Influence Of Insulin On Oocyte Maturation In Invitro Produced Bovine Embryos
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ABSTRACT: Microarray analysis of transcriptome bovine blastocysts Day 8, deriving from oocytes matured under different insulin concentrations as model for metabolic imbalance Three conditions experiment: control, Low Insulin, High Insulin treatments. Biological replicates: 4 for each treatment. One replicate per array.
Project description:IVP blastocysts exposed to hyperglycaemia during early phase of development, i.e from zygote to 8-16 cells stages. Genes expression analysis is done at day 7.5 (7 days of embryo culture). Total RNA form glucose treated blastocysts (4 replicates of 10 embryos each) and control treated blastocysts (4 replicates of 10 embryos each) were compared on a 2-color micro-array design and dye swap.
Project description:Early bovine cleavage (day 0 to 3) was treated with high glucose (5mM) or control glucose (0.2mM) then total day 3 embryos were cultured in control condition until day 5 when gene expression was assessed in developping morula by microarray 4 replicates of 10 morulae/rep was produced for high glucose and control treatment. According to a dye swap design, 4 arrays were used, one array for each comparison between HG and control, i.e replicate 1 from HG were compared to replicate 1 from CTL, rep 2 from HG were compared to rep 2 from CTL, rep 3 HG vs rep 3 CTL, rep 4 HG vs rep 4 CTL.
Project description:In order to understand how in vitro culture affects embryonic quality, we analyzed survival and global gene expression in bovine blastocysts after exposure to increased oxidative stress conditions of IVC. A pro-oxidant agents that act intra-cellularly by inhibiting GSH synthesis (0.4 mM buthionine sulfoximine [BSO]) was added from days 3 to 7, and transcriptomic analysis was then performed in resulting blastocysts. Precisely, after in vitro maturation and fertilization, bovine zygotes were culture in vitro in normal condition, then at day 3, embryos were allocated into culture in control or supplemented with BSO (0.4 mM) until day 7. At this time, blastocysts were harvested and analyzed. Our hypothesis was that BSO treatment will affect blastocyst survival and gene expression associated with low embryo quality 4 replicates of 10 blast/rep was produced for BSO and control treatment. According to a dye swap design with 2 colors, 4 arrays were used to compare BSO (green) against CTL (red) and 4 arrays were used to compared BSO (red) againt CTL (green).
Project description:In order to understand how in vitro culture affects embryonic quality, we analyzed survival and global gene expression in bovine blastocysts after exposure to increased oxidative stress conditions of IVC. A pro-oxidant agents that act extra-cellularly by promoting ROS production (0.01 mM 2,2'-azobis (2-amidinopropane) dihydrochloride [AAPH]) was added from days 3 to 7, and transcriptomic analysis was then performed in resulting blastocysts. Precisely, after in vitro maturation and fertilization, bovine zygotes were culture in vitro in normal condition, then at day 3, embryos were allocated into culture in control or supplemented with AAPH (0.01 mM) until day 7. At this time, blastocysts were harvested and analyzed. Our hypothesis was that AAPH treatment will affect blastocyst survival and gene expression associated with low embryo quality 4 replicates of 10 blast/rep was produced for AAPH and control treatment. According to a dye swap design with 2 colors, 4 arrays were used to compare AAPH (green) against CTL (red) and 4 arrays were used to compared AAPH (red) againt CTL (green).
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:Determining immediate-early response genes to FGF18 treatment. Cells treated for 2 h vs non-treated cells. Three biological replicates.
Project description:Comparative trascriptomic analysis between porcine early-blastocyst and Hatched blastocyst collected around day 5-6 direct comparison with dye-swap; two different arrays with 6 Samples each