ABSTRACT: Cumulus cells (CCs) are biologically distinct from other follicular cells and perform specialized roles, transmitting signals within the ovary and supporting oocyte maturation during follicular development. The Affymetrix 3’ IVT express protocol was used to prepare cRNA (one-cycle amplification) with a starting concentration of 100 ng of total RNA To compare groups of CCs at different stages of oocyte nuclear maturation, a Significance Analysis of Microarrays (SAM) was performed. CC samples were issued from oocyte at germinal vesicle (CCGV), stage metaphase I (CCMI) and stage metaphase II (CCMII)
Project description:Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder that is characterized by increased circulating androgen levels, anovulatory infertility, and frequently, insulin resistance and hyperinsulinemia.The abnormity of oocyte nuclear maturity is the main reason for anovulatory infertility and pregnancy loss in PCOS patients.The bidirectional exchanges between oocyte and contiguous CCs are important for oocyte competence acquisition, early embryonic development and CC expansion.Gene expression profiles of CCs has been suggested to predict embryo development and pregnancy outcome. We used microarrays to detail the global programme of gene expression of CCs isolated from oocytes at metaphase I (CCMI) and metaphase II (CCMII) stage under controlled ovarian stimulation (COS) cycle in PCOS patients. Cumulus cells were isolated from oocyte at stage metaphase 1(MI)and stage metaphase II (MII) of PCOS patients for RNA extraction and hybridization on Affymetrix microarrays. For microarray analysis, we used three chips for each CC category. That is, CCMI1,CCMI2,CCMI3,CCMII1,CCMII2 and CCMII3.
Project description:The bidirectional communication between bovine oocytes and CCs is vital for functioning and development of both cell types. We used microarray to identify genes which are differentially expressed between germinal vesicle (GV)- and metaphase II (MII)-stage oocytes and CCs and those differentially expressed when oocytes mature with or without the other. We also identified genes differentially expressed between CCs at GV and MII stages. Overall design: Slaughterhouse ovaries were collected and GV-stage cumulus oocyte complexes (COCs) were aspirated. Different stages and types of oocytes and CCs were used for total RNA isolation and hybridisation on Affymetrix microarray.
Project description:Purpose: The goal of this study was to identify differentially expressed genes and pathways between the cumulus of compact/unstimulated cumulus-oocyte-complex (COC) and the cumulus of expanded/stimulated COC. Methods: mRNA profiles of Compact/unstimulated cumulus cells (CCs) from germinal vesicle (GV) COC obtained from two patients undergoing unstimulated IVM procedure and expanded/stimulated CCs from metaphase 2 (MII) COC obtained from three patients undergoing IVF/ICSI were generated by deep sequencing using Illumina HiSeq 2000. The sequence reads that passed quality filters were mapped to the human genome (hg19) using Tophat software. Differential expression analysis was done using DESeq bioconductor package. qRT–PCR validation was performed using SYBR Green assays. Results: A total of 40-80 million sequence reads per sample were mapped to the human genome (hg19). A total of 1746 differentially expressed genes between compact and expanded CCs with fold change > 2, and adjusted p value < 0.05 were identified. Gene ontology analysis of differentially expressed genes revealed a number of cellular processes regulated during the periovulatory interval including cellular movement, inflammatory response, immune cell trafficking, tissue development, lipid metabolism, tissue morphology, DNA replication and cell cycle. A total of 116 of the differentially expressed genes were annotated as long non-coding RNAs, 10 of them coded from introns of genes known to be involved in granulosa cell processes suggesting that unique non coding RNA transcripts may contribute to the regulation of cumulus expansion and oocyte maturation. Results were validated using qRT-PCR. Conclusions: Using global transcriptome sequencing we identified new important genes and non coding RNAs involved in COC maturation and cumulus expansion, which may contribute to improve the process of in vitro maturation of immature oocytes utilized in IVM cycles mRNA profiles of Compact/unstimulated cumulus cells (CCs) from germinal vesicle (GV) COC obtained from two patients undergoing unstimulated IVM procedure and expanded/stimulated CCs from metaphase 2 (MII) COC obtained from three patients undergoing IVF/ICSI were generated by deep sequencing using Illumina HiSeq 2000.
Project description:The bidirectional communication between bovine oocytes and CCs is vital for functioning and development of both cell types. We used microarray to identify genes which are differentially expressed between germinal vesicle (GV)- and metaphase II (MII)-stage oocytes and CCs and those differentially expressed when oocytes mature with or without the other. We also identified genes differentially expressed between CCs at GV and MII stages. Slaughterhouse ovaries were collected and GV-stage cumulus oocyte complexes (COCs) were aspirated. Different stages and types of oocytes and CCs were used for total RNA isolation and hybridisation on Affymetrix microarray.
