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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.

ORGANISM(S): Bos taurus

PROVIDER: GSE27872 | GEO | 2011/03/11

SECONDARY ACCESSION(S): PRJNA138021

REPOSITORIES: GEO

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Project description: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.

Project description:Production of embryos in vitro has enormous potential for research and commercial applications. Unfortunately, in vitro production of porcine embryos is extremely inefficient. Despite the characterization of distinct phenotypes, little is known about the molecular mechanisms and altered physiological processes that account for poor IVP development. The objective of the current study was to compare global gene expression patterns from IVO and IVP embryos using small amplified RNA (SAR)-SAGE. Whole-cell RNA from pools of Day 6 in vivo-(IVO) and in vitro-produced (IVP) blastocysts was used to construct SAR-SAGE libraries. Sequence analysis of the IVO and IVP libraries yielded a total of 98,771 and 98,408 tags, respectively. A total of 20,029 and 23,453 unique putative transcripts were detected in the IVO and IVP libraries, respectively. Statistical analyses of SAGE tag frequencies between the IVO and IVP libraries indicated that 938 and 193 tags were differentially expressed at a P < 0.05 and P < 0.001 level of significance, respectively. Tentative annotation of the differentially expressed SAGE tags was determined using BLAST sequence alignment with the TIGR porcine specific gene index (SSGI) and cross-species alignment using RepeatMasker to determine homologous human orthologs. Annotated tags were categorized into functional groupings according to gene ontology annotations. Real-time PCR was used to confirm differential expression for several transcripts from IVO and IVP blastocysts. These results demonstrate compromised gene expression from IVP blastocysts compared with IVO blastocysts for a number of biological processes including cellular metabolism, organization and response to stress; thereby providing potential target pathways for improvement of IVP methods. Keywords: Comparative (in vivo- vs. in vitro-produced porcine embryos) Whole-cell RNA from pools of Day 6 in vivo- and in vitro-produced blastocysts was used to construct small amplified RNA (SAR)-SAGE libraries.

Project description:Embryo transfer is largely used in cattle and classically performed at D7 (or D8) using unsorted D7 (or D8) blastocysts produced in vivo, or in vitro in defined media without serum or feeders. Outdated systems including serum and co-culture were however of interest for research purposes. We thus wondered whether embryos that would form a blastocoel at different times after fertilisation (D6 to D8) and stay in culture for up to 2 additional days (D6+1, D6+2, D7+1) would equally develop in vivo after temporary transfer to oestrus-synchronised recipients. Globally alike, those that survived up to D18 reached primitive streak stages and elongated to filamentous sizes similarly to in vivo (D18) or in vitro controls (classical D7-T7). Recovery rate differed between D6 and D8 embryos that were immediately transferred (58 vs 25% in D6-T6 vs D8-T8). With a reduced but intermediate survival (33%), the D6 embryos that stayed 2 more days in culture produced 7 times more IFN-tau at D18 than the immediately transferred D6 embryos. At the end of the culture, D6+2 embryos also displayed the higher number of differences with the D6 blastocysts. A “+1” phenotype emerged from the D6+1 and D7+1 embryos, that shared a larger gene set enrichment than the blastocysts they derived from, possible sign of a similar adaption to the in vitro environment. To the best of our knowledge, this is the first time that this culture system (B2, serum, co-culture) is used to study its impacts on the embryonic transcriptome prior to transfer. Initially reputed as beneficial to produce more expanding and hatching blastocysts, this culture system generated blastocysts that all dissembled in vivo developed ones (D7). Despite a loss of 40 to 60% in the two weeks after ET, no dying (vs surviving) signature was detectable in any of the transferred groups (D6, D6+2, D7+1, D8); was it rather a matter of developmental “pause”? Whether molecular differences prevailing to transfer partly reflected those induced by unfavourable conditions (or diapause) was therefore assessed.

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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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