Lineage specification and pluripotency revealed by transcriptome analysis from oocyte to blastocyst in pig

Kong, Qingran; Xu, Yang; Zhang, Heng; Liu, Shichao; Zhao, Jiying; Zhang, Jiaming; Weng, Xiaogang; Jin, Jun-Xue

doi:10.1096/fj.201901818rr

Cited by 50 publications

(74 citation statements)

References 43 publications

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“…Publicly available datasets were analyzed in this study ( Kim et al, 2018 ; Kong et al, 2020 ). These data can be found in the Gene Expression Omnibus database 1 under accession number GSE139512 and GSE108570 .…”

Section: Methodsmentioning

confidence: 99%

Maternal Cytokines CXCL12, VEGFA, and WNT5A Promote Porcine Oocyte Maturation via MAPK Activation and Canonical WNT Inhibition

Liu

Hao

et al. 2020

Front. Cell Dev. Biol.

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Maternal regulatory factors endow the oocyte with developmental competence in vivo , which might be absent in current in vitro maturation (IVM) systems, thereby compromising oocyte quality. In the present study, by employing RNA sequencing data analysis, we expect to identify potential contributing factors to support porcine oocyte maturation through binding to their receptors on the oolemma. Here, C-X-C motif chemokine ligand 12 (CXCL12), vascular endothelial growth factor A (VEGFA), and Wingless-type MMTV integration site family member 5A (WNT5A), termed CVW, are selected and confirmed to be important maternal cytokines for porcine oocyte maturation. Combined supplementation of CVW promotes the nuclear maturation percentage from 57.2% in controls to 75.9%. More importantly, these maternal cytokines improve the developmental potential of matured oocytes by parthenogenesis, fertilization, and cloning, as their blastocyst formation efficiencies and total cell numbers are increased. CVW supplementation also enlarges perivitelline space and promotes cumulus expansion, which results in a more complete transzonal projection retraction on the zona pellucida, and a reduced incidence of polyspermy in fertilized oocytes. Meanwhile, inhibiting the CVW receptor-mediated signaling pathways severely impairs oocyte meiotic resumption and cumulus expansion during IVM. We further determine that maturation improvement by CVW is achieved through activating the MAPK pathway in advance and inhibiting the canonical WNT pathway at the end of the IVM period. These findings provide a new combination of three cytokines to promote the porcine IVM process, which also holds potential to be used in human assisted reproduction technologies as well as in other species.

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Section: Methodsmentioning

confidence: 99%

Maternal Cytokines CXCL12, VEGFA, and WNT5A Promote Porcine Oocyte Maturation via MAPK Activation and Canonical WNT Inhibition

Liu

Hao

et al. 2020

Front. Cell Dev. Biol.

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“…Recent studies of porcine embryo had verified a number of common markers and discovered pig-specific lineage markers [ 20 , 21 , 22 ]. We have identified the trends of the marker gene expression in the porcine embryos ( Figure 1 A).…”

Section: Resultsmentioning

confidence: 99%

“…Using single cell qPCR, stage- and lineage-specific genes were identified, and ICM/TE cells were sorted according to the patterns of the marker gene expression [ 20 ]. Cells were categorized into lineage groups based on the single cell RNA-seq, and it has been demonstrated that certain conventional lineage markers (e.g., CDX2 and TEAD4 ) do not correspond to markers detected in porcine embryos [ 21 , 22 ]. A number of studies have been investigating in vivo embryos; however, detailed information about in vitro fertilized (IVF) porcine embryos remains unavailable.…”

Section: Introductionmentioning

confidence: 99%

Identification of the Lineage Markers and Inhibition of DAB2 in In Vitro Fertilized Porcine Embryos

