- Transcriptome dynamics in early in vivo developing and in vitro produced porcine embryos. - 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 in vivo developed and in vitro produced hatched blastocysts further were compared to identify molecular signaling pathways indicative of lower developmental competence of in vitro produced hatched blastocysts. - transporter SLC7A1 were found in in vitro produced hatched blastocysts.. - 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.. - Keywords: Transcriptomics, Porcine, Embryo development, In vivo embryo development, in vitro fertilization. - Up to the blastocyst stage, embryos can be produced and cultured in vitro. - Despite ongoing efforts to improve the quality of in vitro produced blastocysts, these embryos are less competent than in vivo developed blastocysts [5]. - Therefore, it is important to understand which molecular pathways are affected by the in vitro embryo production pipelines. - have been investigated in both in vivo developed and in vitro produced whole embryos, aiming at gaining insights into the mechanisms that lead to reduced devel- opmental potential of in vitro produced embryos [13]. - In vitro produced embryos displayed altered transcript levels for apoptotic factors, cell cycle regulation factors and spindle components, as well as transcription factors, collectively contributing to reduced developmental com- petence of in vitro produced embryos [13]. - To date, the developmental competence, as well as pregnancy rates after transferring in vitro produced porcine embryos remain low [17]. - Com- pared to in vivo produced embryos, in vitro produced embryos showed developmental stage-dependent altered chromatin dynamics. - In vitro produced embryos showed global chromatin remodeling imperfections and failed to estab- lish a proper first lineage segregation at the blastocyst stage [18]. - To improve the developmental competence of in vitro embryos, it is crucial to elucidate their tran- scriptional profile during pre-implantation development.. - In this study, we aimed at furthering the understanding of early embryo development, and to identify molecular pathways that could explain lower developmental com- petence of in vitro produced hatched blastocysts.. - 0.1, increased with developmental progression for the in vivo produced em- bryos, while it decreased for the in vitro produced em- bryos (Additional file 1). - Given the differ- ences in RNA quality as assessed by the cDNA profile, library smear analyses, and read alignment at the 4-cell, as well as at the morula stage (Additional file 2 and 3), the in vivo developed and in vitro produced embryos were analyzed separately and were not compared to each other. - To identify in vitro fertilization pipeline-induced transcriptome differences, the hatched blastocysts were used for an in vivo developed versus in vitro produced comparison.. - Principal component analyses (PCA) were performed separately for the in vivo developed and in vitro produced embryos and showed a clear develop- mental stage-specific clustering of the embryos (Fig. - For the in vitro produced embryos, PC1 and PC2 explained 71.8 and 17.3% of the variance. - The in vivo 4- cell embryos displayed a larger degree of transcriptional heterogeneity than the in vitro 4-cell embryos. - In vivo and in vitro embryonic developmental dynamics The developmental transcriptome dynamics were further analyzed by identifying differentially expressed genes (DEGs) between the 4-cell and morula stage, and the morula and hatched blastocyst stage for both the in vivo developed and in vitro produced embryos. - For the in vitro embryos, 8152 and 4023 DEGs were identified between the 4-cell to the morula stage and the morula stage to the hatched blastocyst stage, re- spectively (Fig. - For both the in vivo and in vitro produced embryos, the detected transcript expression changed from the 4-cell to the morula stage. - In both the in vivo and the in vitro produced 4-cell to morula stage embryos, there was a significant enrichment of oxidative phosphorylation and EIF2 signaling. - all higher expressed at the hatched blastocyst stage for both the in vivo and in vitro produced embryos.. - In vivo and in vitro differences at the hatched blastocyst stage. - The in vivo and in vitro hatched blastocysts were com- pared, as the embryos displayed similar cDNA profiles, library smears and alignment coverages for the most abundant transcripts at this developmental stage. - Yet, we unraveled in vitro fertilization pipeline- induced sex-specific differences. - The in vivo. - In vivo developed embryos, and b. - In vitro produced embryos. - They were separated from the in vitro hatched blastocyst in a sex-specific manner by principal component 1. - in vitro produced embryos, 241 DEGs were identified between the male in vivo and in vitro produced em- bryos. - Figure 6b displays the difference between in vivo developed and in vitro produced embryos in a