Unknown,Transcriptomics,Genomics,Proteomics

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Cumulus High Vs. Low Potential

ABSTRACT: Differences between high- and low-potential COCs were examined by transcriptomic analysis of CC biopsies obtained from COCs of 2-6mm follicles from slaughterhouse ovaries before individual in vitro maturation, fertilization and culture until day 8 post-fertilization. Each COC was individually tracked and categorized based on his fate: embryo at blastocyst stage (CC-Blast) or embryo arrested at 2- to 8-cell stage (CC-2-8-cells). Average blastocyst rates were 27.7% for individual culture, and 31.2% for group control (not significantly different). Five cumulus biopsies per replicate were pooled for each fate. Three CC replicates underwent transcriptomic analysis using RNA microarray assay. Each cumulus cell sample is composed of 5 COC biopsies, 3 samples or biological replicates for each fate (CC-blast or CC-2-8-cells) were analysed by microarray. CC-2-8-cells condition was used as reference.

ORGANISM(S): Bos taurus

SUBMITTER: Audrey BUNEL

PROVIDER: E-GEOD-55368 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background The domestic pig is an important livestock species for meat production worldwide and is becoming an established biomedical research model. As a result, there is a strong interest in the factors that affect the efficient production of viable embryos and offspring in this species using either in vivo or in vitro production methods. A limited understanding of the molecular mechanisms involved in this critical physiological process has inhibited our ability to fully elucidate these factors. The use of next generation deep sequencing and microarray technology are powerful tools for delineation of molecular pathways during early embryonic development of mammals. Here, we report on the assessment of a porcine-embryo-specific microarray platform created from a large expressed sequence tag (EST) analysis generated by Roche/454 next-generation sequencing of cDNAs constructed from critical stages of in vivo or in vitro porcine preimplantation embryos. Results Two cDNA libraries constructed from in vitro and in vivo produced preimplantation porcine embryos were normalized and sequenced using the 454 Titanium pyrosequencing technology. Treatment of cDNA libraries with BAL 31 nuclease digestion resulted in a 2 fold improvement of sequencing quality compared with untreated libraries. Over one million high quality EST sequences were obtained from this process and used to create an augmented porcine genome catalogue. Using the resulting dataset the EMbryogene Porcine Version 1 (EMPV1) microarray was developed and is composed of 43,795 probes printed onto a 4 × 44 K Agilent array. Based on the initial probe sequences annotation, the EMPV1 featured 17,409 protein-coding, 473 pseudogenes, 46 retrotransposed, 2,359 non-coding RNA (snRNA, snoRNA, etc.), 4,121 splice variants in 2,862 genes and a total of 12,324 Novel Transcript Regions (NTR). After re-annotation, the total unique genes increased from 11,961 to 16,281 and 1.9% of them belonged to a large olfactory receptor (OR) gene family. Quality control of EMPV1 was performed using porcine cumulus–oocyte complexes (COC) as well as early developmental stages of embryos. This revealed an even distribution of ten clusters of spike-in control spots and array to array (dye-swap) correction was 0.97. Further bioinformatics analysis revealed that our microarray probes hybridized with more developmental related transcripts from embryonic labelled targets when compared to COC. Conclusions Using next-generation deep sequencing we have produced a large EST dataset to provide the selection of probe sequences for the development of the EMPV1 microarray platform. The quality of this embryo- specific array was confirmed with the high level of reproducibility using current Agilent microarray technology. Despite the current limitations for full NTR annotation, due to the incomplete porcine genome sequencing project, a significant number of NTR were annotated using Version 10 of porcine genome and human RefSeq RNA database to enrich the orthologous genes with unique gene symbol (GS) for Gene Ontology (GO) search. GO terms confirmed that many are related relevant developmental processes. With more than an estimated 20 thousands unique genes represented on the EMPV1, this platform will provide the foundation for future research into the in vivo and in vitro factors that affect the viability of the porcine embryos, as well as the effects of these factors on the live offspring that result from these embryos. Two biological samples.

Project description:In cultured cumulus oocyte complexes (COC) FSH induces gene transcription required for germinal vesicle breakdown (GVBD). Experiments were performed to determine the critical period when gene transcription is required for GVBD and identify candidate mRNAs involved. Experiment I: murine COC were cultured 4h in the presence of FSH with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) added at different intervals after the start of culture. COC cultured with FSH underwent GVBD (82+/-7%). When DRB was added at 0, 5 or 10 min after culture initiation, oocyte maturation was blocked (17+/-7%, 14+/-6% and 21+/-6% GVBD, respectively). When DRB was added after 15, 20 or 30 min, progressively more COC underwent GVBD (37+/-6%, 39+/-6% and 66+/-6%, respectively). The critical period of transcription required for GVBD occurred between 15 and 30 min after culture initiation. Experiment II: COC were cultured for 25 min in the presence (plusDRB) or absence (minusDRB) of DRB. SAGE libraries were generated from COC RNA of each treatment group. A total of 48,431 and 45,367 tags were sequenced for the plusDRB and minusDRB libraries, respectively. Criteria used to identify transcripts of interest included a total tag count of at least 10 across both libraries and a 3-fold or greater difference in expression between libraries. Using these criteria, 39 and 27 transcripts were identified as differentially expressed at the P</=0.01 and P</=0.001 levels, respectively. Differentially expressed transcripts were classed into major categories that included cell growth, development and regulation of gene expression. Differentially expressed transcripts represent candidates potentially involved in regulating maturation of murine COC. Keywords: Analysis of treatment effect using SAGE library comparisons Mouse cumulus oocyte complexes (COC) cultured 25 min in presence (plusDRB) or absence (minusDRB) of 120 uM DRB, a transcriptional inhibitor. SAGE libraries were constructed from 9 to 10 ug RNA from each treatment group. Approx. 48,000 tags seqenced from each library. Comparison of these 2 libraries used to identify potential candidate mRNAs associated with regulation of germinal vesicle breakdown in murine COC.

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Cumulus High Vs. Low Potential

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