Lee

Choe

et al. 2020

IJMS

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Specification of embryonic lineages is an important question in the field of early development. Numerous studies analyzed the expression patterns of the candidate transcripts and proteins in humans and mice and clearly determined the markers of each lineage. To overcome the limitations of human and mouse embryos, the expression of the marker transcripts in each cell has been investigated using in vivo embryos in pigs. In vitro produced embryos are more accessible, can be rapidly processed with low cost. Therefore, we analyzed the characteristics of lineage markers and the effects of the DAB2 gene (trophectoderm marker) in in vitro fertilized porcine embryos. We investigated the expression levels of the marker genes during embryonic stages and distribution of the marker proteins was assayed in day 7 blastocysts. Then, the shRNA vectors were injected into the fertilized embryos and the differences in the marker transcripts were analyzed. Marker transcripts showed diverse patterns of expression, and each embryonic lineage could be identified with localization of marker proteins. In DAB2-shRNA vectors injected embryos, HNF4A and PDGFRA were upregulated. DAB2 protein level was lower in shRNA-injected embryos without significant differences. Our results will contribute to understanding of the mechanisms of embryonic lineage specification in pigs.

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“…NANOG knock-in positive PC-iPS cells were positive to AP staining (Additional file 3: Figure S2C), the method of AP staining is reference for our publication data [14]. Clustering analysis showed that knock-in positive PC-iPS cells could be clustered with pig EPS cells [12], but were separate from trophoblast cell (TE), inner cell mass (ICM), and early blastocyst (SB) [43] ( Fig. 2A) (further detail provided in Additional file 4: Table S2); knock-in positive PC-iPS were expressed OCT4 and SOX2 pluripotent markers (Fig.…”

Section: Verification and Transcriptome Analysis Of Nanog Tdtomato Knmentioning

confidence: 95%

“…KEGG pathway analyses were performed using ClusterProfiler [42]. Reanalyzed previously published data are available under the accession codes GSE139512 [43] for pig preimplantation embryos and E-MTAB-7253 [12] for pig EPS cells.…”

Section: Rna Sequencing and Transcriptome Analysismentioning

confidence: 99%

A cytokine screen using CRISPR-Cas9 knock-in reporter pig iPS cells reveals that Activin A regulates NANOG

Zhang

et al. 2020

Stem Cell Res Ther

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Background: NANOG functions as the gateway for the generation of pluripotent stem cells (PSCs) in mice and humans. NANOG is a transcription factor highly expressed in pig pre-implantation embryos, indicating that it is a conserved pluripotency-associated factor. However, pig NANOG reporter PSCs have yet to be established, and the regulation of pluripotency by NANOG is not fully understood in this animal.Methods: In this study, pig NANOG tdTomato knock-in reporter positive PC-iPS cells were established using CRISPR/ Cas9. The resulting cell line was treated with several cytokines and their corresponding inhibitors to identify pathways that regulate NANOG expression. The pathways examined were LIF (leukemia inhibitory factor)/IL6 (interleukin 6)-STAT3, FGF (fibroblast growth factor)/ERK, IGF1 (insulin-like growth factor 1)/PIP3 (phosphoinositide 3kinase)-AKT, Activin A/SMAD, and BMP4 (bone morphogenetic proteins)/SMAD. Results:Our experiments showed that the Activin A/SMAD pathway is directly associated with activation of NANOG expression in the pig, as is also the case in mice and humans. Activin A directly regulates the expression of pig NANOG via SMAD2/3; inhibition of this pathway by SB431542 resulted in inhibition of NANOG expression. Conclusions:Our results show that Activin A plays an important regulatory role in NANOG-mediated pluripotency in pig iPS cells. Activin A treatment may be therefore an effective method for de novo derivation of authentic embryonic stem cells (ESCs) from pig pre-implantation embryos.

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Lineage specification and pluripotency revealed by transcriptome analysis from oocyte to blastocyst in pig

Cited by 50 publications

References 43 publications

Maternal Cytokines CXCL12, VEGFA, and WNT5A Promote Porcine Oocyte Maturation via MAPK Activation and Canonical WNT Inhibition

Maternal Cytokines CXCL12, VEGFA, and WNT5A Promote Porcine Oocyte Maturation via MAPK Activation and Canonical WNT Inhibition

Identification of the Lineage Markers and Inhibition of DAB2 in In Vitro Fertilized Porcine Embryos

A cytokine screen using CRISPR-Cas9 knock-in reporter pig iPS cells reveals that Activin A regulates NANOG

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