Background: Mammalian first lineage segregation generates trophectoderm (TE) and pluripotent inner cell mass (ICM), which provides an ideal model for studying the mechanisms of maintenance and loss of pluripotency. In mouse, the transcription factor OCT4 restricts to ICM and plays a key role in TE/ICM specification and pluripotent regulatory networks. However, in pig, OCT4 does not restrict to ICM cells, suggesting a different molecular basis in TE/ICM specification and pluripotent regulatory networks. Results: To explore molecular basis of porcine TE/ICM specification and pluripotent regulatory networks, we examined expression pattern of pluripotency factors, including SOX2, REX1, SALL4, ESG1, NANOG, TBX3, LIN28, KLF2, and KLF5, in porcine blastocysts. We found that SOX2 is a faithful pluripotent marker that anchored to the pluripotent cells including embryonic part cells, ICM cells and newly EPI cells along with developmental progress, whereas OCT4 expressed in almost all the cells at the same time. Consistently, analysis of spatiotemporal distribution of SOX2 and the TE marker CDX2 revealed an exclusive expression pattern in D6 blastocysts, whereas no correlation was observed between OCT4 and CDX2 at the same stage. Conclusions: Our results provide a molecular basis in porcine embryonic patterning and a clue for further studying porcine pluripotent regulatory networks. Developmental Dynamics 244:619-627, 2015. V C 2015 Wiley Periodicals, Inc.
Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC) line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP) positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs) could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs.
Aberrant epigenetic reprogramming is the main obstacle to the development of somatic cell nuclear transfer (SCNT) embryos and the generation of induced pluripotent stem (iPS) cells, which results in the low reprogramming efficiencies of SCNT and iPS. Histone H3 lysine 27 trimethylation (H3K27me3), as a repressive epigenetic mark, plays important roles in mammalian development and iPS induction. However, the reprogramming of H3K27me3 in pig remains elusive. In this study, we showed that H3K27me3 levels in porcine early cloned embryos were higher than that in IVF embryos. Then GSK126 and GSK-J4, two small molecule inhibitors of H3K27me3 methylase (EZH2) and demethylases (UTX/JMJD3), were used to regulate the H3K27me3 level. The results showed that H3K27me3 level was reduced in cloned embryos after treatment of PEF with 0.75 mM GSK126 for 48 h, incubation of one-cell reconstructed oocytes with 0.1 mM GSK126 and injection of antibody for EZH2 into oocyte. Meanwhile, the development of the cloned embryos was significantly improved after these treatments. On the contrary, GSK-J4 treatment increased the H3K27me3 level in cloned embryos and decreased the cloned embryonic development. Furthermore, iPS efficiency was both increased after reducing the H3K27me3 level in donor cells and in early reprogramming phase. In summary, our results suggest that H3K27me3 acts as an epigenetic barrier in SCNT and iPS reprogramming, and reduction of H3K27me3 level in donor cells and in early reprogramming phase can enhance both porcine SCNT and iPS efficiency.
Endogenous retroviruses (ERVs) are transcriptionally active in cleavage stage embryos, yet their functions are unknown. ERV sequences are present in the majority of long intergenic noncoding RNAs (lincRNAs) in mouse and humans, playing key roles in many cellular processes and diseases. Here, we identify LincGET as a nuclear lincRNA that is GLN-, MERVL-, and ERVK-associated and essential for mouse embryonic development beyond the two-cell stage. LincGET is expressed in late two- to four-cell mouse embryos. Its depletion leads to developmental arrest at the late G2 phase of the two-cell stage and to MAPK signaling pathway inhibition. LincGET forms an RNA-protein complex with hnRNP U, FUBP1, and ILF2, promoting the cis-regulatory activity of long terminal repeats (LTRs) in GLN, MERVL, and ERVK (GLKLTRs), and inhibiting RNA alternative splicing, partially by downregulating hnRNP U, FUBP1, and ILF2 protein levels. Hnrnpu or Ilf2 mRNA injection at the pronuclear stage also decreases the preimplantation developmental rate, and Fubp1 mRNA injection at the pronuclear stage causes a block at the two-cell stage. Thus, as the first functional ERV-associated lincRNA, LincGET provides clues for ERV functions in cleavage stage embryonic development.
Background: Oocyte factors can reprogram the somatic nucleus efficiently, but these factors still need to be defined. Results: Maternal vimentin acts as a genomic protector and results in p53 down-regulation during nuclear reprogramming. Conclusion: Maternal vimentin is crucial for nuclear reprogramming. Significance: We report the first evidence of vimentin as a reprogramming factor.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.