Effects of Downregulating Oct-4 Transcript by RNA Interference on Early Development of Porcine Embryos

Sakurai, Nobuyuki; Fujii, Takashi; Hashizume, Takumi; Sawai, Ken

doi:10.1262/jrd.2013-003

Cited by 17 publications

(24 citation statements)

References 46 publications

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“…In porcine embryos derived from IVF, OCT-4 is required for embryonic development from the morula to the blastocyst stage [5]. In this study, OCT-4 downregulation inhibited the transformation of morula to blastocyst in porcine embryos derived by parthenogenetic treatment.…”

Section: Discussionmentioning

confidence: 57%

OCT-4 expression is essential for the segregation of trophectoderm lineages in porcine preimplantation embryos

Emura

Sakurai

Takahashi

et al. 2016

Journal of Reproduction and Development

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Oct-4, a member of the POU family of transcription factors, is a key factor that regulates the segregation of the inner cell mass (ICM) and the trophectoderm (TE) during the transition from morula to blastocyst in mice. However, little is known about its role in porcine early embryogenesis. To determine the function of OCT-4 in the ICM and TE segregation of porcine embryos, we studied the developmental morphology of porcine embryos using RNA interference technology. Our experiments demonstrated that when 1-cell stage embryos were co-injected with the small interfering RNA (siRNA)for targeted knockdown of OCT-4 (OCT-4-siRNA) and tetramethylrhodamine isothiocyanate (TRITC)-dextran conjugate (Dx), they failed to form blastocysts. Therefore, in this study, we constructed chimeric embryos comprising blastomeres that either expressed OCT-4 normally or showed downregulated OCT-4 expression by co-injection of OCT-4-siRNA and Dx into one blastomere in 2- to 4-cell stage embryos. In control embryos, which were co-injected with control siRNA and Dx, Dx-positive cells contributed to the TE lineage in almost all the blastocysts examined. In contrast, Dx-positive cells derived from a blastomere co-injected with OCT-4-siRNA and Dx were degenerated in almost half the blastocysts. This was probably due to the inability of these cells to differentiate into the TE lineage. Real-time RT-PCR analysis revealed no difference in the levels of SOX2, TEAD4, FGF4 and FGFR1-IIIc, all of which are known to be regulated by OCT-4, between the OCT-4-siRNA-injected morulae and the control ones. However, the level of CDX2, a molecule specifically expressed in the TE lineage, was significantly higher in the former than in the latter. Our results indicate that continuous expression of OCT-4 in blastomeres is essential for TE formation of porcine embryos.

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Section: Discussionmentioning

confidence: 57%

OCT-4 expression is essential for the segregation of trophectoderm lineages in porcine preimplantation embryos

Emura

Sakurai

Takahashi

et al. 2016

Journal of Reproduction and Development

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“…is essential for porcine TE segregation (Emura et al, 2016;Sakurai et al, 2013). Therefore, OCT-4 functions in the formation of both ICM and TE in porcine embryos.…”

Section: Yap1 and Lats2 Knockdown In Porcine Embryosmentioning

confidence: 99%

“…Moreover, there are evidences for differences in molecular mechanisms of the ICM/TE segregation between mice and pigs. For example, our previous report has demonstrated that OCT-4 is essential for both ICM and TE segregation in porcine embryos, while it is needed only for ICM formation in mice (Emura et al, 2016;Nichols et al, 1998;Sakurai et al, 2013). In addition, CDX2 is dispensable for blastocyst formation in porcine embryos, but not in murine embryos (Bou et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Effect of Downregulating the Hippo Pathway Members YAP1 and LATS2 Transcripts on Early Development and Gene Expression Involved in Differentiation in Porcine Embryos

Emura

Saito

Miura

et al. 2020

Cellular Reprogramming

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In mouse development, differentiation of the inner cell mass (ICM) and trophectoderm (TE) during the transition from the morula to blastocyst stage is regulated by the Hippo pathway; however, the functions of the Hippo pathway in porcine embryogenesis have not been investigated. In the present study, we examined the gene expression patterns of the Hippo pathway members yes-associated protein 1 (YAP1) and large tumor suppressor 2 (LATS2) and the functions of these genes during porcine preimplantation development using RNA interference. Both YAP1 and LATS2 mRNA levels were shown high in the in vitro matured oocytes and 1-cell stage embryos and fell progressively with development. YAP1 nuclear localization was detected at the morula and blastocyst stages. Downregulation of either YAP1 or LATS2 inhibited porcine preimplantation development and affected the expression levels of POU class 5 homeobox 1 (OCT-4) and SRY-related HMG-box gene 2 (SOX2), transcription factors necessary for the ICM/TE differentiation. Taken together, YAP1 and LATS2 are essential for porcine preimplantation development, and it is possible that the Hippo pathway has important roles in porcine ICM/TE segregation.

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“…In previous studies using mouse embryos, knockout or of OCT4 itself did not impair early blastocyst formation [40, 44] although formation of the inner cell mass and its pluripotency were impaired [25]. To test targeting efficiency of the porcine OCT4 gene, we chose exons 2 and 5 and selected two target regions to introduce indels via non-homologous end repair(NHEJ), as shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

CRISPR/Cas9 as Tool for Functional Study of Genes Involved in Preimplantation Embryo Development

2015

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The CRISPR/Cas9 system has proven to be an efficient gene-editing tool for genome modification of cells and organisms. However, the applicability and efficiency of this system in pig embryos have not been studied in depth. Here, we aimed to remove porcine OCT4 function as a model case using the CRISPR/Cas9 system. Injection of Cas9 and single-guide RNA (sgRNA) against OCT4 decreased the percentages of OCT4-positive embryos to 37–50% of total embryos, while ~100% of control embryos exhibited clear OCT4 immunostaining. We assessed the mutation status near the guide sequence using polymerase chain reaction (PCR) and DNA sequencing, and a portion of blastocysts (20% in exon 2 and 50% in exon 5) had insertions/deletions near protospacer-adjacent motifs (PAMs). Different target sites had frequent deletions, but different concentrations of sgRNA made no impact. OCT4 mRNA levels dramatically decreased at the 8-cell stage, and they were barely detectable in blastocysts, while mRNA levels of other genes, including NANOG, and CDX2 were not affected. In addition, the combination of two sgRNAs led to large-scale deletion (about 1.8 kb) in the same chromosome. Next, we injected an enhanced green fluorescent protein (eGFP) vector targeting the OCT4 exon with Cas9 and sgRNA to create a knockin. We confirmed eGFP fluorescence in blastocysts in the inner cell mass, and also checked the mutation status using PCR and DNA sequencing. A significant portion of blastocysts had eGFP sequence insertions near PAM sites. The CRISPR/CAS9 system provides a good tool for gene functional studies by deleting target genes in the pig.

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Effects of Downregulating Oct-4 Transcript by RNA Interference on Early Development of Porcine Embryos

Cited by 17 publications

References 46 publications

OCT-4 expression is essential for the segregation of trophectoderm lineages in porcine preimplantation embryos

OCT-4 expression is essential for the segregation of trophectoderm lineages in porcine preimplantation embryos

Effect of Downregulating the Hippo Pathway Members YAP1 and LATS2 Transcripts on Early Development and Gene Expression Involved in Differentiation in Porcine Embryos

CRISPR/Cas9 as Tool for Functional Study of Genes Involved in Preimplantation Embryo Development

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