Impact of media supplementation on the transcriptional activity of porcine embryos produced in vitro
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1021197 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR26182946_1.fastq.gz | Fastqsanger.gz | |||
| SRR26182946_2.fastq.gz | Fastqsanger.gz | |||
| SRR26182947_1.fastq.gz | Fastqsanger.gz | |||
| SRR26182947_2.fastq.gz | Fastqsanger.gz | |||
| SRR26182948_1.fastq.gz | Fastqsanger.gz |
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Project description:In this article, we focused on the impact of precisely chemically modified FLI maturation medium and its potential to improve the efficiency of in vitro production of porcine embryos. Based on our findings, we demonstrated the positive influence of FLI medium in the evaluation of in vitro maturation, in vitro embryo production, EGA detection, transcriptomic and proteomic profile, which was confirmed by the positive activation of the embryonal genome in the 4-cell stage of parthenogenetically activated embryos.
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Project description:The production of embryos in vitro caused a revolution in the field of assisted reproduction technologies, which currently allows the manipulation and selection of embryos before implantation. One of the key aspects of this process is the maturation of oocytes in a controlled in vitro environment. In this article, we focused on the impact of precisely chemically modified FLI maturation medium and its potential in improving the efficiency of in vitro production of porcine embryos. Based on our findings, we demonstrated the positive influence of FLI medium in the evaluation of in vitro maturation, in vitro embryo production, EGA detection, transcriptomic profile and proteomic analysis, which was confirmed by the positive activation of the embryonal genome in the 4blastoembry stage of parthenogenetically activated embryos.
2023-09-25 | PXD045481 | Pride
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.
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Project description:In this article, we focused on the impact of precisely chemically modified FLI maturation medium enriched with fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), insulin-like growth factor 1 (IGF1), and polyvinyl alcohol (PVA) and its potential to improve the efficiency of in vitro production of porcine embryos. We hypothesized that enhancing the composition of the maturation medium could result in an elevated production of embryos in vitro and can affect EGA. FLI medium resulted in a significantly higher rate of oocyte blastocyst maturation and formation compared to the control DMEM medium. In addition, immunocytochemical labelling confirmed the detection of UBF in 4-cell FLI parthenogenic embryos, suggesting similarities with natural embryo development. Through RNAseq analysis, upregulated genes present in 4-cell FLI embryos were found to play key roles in important biological processes such as cell proliferation, cell differentiation, and transcriptional regulation. Based on our findings, we demonstrated the positive influence of FLI medium in the evaluation of in vitro embryo production, EGA detection, transcriptomic and proteomic profile, which was confirmed by the positive activation of the embryonal genome in the 4-cell stage of parthenogenetically activated embryos.
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Project description:Transcriptome dynamics in early in vivo developing and in vitro produced porcine embryos
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Project description:To investigate the interaction between two porcine isolates of P. multocida (PM2 for type D and PM7 for type A) with Aeromonas caviae by co-culturing P. multocida in the conditioned media prepared from A. caviae
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Project description:In the field of in vitro embryo production it is generally thought that the culture media are not as good as the oviductal fluid, resulting in detrimental changes of embryonic gene expression. Yet in vitro embryo culture (IVC) is one of the pillars of the assisted reproductive technologies. Therefore, unintended effects on gene expression may impact on the health of ART babies (PMID 27554442). Granted, human ART has many more components than just the culture media, but, the practice of embryo culture superimposes with the phase of life when cellular totipotency is present - that's why embryo culture media receive special attention. Discerning what culture media do is difficult because several confounders are present, including but not limited to oocyte heterogeneity, sperm heterogeneity as well as time of fertilization. We propose to take advantage of experimentally produced parthenogenetic embryos, and thereby remove two confounders (sperm variability, time of fertilization), in order to assess the effects of specific culture media of embryonic gene expression, in the mouse model of human reproduction. After synchronous parthenogenetic activation of MII oocytes, pronuclear oocytes were cultured in parallel in ART medium vs KSOM(aa) medium. Resultant blastocysts were compared and contrasted for gene expression among the parthenotes, as well as against zygotic blastocysts.
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Project description:Improper composition of culture medium contributes to reduced viability of in vitro-produced embryos. Glutamine (Gln) is a crucial amino acid for preimplantation embryos as it supports proliferation and is involved in many different biosynthetic pathways. Previous transcriptional profiling revealed several upregulated genes related to Gln transport and metabolism in in vitro-produced porcine blastocysts compared to in vivo-produced counterparts, indicating a potential deficiency in the culture medium. Therefore, the objective of this study was to determine the effects of Gln supplementation on in vitro-produced porcine embryo development, gene expression, and metabolism. Cleaved embryos were selected and cultured in MU2 medium supplemented with 1 mM Gln (control), 3.75 mM Gln (+Gln), 3.75 mM GlutaMAX (+Max), or 3.75 mM alanine (+Ala) until day 6. Embryos cultured with +Gln or +Max had increased development to the blastocyst stage and total number of nuclei compared to the control (P < 0.05). Moreover, expression of misregulated transcripts involved in glutamine and glutamate transport and metabolism was corrected when embryos were cultured with +Gln or +Max. Metabolomics analysis revealed increased production of glutamine and glutamate into the medium by embryos cultured with +Max and increased consumption of leucine by embryos cultured with +Gln or +Max. As an indicator of cellular health, mitochondrial membrane potential was increased when embryos were cultured with +Max which was coincident with decreased apoptosis in these blastocysts. Lastly, two embryo transfers by using embryos cultured with +Max resulted in viable piglets, confirming that this treatment is consistent with in vivo developmental competence.
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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.
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