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: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.
Project description:We have been able to derive EpiSC-like pESC lines from in vivo produced porcine blastocysts. Our cell lines showed AP activity, expressions of the genes Oct4, Sox2, Nanog, Rex1, TDGF1, bFGF, FGFR1, FGFR2, Nodal and Activin-A involved in pluripotency and signaling pathways and in vitro differentiation potential, displaying similarities to epiblast stem cells or hES cells. Porcine blastocysts were cultured on mitotically inactivated mouse embryonic fibroblasts (MEFs) in pESC medium. Following 5-7 days of culture, we observed EpiSC-like primary colonies derived from day 7 in vivo-produced. These EpiSC-like pESC colonies were mechanically dissociated into several clumps using pulled glass pipettes 10-15 days after seeding. Dissociated clumps were then re-seeded on fresh MEFs, and subsequent EpiSC-like pESC lines were routinely passaged via the pulled glass pipette method every 5-7 days. Our cell lines maintained stemness and a stable morphology for more than 56 passages. The main purpose of the present study was to investigate gloval gene expression from porcine embryonic stem cells.
Project description:Comparison of gene expression from expanded bovine blastocysts collected 7 days after fertilization and produced in vivo vs in vitro-SOF-OPU Two kinds of 7 days post fertilization bovine embryos, in-vivo 7 days blastocysts vs. In vitro 7 days blastocysts, Biological replicates: 4 in-vitro, 4 in-vivo, protocol,extract and semen shared. Dye swap.
Project description:Comparison of gene expression from expanded bovine blastocysts collected 7 days after fertilization and produced in vivo vs in vitro-SOF-OPU
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)