal tract, as determined by two statistical models (a global error assessment model and a classic ANOVA, both with a P < 0.01). Concordance with semiquantitative real-time PCR was high. Analyzing the promoters of the differentially expressed ABC transporters did not identify common transcriptional motifs between family members or with other genes; however, the expression profile for Abcb9 was highly correlated with fibulin-1, and both genes share a common complex promoter model involving the NFκB, zinc binding protein factor (ZBPF), GC-box factors SP1/GC (SP1F), and early growth response factor (EGRF) transcription binding motifs. The cellular location of another of the differentially expressed ABC transporters, Abcc3, was examined by immunohistochemistry. Staining revealed that the protein is consistently expressed in the basolateral compartment of enterocytes along the anterior-posterior axis of the intestine. Furthermore, the intensity of the staining pattern is concordant with the expression profile. This agrees with previous findings in which the mRNA, protein, and transport function of Abcc3 were increased in the rat distal intestine. These data reveal regional differences in gene expression profiles along the intestinal tract and demonstrate that a complete understanding of intestinal ABC transporter function can only be achieved by examining the physiologically distinct regions of the gut.

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Is gene expression among women with rheumatoid arthritis dysregulated during a postpartum flare?

Arthritis Research & Therapy ◽

10.1186/s13075-021-02418-w ◽

2021 ◽

Vol 23 (1) ◽

Author(s):

Matthew Wright ◽

Mette K. Smed ◽

J. Lee Nelson ◽

Jørn Olsen ◽

Merete L. Hetland ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Gene Expression ◽

Disease Activity ◽

Activity Index ◽

Expression Profiles ◽

Expression Patterns ◽

Time Frame ◽

Differentially Expressed ◽

Healthy Women ◽

Immune Related Genes

Abstract Background To evaluate our hypotheses that, when rheumatoid arthritis (RA) flares postpartum, gene expression patterns are altered compared to (a) healthy women, (b) RA women whose disease activity is low or in remission postpartum, and (c) pre-pregnancy expression profiles. Methods Twelve women with RA and five healthy women were included in this pilot study. RA disease activity and postpartum flare were assessed using the Clinical Disease Activity Index (CDAI). Total RNA from frozen whole blood was used for RNA sequencing. Differential gene expression within the same women (within-group) over time, i.e., postpartum vs. third trimester (T3) or pre-pregnancy (T0), were examined, using a significance threshold of q < 0.05 and fold-change ≥ 2. Results Nine of the women with RA experienced a flare postpartum (RAFlare), while three had low disease activity or were in remission (RANoFlare) during that time frame. Numerous immune-related genes were differentially expressed postpartum (vs. T3) during a flare. Fold-changes in expression from T3 to postpartum were mostly comparable between the RAFlare and healthy groups. At 3 months postpartum, compared to healthy women, several genes were significantly differentially expressed only among the RAFlare women, and not among the RANoFlare women. Some of these genes were among those whose “normal” expression was significantly modulated postpartum, and the postpartum expression patterns were significantly altered during the RA flare. There were also some genes that were significantly differentially expressed in RAFlare compared to both healthy and RANoFlare women, even though their expression was not significantly modulated postpartum. Furthermore, while postpartum expression profiles were similar to those at pre-pregnancy among healthy women, significant differences were found between those time points among the RAFlare women. Conclusions The large majority of gene expression changes between T3 and 3 months postpartum among RA women who flared postpartum reflected normal postpartum changes also seen among healthy women. Nonetheless, during a postpartum flare, a set of immune-related genes showed dysregulated expression compared to healthy women and women with RA whose disease activity was low or in remission during the same time frame, while other genes demonstrated significant differences in expression compared to RA pre-pregnancy levels.

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136 FUNCTIONAL ANALYSIS OF RHOPHILIN-2 GENE IN PRE-IMPLANTATION MOUSE EMBRYO

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab136 ◽

2006 ◽

Vol 18 (2) ◽

pp. 176

Author(s):

T. Matsuoka ◽

Y. Sono ◽

K. Matsumoto ◽

T. Amano ◽

S. Mizuno ◽

...

