Dataset Information

Transcriptome dynamics in early in vivo developing and in vitro produced porcine embryos

ABSTRACT: Background: The transcriptional changes around the time of embryonic genome activation in pre-implantation embryos indicate that this process is highly dynamic. In vitro produced porcine blastocysts are known to be less competent than in vivo developed blastocysts. To understand the conditions that compromise developmental competence of in vitro embryos, it is crucial to evaluate the transcriptional profile of porcine embryos during pre-implantation stages. In this study, we investigated the transcriptome dynamics in in vivo developed and in vitro produced 4-cell embryos, morulae and hatched blastocysts. Results: In vivo developed and in vitro produced embryos displayed largely similar transcriptome profiles during development. Enriched canonical pathways from the 4-cell to the morula transition that were shared between in vivo developed and in vitro produced embryos included oxidative phosphorylation and EIF2 signaling. The shared canonical pathways from the morula to the hatched blastocyst transition were 14-3-3-mediated signaling, xenobiotic metabolism general signaling pathway, and NRF2-mediated oxidative stress response. The in vivo developed and in vitro produced hatched blastocysts were compared to identify molecular signaling pathways indicative of lower developmental competence of in vitro produced hatched blastocysts. A higher metabolic rate and expression of the arginine transporter SLC7A1 were found in in vitro produced hatched blastocysts. Conclusions: Our findings suggest that embryos with compromised developmental potential are arrested at an early stage of development, while embryos developing to the hatched blastocyst stage display largely similar transcriptome profiles, irrespective of the embryo source. The hatched blastocysts derived from the in vitro fertilization-pipeline showed an enrichment in molecular signaling pathways associated with lower developmental competence, compared to the in vivo developed embryos.

ORGANISM(S): Sus scrofa

PROVIDER: GSE155043 | GEO | 2021/01/30

REPOSITORIES: GEO

ACCESS DATA

Shared Molecules

Only show the datasets with similarity scores above: 0.5

Threshold

0.5

Similar Datasets

Project description:Despite many improvements with in vitro culture systems, the quality and developmental ability of mammalian embryos produced in vitro is still lower than their in vivo counterparts. To bring knowledge to answer this question, we used a mass spectrometry (MS) approach to compare the protein content of bovine embryos that were conceived in vivo or produced in vitro. A total of 38 pools of grade-1 quality bovine embryos at the 4-6 cell, 8-12 cell, morula, compact morula and blastocyst stages developed either in vivo or in vitro were analyzed by nano-liquid chromatography coupled with label-free quantitative mass spectrometry, allowing the identification of 3,028 proteins. Multivariate analysis of quantified proteins showed a clear separation of embryo pools according to their in vivo or in vitro origin at all stages. Three clusters of differentially abundant proteins (DAPs) according to embryo origin were evidenced, including 463 proteins more abundant in vivo than in vitro across development, and 314 and 222 proteins more abundant in vitro than in vivo before and after the morula stage, respectively. The functional analysis of proteins found more abundant in vivo showed an enrichment in carbohydrate metabolism and cytoplasmic cellular components. Proteins found more abundant in vitro before the morula stage were mostly localized in mitochondrial matrix and involved in ATP-dependent activity while those overabundant after morula stage were mostly localized in the ribonucleoprotein complex and involved in protein synthesis. Oviductin and other proteins previously shown to interact with early embryos in vitro were among the most overabundant proteins after in vivo development. In conclusion, the maternal environment led to faster degradation of mitochondrial proteins at early embryo developmental stages, lower abundance of proteins involved in protein synthesis at the time of embryonic genome activation and a global upregulation of carbohydrate and small molecule metabolic pathways compared to in vitro produced embryos. Furthermore, our data confirm that embryos developed in vivo uptake large amounts of oviduct fluid-derived proteins as soon as the 4-6 cell stage. These data provide new insight into the molecular contribution of the mother to the developmental ability of early embryos and will help designing better in vitro culture systems.

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: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.

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.