sex-independent manner. - comparing male and female embryos for either in vivo developed or in vitro produced embryos. - By comparing the female in vivo developed versus in vitro produced embryos, the DEGs inositol poly- phosphate multikinase (IPMK) and Rac family small GTPase 1 (RAC1) were specific to this comparison.. - The other 31 DEGs were also discovered by compar- ing the in vivo and in vitro male hatched blastocysts.. - Both male and female in vivo derived embryos had a lower ex- pression of genes involved in amino acid transport, synthesis and metabolism compared to the male and female in vitro produced embryos.. - The persistent difference between in vivo developed and in vitro produced embryos at the hatched blastocyst stage were illustrated by an en- richment of four canonical pathways (Fig. - Except for a higher expression in in vivo versus in vitro hatched blastocysts of DEGs involved in cyclins and cell cycle regulation and LXR/RXR activation, the DEGs involved in tRNA charging and xenobiotic me- tabolism AHR signaling pathways were higher expressed in in vitro than in in vivo hatched blastocysts.. - Early developing porcine embryos displayed a great adaptive capacity towards their environment, evidenced by largely similar transcriptome dynamics observed in both in vivo developed and in vitro produced embryos.. - in vitro produced embryos offer the opportunity to study molecular pathways of interest in a developmental-stage specific manner, as there is a higher degree of certainty re- garding the time of fertilization compared to in vivo devel- oped embryos. - However, developmental rates and embryo competence of in vitro produced embryos are still lower compared to their in vivo developed counterparts [5]. - The discrepancy in embryo de- velopment between in vivo developed and in vitro pro- duced embryos at early post-fertilization developmental stages might be explained by the use of a pool of non- selected oocytes of overall lower competence for in vitro maturation, compared to those selected for ovulation, and the effects of in vitro maturation on oocyte quality. - In addition, the blastocyst rates of in vivo and in vitro matured rabbit oocytes did not significantly differ, while at earlier developmental stages, the in vivo embryo development rates were signifi- cantly higher than observed for embryos produced with in vitro matured oocytes [22]. - The early embryo development was studied at the 4-cell, morula and hatched blastocyst stage for both in vivo de- veloped and in vitro produced embryos. - Both the in vivo developed and in vitro produced 4-cell to morula transition was characterized by an enrichment of oxidative phosphoryl- ation and EIF2 signaling. - Shared enriched canonical pathways in both in vivo developed and in vitro produced embryos at the 4-cell versus morula or morula versus hatched blastocyst stage are indicated in purple. - Yet, in vitro produced embryos displayed a higher oxygen con- sumption, which was related to lower pregnancy rates [25]. - Thus, in vitro morulae seem developmentally competent, as they display increased transcription of genes related to oxidative phosphorylation, as ob- served for the in vivo embryos. - During the morula to the hatched blastocyst transition, both in vivo developed and in vitro produced embryos displayed an enrichment of the pathways 14–3-3-medi- ated signaling, xenobiotic metabolism general signaling pathways, and NRF2-mediated oxidative stress response.. - The enrichment of the shared signaling pathways in both in vivo developed and in vitro produced embryos during development from the morula to hatched blastocyst stage appeared to be indi- cative of largely similar developmental transcriptional profiles, potentially related to embryo competence.. - Between group analysis of in vivo and in vitro female and male hatched blastocysts. - Heatmap displaying the 243 DEGs observed by comparing in vivo produced versus in vitro developed females and the in vivo produced versus in vitro developed males. - Significantly enriched canonical pathways between the in vivo developed and in vitro produced hatched blastocysts. - In vivo developed versus in vitro produced hatched blastocysts. - The differences between in vivo developed and in vitro produced hatched blastocysts were investigated to understand persisting transcriptional differences and their relationship to embryo competence. - Unlike the difference in expression of HMGB1 they reported, we did not find a difference in its expression between in vivo developed and in vitro produced hatched blastocysts. - The expres- sion of HMGB1 has been associated with the number of nuclei per embryo [23], suggesting that the stage of our hatched blastocysts is likely similar, thereby allowing the comparison between in vivo developed and in vitro pro- duced embryos at this developmental stage. - Likewise, there was no significant difference in the expression of ATP5A1 between in vivo developed