Project description:Aging is the primary risk factor of neurodegenerative disorders such as Alzheimer's disease (AD). However, the molecular events occurring during brain aging are extremely complex and still largely unknown. For a better understanding of these age-associated modifications, animal models as close as possible to humans are needed. We thus analyzed the transcriptome of the temporal cortex of the primate Microcebus murinus using human oligonucleotide microarrays (Affymetrix). Gene expression profiles were assessed in the temporal cortex of 6 young adults, 10 healthy old animals and 2 old, "AD-like" animals that presented b-amyloid plaques and cortical atrophy, which are pathognomonic signs of AD in humans. Gene expression data of the 14,911 genes that were detected in at least 3 samples were analyzed. By SAM (significance analysis of microarrays), we identified 47 genes that discriminated young from healthy old and "AD-like" animals. These findings were confirmed by principal component analysis (PCA). ANOVA of the expression data from the three groups identified 695 genes (including the 47 genes previously identified by SAM and PCA) with significant changes of expression in old and "AD-like" in comparison to young animals. About one third of these genes showed similar changes of expression in healthy aging and in “AD-like” animals, whereas more than two thirds showed opposite changes in these two groups in comparison to young animals. Hierarchical clustering analysis of the 695 markers indicated that each group had distinct expression profiles which characterized each group, especially the "AD-like" group. Functional categorization showed that most of the genes that were up-regulated in healthy old and down-regulated in "AD-like" animals belonged to metabolic pathways, particularly protein synthesis. These data suggest the existence of compensatory mechanisms during physiological brain aging that disappear in “AD-like” animals. These results open the way to new exploration of physiological and “AD-like” aging in primates. Microcebus murinus were divided in 3 groups: the first group included 6 young adults (4 females and 2 males), the second included 10 healthy old animals (7 females and 3 males) and the third one was composed by 2 "AD-like" old females. Since Microcebus murinus microarrays do not exist, we decided to use Affymetrix human genome chips (HG U133 plus 2), since studies have illustrated the feasibility of detecting non-human primate brain transcripts using human genome chips.
Project description:Pluripotent stem cells, which are capable to generate any cell type of the human body, such as human embryonic stem cells (hESC) or human induced pluripotent stem cells (hiPS) are a very promising source of cells for regenerative medicine. However, the genesis, the in vitro amplification and the differentiation of these cells still need improvement before clinical use. This study aimed to improve our knowledge on these critical steps in pluripotent stem cell generation. We derived new hESC lines, generated hiPS and compared these cell types with human foreskin fibroblasts and partially reprogrammed fibroblasts. We included in the overall study hESC, hiPS, human foreskin fibroblasts and partially reprogrammed fibroblasts. Here, hESC lines derived from embryos were hybridized on U133 Plus 2.0 GeneChips (Affymetrix). All samples were normalized using the MAS5 (GCOS 1.2) algorithm, using the default analysis settings and global scaling as normalization method, with a trimmed mean target intensity value (TGT) of each array arbitrarily set to 100. Human pluripotent stem cells were compared with somatic samples and partially reprogrammed cells.
Project description:In humans, the embryonic genome activation (EGA) program is functional by day 3 after fertilization. The 6-8 cell stage embryo (day 3 post-fertilization) starts the process of “compaction” that leads to the generation of the tightly organized cell mass of the morula and is followed by differentiation of the morula into a blastocyst. The transition from day 3 embryos to day 5 blastocysts is likely to be controlled by many and specific changes in the expression of different genes. We used mRNA amplification technique and compared the transcriptomes of day 3 human embryos and trophectoderm (TE) cells from day 5 human blastocysts to identify transcripts that are differentially expressed during the embryo-to-TE transition and involved in the TE specification. These six embryo samples were compared to our five human trophectoderm samples that are available in GEO under the following accession numbers: GSM706168 GSM706169 GSM706170 GSM706171 GSM706172
Project description:Pluripotent stem cells have the potential to differentiate in vitro in many, if not all, functional cell types. Induced pluripotent stem cells (iPS) have recently emerged as a reproducible model of pluripotent stem cells that can be generated from post-natal tissues. To understand this process at the transcriptome level, we generated iPS cell lines, partially reprogrammed cell lines and compared their transcriptome with that of the partental human foreskin fibroblasts and human embryonic stem cell lines. Four categories of samples, comprising human foreskin fibroblasts, fully reprogrammed iPS cell lines, partially reprogrammed iPS cell lines and human embryonic stem cell lines were compared using Significance Analysis of Microarrays (SAM). These data were also compared to a compendium of differentiated human samples and a pluripotency classifier was computed.