Keyword(s):

Real Time ◽

Hybrid System ◽

Real Time Pcr ◽

Expression Profiles ◽

Subsequent Development ◽

Taqman Probe ◽

Cell Stage ◽

Transcript Levels ◽

Cytoskeletal Organization ◽

G2 Phase

Zygotic gene activation (ZGA), which starts at the G2 phase at the 1-cell stage (Latham 1999), promotes the reprogramming of gene expression and is critical for the subsequent development of pre-implantation embryos. We have investigated the molecule function of many gene clusters, DD clones obtained by Differential-Display assays for ovulated eggs at the M II stage, and 1-cell embryos at the G2 phase. The differential expression of rhophilin-2 shown in DD assays was also confirmed by 3 independent real-time PCR analyses (P < 0.05). For these reasons, in this study, we focused on the rhophilin-2 gene, which regulates cytoskeletal organization (Peck et al. 2002). At first, we identified a protein that interacts with the Rhophilin-2 protein by a yeast 2-hybrid system. To confirm the interaction between Rhophilin-2 and the putative protein obtained by a yeast two-hybrid system, we used a co-immunoprecipitaion assay. We also investigated the expression profiles of rhophilin-2 and the transcripts of the identified protein in ovary and pre-implantation embryos using real-time PCR and immunofluorescence (IF) analysis. The ICR mice at 48 h after PMSG priming were primed with hCG, and ovaries were collected at 7 h after hCG priming. Pre-implantation embryos were collected at 1-cell, 2-cell, and 4-cell stages, and cDNA was produced by mRNA isolated from 10 oocytes or embryos in each group and was subjected to real-time PCR using a TaqMan Probe system (ABI). Sectioned ovaries and pre-implantation embryos were analyzed by IF analysis using antibody of Rhophilin-2 and the identified protein. This is the first report that GABA receptor-association protein (GABARAP) was identified as a protein that interacts with Rhophilin-2, as a result of using the yeast 2-hybrid system and subsequent co-immunoprecipitation assay. After fertilization, transcript levels of rhophilin-2 significantly decreased from the 1-cell stage to the 2-cell stage (P < 0.05), but transcript levels of GABARAP significantly increased from the 1-cell stage to the 2-cell stage (P < 0.05). The IF analysis revealed localization of Rhophilin-2 and GABARAP at the nucleolus of all follicle stage in the ovary. Moreover, Rhophiln-2 and GABARAP were found to be localized on the microtubules of 1-cell and 2-cell embryos, but no signal of Rhophilin-2 was detected in 4-cell embryos. These results suggest that Rhophilin-2 protein regulates the cytoskeletal organization in 1-cell to 2-cell embryos and is involved in the molecular mechanism of cell division by coupling with GABARAP. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST.

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The Expression of Human Placental Genes Related to Carotenoid/retinoid Metabolism and Pathways (P02-005-19)

Current Developments in Nutrition ◽

10.1093/cdn/nzz029.p02-005-19 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

Author(s):

Xiaoming Gong ◽

Lewis Rubin

Keyword(s):

Gene Expression ◽

Microarray Analysis ◽

Fetal Development ◽

Expression Profiles ◽

Expression Patterns ◽

Gene Set Enrichment Analysis ◽

Differentially Expressed ◽

Retinoid Metabolism ◽

Expression Of Genes ◽

Β Carotene

Abstract Objectives Carotenoid/retinoids status and metabolism are essential for normal placental and fetal development. Both deficiencies and excess of retinoids and some carotenoids are associated with adverse pregnancy outcomes, such as preeclampsia and preterm birth. A group of important genes involved in regulating carotenoid/retinoid metabolism and maternal to fetal transfer in human placenta. The objective of this study is to analyze (a) the expression of genes critical for regulating carotenoid/retinoid metabolism and maternal-fetal transport in human trophoblasts and (b) placental transcriptional profiles of these pathways in response to carotenoid exposure. Methods Human cytotrophoblasts (CTBs) were isolated from term placentas. CTB RNA was used to analyze the expression of genes involved in carotenoid/retinoid metabolism and pathways by qRT-PCT. First trimester-like trophoblasts (HTR-8/SVneo) were treated with either β-carotene or lycopene. RNAs were isolated and gene expression were analyzed by DNA microarrays. Results Human CTBs express retinoid metabolism and pathways-related genes, including Stra6, Lrat, Rdh5, Rdh10, Aldh1a1, Aldh1a2, Aldh1a3, Aldh8a1, Cyp26a1, and Cyp26b1, but not carotenoid metabolism genes, BCO1 and BCO2. Microarray analysis of placental gene expression profile revealed a total of 872 and 756 differentially expressed genes, respectively, compared to the control. Gene set enrichment analysis and functional annotation clustering was performed to characterize the genes differentially expressed in either β-carotene or lycopene-treated HTR-8/SVneo cells. Many known retinoid metabolism related genes and genes involved in regulation of retinoid signaling were found, and the expression profiles of these genes were markedly different in response to β-carotene treatments. Finally, the qRT-PCR and microarray analysis results showed similar gene expression patterns of carotenoid/retinoid metabolism and pathways. Conclusions These findings suggest that placental expression of genes involved in retinoid metabolism and transport in trophoblasts is critical for regulating retinoid homeostasis during placental and fetal development. Carotenoid exposure in early placental development, significantly modify the placenta gene expression related to retinoid pathways and maternal to fetal transfer. Funding Sources NIH HD421174.

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