and in vitro pro- duced hatched blastocysts. - The expression of ATP5A1 has previously been used to indicate differences in meta- bolic rates in in vivo developed and in vitro produced blastocysts [23]. - In addition, 71% of genes related to cel- lular metabolism were reported to be upregulated in in vivo developed compared to in vitro produced porcine blastocysts [33]. - The in vitro hatched blastocysts in this study displayed a significant increase in amino acid me- tabolism. - Among the genes related to amino acid metab- olism, the arginine transporter SLC7A1 has previously been reported to be significantly upregulated in in vitro produced embryos compared to in vivo developed em- bryos [34]. - It has previously been shown that adding arginine to a final concentration of 0.36 mM to the embryo culture medium decreased the SLC7A1 transcript level in in vitro produced embryos to a level comparable to the in vivo developed embryos [34]. - In our study, the in vitro produced hatched blasto- cyst displayed a higher transcript expression of genes re- lated to tRNA charging and xenobiotic metabolism AHR signaling pathways. - in vitro produced porcine blastocyst have previ- ously been reported to display a higher transcript expression of genes involved in, among others, mRNA transcription, nucleotide metabolism, DNA metabolism, amino acid metabolism, and lipid metabolism [34]. - The higher metabolic rate of in vitro produced embryos is evidenced in our in vitro hatched blastocysts by an en- richment in tRNA charging and amino acid metabolism.. - Thus, we propose that the transcriptome of in vitro produced hatched blastocysts is indicative of an increased level of DNA damage, as evidenced by the higher degree of amino acid metabolism. - Thereby, an improvement of the currently employed in vitro fertilization pipe- lines can be assessed.. - Taken together, we show that early developing in vivo and in vitro produced embryos display largely similar transcriptome profiles. - The in vitro produced hatched blastocysts displayed the expression of transcripts indi- cative of a higher metabolic rate and the arginine trans- porter, suggesting a lower developmental competence compared to the in vivo developed embryos.. - Porcine embryos were allowed to develop in vivo and were produced in vitro (Fig. - At the hatched blastocyst stage, male and fe- male in vivo embryos were compared to the respective in vitro produced embryos.. - In vivo. - The in vivo embryos were produced as described previ- ously [16]. - In vitro. - The in vitro embryos were produced as previously de- scribed [40, 41]. - The in vitro fertilization was performed using frozen sperm derived from the same Duroc boar as used for the in vivo devel- oped embryos to reduce an influence on genetic variation [40]. - Single 4-cell stage embryos (n = 5/production method), morulae (n = 10/production method) and hatched blasto- cysts (n = 10/production method) were obtained from in vivo flushing or were in vitro produced (Fig. - An absolute log 2 FC cut-off of 4 was ap- plied to obtain 2616 DEGs for canonical pathway analysis for the in vitro 4-cell to morula stage, while a log 2 FC cut- off of 1.7 was applied to obtain 2656 DEGs for canonical pathway analysis for the in vitro morula to hatched blasto- cyst stage. - An absolute log 2 FC cut-off of 0.8 was applied to obtain 377 DEGs for canonical pathway analysis for the in vivo versus in vitro hatched blastocysts. - cDNA profiles and library smear analysis of in vivo developed and in vitro produced 4-cell embryos, morulae and hatched blastocysts.. - MK performed the in vitro embryo production. - Consequences of in vitro culture conditions on embryo development and quality. - RNA profiles of porcine embryos during genome activation reveal complex metabolic switch sensitive to in vitro conditions. - In vitro development to blastocysts of early porcine embryos produced in vivo or in vitro. - Early aberrations in chromatin dynamics in embryos produced under in vitro conditions. - Gene expression profile of cumulus cells derived from cumulusoocyte complexes matured either in vivo or in vitro. - Developmental competence of porcine oocytes after in vitro maturation and in vitro culture under different oxygen concentrations. - In vivo and in vitro maturation of rabbit oocytes differently affects the gene expression profile, mitochondrial distribution, apoptosis and early embryo development. - Investigation of respiration of individual bovine embryos produced in vivo and in vitro and correlation with viability following transfer. - Comparative transcriptome analysis of in vivo- and in vitro- produced porcine blastocysts by small amplified RNA-serial analysis of gene expression (SAR-SAGE). - Transcriptional profiling by deep sequencing identifies differences in mRNA transcript abundance in in vivo-derived versus in vitro- cultured porcine blastocyst stage embryos
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