Unknown,Transcriptomics,Genomics,Proteomics

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Comprehensive across production systems assessment of the impact of in vitro micro-environment on the expression of messengers and long non-coding RNAs in the bovine blastocyst

ABSTRACT: In vitro production of cattle embryos over the past two decades has revealed several negative impacts, which have been attributed to the artificial microenvironment. Studies of embryos produced in vitro clearly point to aberrant gene expression levels. So far, the causal association between phenotype and measured gene expression has not led to substantial improvement of in vitro production systems. The aim of the present study was to generate a unique dataset composed of microarray-derived relative transcript abundance values for blastocysts produced in ten in vitro systems differing primarily in culture medium formulation. Between-group comparisons allowed us to determine the level of overall similarity between systems relative to in vivo reference embryos. The use of the dataset to contrast all in vitro treatments with the in vivo blastocysts pointed to a single common gene network. The “boutique” array contained a panel of novel uncharacterized transcripts that were expressed variably depending on the medium in which the blastocysts were produced. These novel transcripts were expressed differentially in blastocysts even as carry-over from conditions encountered seven days earlier during oocyte maturation. All of the selected novel candidates that were thus expressed were from intergenic regions. The function of this long non-coding RNA remains unknown but points clearly to an additional level of complexity in early embryo development. For all ten in vitro systems and the in vivo counterparts, three biological replicates were done. Technical duplicates for each of these 33 samples were analyzed to generate a dataset composed of 65 microarray hybridizations (one of the replicated array failed). During analysis, the in vivo derived blastocysts were used as reference. Following data pre-processing (that includes, background subtraction, intra-array normalization (Loess) and inter-array normalization (Quantile), all technical replication (replicated spots on the array and technically replicated arrays) were combined to generate a single value (duplicated spots were averaged and the median was drawn from duplicated arrays) that was used for dowstream significance analysis.

ORGANISM(S): Bos taurus

SUBMITTER: Claude Robert

PROVIDER: E-GEOD-27872 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Blastocyst formation is a primordial event of pre-implantation development since it is required for pregnancy establishment and progression. The blastocyst plays a pivotal role because it is the stage at which the embryo is transferred and starts coordinated cross-talks with the mother. It is also the terminal step of pre-implantation developmentbefore transfer; it reflects all stresses the embryo may have faced during the process of in-vitro treatment. Achieving the formation of a morphologically healthy blastocyst following normal kinetics is a good sign but remains a poor indicator of embryo quality. Considering the limitation of the invasive methods for competence assessment, the analysis of blastocysts’ gene expression is a promising way to improve our understanding of blastocyst formation and to study the effects of different treatments on gene expression. Methods: Early, expanded and hatched blastocysts were collected for RNA extraction, amplification, and cDNA microarray hybridization. Gene candidates (IFNt, PLAC8, SSLP1, AKR1B1, HNRNPA2B1, ARGFX, NANOS, CCNB1) were selected and confirmed using Q-RT-PCR to validate the microarray data. Results: Our analysis show that hatched blastocysts are enriched in genes transcripts implicated in implantation, cell adhesion and extracellular matrix digestion. Early blastocysts expressed genes mainly involved in cell cycle control, transcription and translation. Q-RT-PCR validated most microarray results (87.5%). Some of the differentially expressed genes are interesting as potential markers of competence. Conclusions: Overall, our study provides new insights into the molecular regulation of blastocyst formation. In addition, it could help assess blastocyst staging and select better embryos based on the expression of quality markers. In the present study the Laval/Sirard_bovine_embryo_3K_V3.0 array was used to conduct a series of 18 hybridizations (for three Blastocysts stages (early, expended, hatched) with three biological replicates and dye swaps) in a loop design experiment.

Project description:Metabolic stress associated with negative energy balance in high producing dairy cattle and obesity in women is a risk factor for decreased fertility. Non-esterified fatty acids (NEFA) are involved in this pathogenesis as they jeopardize oocyte and embryo development. Growing evidence indicates that maternal metabolic disorders can disturb epigenetic programming, and more specifically DNA methylation, in the offspring. Final oocyte maturation and early embryo development coincide with specific methylation changes and both time periods are sensitive to adverse environments. Therefore, we investigated whether elevated NEFA concentrations affect the establishment and maintenance of DNA methylation patterns in oocytes and embryos and subsequently alter transcriptomic profiles and developmental competence of the resultant blastocysts. To do this, bovine oocytes and embryos were exposed to different NEFA concentrations in two separate experiments. In the first experiment, oocytes were matured in vitro for 24 hours in medium containing: 1) physiological (âBASALâ) concentrations of oleic (OA), palmitic (PA) and stearic (SA) or 2) elevated (pathophysiological or âHIGH COMBIâ) concentrations of OA, PA and SA. In the second experiment, zygotes were cultivated in vitro for 6.5 days under BASAL or HIGH COMBI conditions. The developmental competence was evaluated by assessing cleavage and blastocyst rate. Overall gene expression and DNA methylation of resultant blastocysts were analyzed using microarray techniques. Data regarding DNA methylation were re-evaluated by pyrosequencing. HIGH COMBI-exposed oocytes and embryos displayed a lower competence to develop into blastocysts compared to their BASAL-exposed counterparts (19.3 % compared to 23.2 % and 18.2 % compared to 25.3 %, respectively) (P < 0.05). HIGH COMBI-exposed oocytes and embryos resulted in blastocysts with altered DNA methylation profiles and transcriptomic fingerprints, compared to BASAL-exposed counterparts. Differences in gene expression and methylation were more pronounced after exposure during culture compared to maturation suggesting that zygotes are more susceptible to an adverse environment. Main gene networks affected were related to lipid and carbohydrate metabolism, cell death, immune response and metabolic disorders. This may offer clues regarding the high rate of embryonic loss and metabolic diseases during later stages of life observed in offspring from mothers displaying lipolytic disorders. The effect of elevated NEFA exposure during embryo culture (6.5 days) on gene expression was evaluated by exposing embryos to the BASAL or HIGH COMBI conditions. A total of 1412 oocytes were used, equally distributed between treatments in 4 replicates. Resultant day 7.5 blastocysts were snap frozen for subsequent transcriptome analysis (80 blastocysts in each experiments, equally collected between treatments in 4 replicates).

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Comprehensive across production systems assessment of the impact of in vitro micro-environment on the expression of messengers and long non-coding RNAs in the bovine blastocyst

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