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Inhibition of MEK1/2 and GSK3 (2i system) affects blastocyst quality and early differentiation of porcine parthenotes.

ABSTRACT: Inhibition of both MEK1/2 and glycogen synthase kinase-3 (GSK3; 2i system) facilitates the maintenance of naïve stemness for embryonic stem cells in various mammalian species. However, the effect of the inhibition of the 2i system on porcine early embryogenesis is unknown. We investigated the effect of the 2i system on early embryo development, expression of pluripotency-related genes, and epigenetic modifications. Inhibition of MEK1/2 (by PD0325901) and/or GSK3 (by CHIR99021) did not alter the developmental potential of porcine parthenogenetic embryos, but improved blastocyst quality, as judged by the blastocyst cell number, diameter, and reduction in the number of apoptotic cells. The expression levels of octamer-binding transcription factor 4 and SOX2, the primary transcription factors that maintain embryonic pluripotency, were significantly increased by 2i treatments. Epigenetic modification-related gene expression was altered upon 2i treatment. The collective results indicate that the 2i system in porcine embryos improved embryo developmental potential and blastocyst quality by regulating epigenetic modifications and pluripotency-related gene expression.

SUBMITTER: Kwon J

PROVIDER: S-EPMC6327883 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Inhibition of MEK1/2 and GSK3 (2i system) affects blastocyst quality and early differentiation of porcine parthenotes.

Kwon Jeongwoo J Li Ying-Hua YH Jo Yu-Jin YJ Oh YoungJin Y Namgoong Suk S Kim Nam-Hyung NH

PeerJ 20190107

Inhibition of both MEK1/2 and glycogen synthase kinase-3 (GSK3; 2i system) facilitates the maintenance of naïve stemness for embryonic stem cells in various mammalian species. However, the effect of the inhibition of the 2i system on porcine early embryogenesis is unknown. We investigated the effect of the 2i system on early embryo development, expression of pluripotency-related genes, and epigenetic modifications. Inhibition of MEK1/2 (by PD0325901) and/or GSK3 (by CHIR99021) did not alter the ...[more]

PMID: 30643672

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Project description:BackgroundCurrently, there is a high demand for efficient pig embryo cryopreservation procedures in the porcine industry as well as for genetic diversity preservation and research purposes. To date, vitrification (VIT) is the most efficient method for pig embryo cryopreservation. Despite a high number of embryos survives in vitro after vitrification/warming procedures, the in vivo embryo survival rates after embryo transfer are variable among laboratories. So far, most studies have focused on cryoprotective agents and devices, while the VIT effects on porcine embryonic gene expression remained unclear. The few studies performed were based on vitrified/warmed embryos that were cultured in vitro (IVC) to allow them to re-expand. Thus, the specific alterations of VIT, IVC, and the cumulative effect of both remained unknown. To unveil the VIT-specific embryonic alterations, gene expression in VIT versus (vs.) IVC embryos was analyzed. Additionally, changes derived from both VIT and IVC vs. control embryos (CO) were analyzed to confirm the VIT embryonic alterations. Three groups of in vivo embryos at the blastocyst stage were analyzed by RNA-sequencing: (1) VIT embryos (vitrified/warmed and cultured in vitro), (2) IVC embryos and (3) CO embryos.ResultsRNA-sequencing revealed three clearly different mRNA profiles for VIT, IVC and CO embryos. Comparative analysis of mRNA profiles between VIT and IVC identified 321, differentially expressed genes (DEG) (FDR < 0.006). In VIT vs. CO and IVC vs. CO, 1901 and 1519 DEG were found, respectively, with an overlap of 1045 genes. VIT-specific functional alterations were associated to response to osmotic stress, response to hormones, and developmental growth. While alterations in response to hypoxia and mitophagy were related to the sum of VIT and IVC effects.ConclusionsOur findings revealed new insights into the VIT procedure-specific alterations of embryonic gene expression by first comparing differences in VIT vs. IVC embryos and second by an integrative transcriptome analysis including in vivo control embryos. The identified VIT alterations might reflect the transcriptional signature of the embryo cryodamage but also the embryo healing process overcoming the VIT impacts. Selected validated genes were pointed as potential biomarkers that may help to improve vitrification.

Project description:Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes serious diarrhea in suckling piglets and has the potential for cross-species transmission. Although extensive studies have been reported on the biology and pathogenesis of PDCoV, the mechanisms by which PDCoV enters cells are not well characterized. In this study, we investigated how PDCoV enters IPI-2I cells, a line of porcine intestinal epithelial cells derived from pig ileum. Immunofluorescence assays, small interfering RNA (siRNA) interference, specific pharmacological inhibitors, and dominant negative mutation results revealed that PDCoV entry into IPI-2I cells depended on clathrin, dynamin, and a low-pH environment but was independent of caveolae. Specific inhibition of phosphatidylinositol 3-kinase (PI3K) and the Na+/H+ exchanger (NHE) revealed that PDCoV entry involves macropinocytosis and depends on NHE rather than on PI3K. Additionally, Rab5 and Rab7, but not Rab11, regulated PDCoV endocytosis. This is the first study to demonstrate that PDCoV uses clathrin-mediated endocytosis and macropinocytosis as alternative endocytic pathways to enter porcine intestinal epithelial cells. We also discussed the entry pathways of PDCoV into other porcine cell lines. Our findings reveal the entry mechanisms of PDCoV and provide new insight into the PDCoV life cycle. IMPORTANCE An emerging enteropathogenic coronavirus, PDCoV, has the potential for cross-species transmission, attracting extensive attenuation. Characterizing the detailed process of PDCoV entry into cells will deepen our understanding of the viral infection and pathogenesis and provide clues for therapeutic intervention against PDCoV. With the objective, we used complementary approaches to dissect the process in PDCoV-infected IPI-2I cells, a line of more physiologically relevant intestinal epithelial cells to PDCoV infection in vivo. Here, we demonstrate that PDCoV enters IPI-2I cells via macropinocytosis, which does not require a specific receptor, and clathrin-mediated endocytosis, which requires a low-pH environment and dynamin, while a caveola-mediated endocytic pathway is used by PDCoV to enter swine testicular (ST) cells and porcine kidney (LLC-PK1) cells. These findings provide a molecular detail of the cellular entry pathways of PDCoV and may direct us toward novel antiviral drug development.

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Inhibition of MEK1/2 and GSK3 (2i system) affects blastocyst quality and early differentiation of porcine parthenotes.

Publications

Inhibition of MEK1/2 and GSK3 (2i system) affects blastocyst quality and early differentiation of porcine parthenotes.

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