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Interleukin-6 increases inner cell mass numbers in bovine embryos.

ABSTRACT: BACKGROUND:Work in other species suggests that interleukin-6 (IL6) promotes early embryo development. It was unclear whether IL6 serves as an embryokine in cultured bovine embryos. This work was undertaken to elucidate the role of IL6 during in vitro bovine embryo production. RESULTS:Transcripts for IL6 and its two cognate receptor subunits (IL6R, IL6ST) were confirmed in bovine embryos from the 1-cell to blastocyst stages. Supplementing 100?ng/ml recombinant bovine IL6 to in vitro-produced bovine embryos at day 1, 3 or 5 increased (P?

SUBMITTER: Wooldridge LK

PROVIDER: S-EPMC6359871 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Interleukin-6 increases inner cell mass numbers in bovine embryos.

Wooldridge Lydia K LK Ealy Alan D AD

BMC developmental biology 20190201 1

<h4>Background</h4>Work in other species suggests that interleukin-6 (IL6) promotes early embryo development. It was unclear whether IL6 serves as an embryokine in cultured bovine embryos. This work was undertaken to elucidate the role of IL6 during in vitro bovine embryo production.<h4>Results</h4>Transcripts for IL6 and its two cognate receptor subunits (IL6R, IL6ST) were confirmed in bovine embryos from the 1-cell to blastocyst stages. Supplementing 100 ng/ml recombinant bovine IL6 to in vitr ...[more]

PMID: 30709330

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Addition of interleukin-6 to mouse embryo culture increases blastocyst cell number and influences the inner cell mass to trophectoderm ratio.

Project description:ObjectiveIn vitro culture of preimplantation embryos is improved by grouping embryos together in a drop of media. Individually cultured embryos are deprived of paracrine factors; with this in mind, we investigated whether the addition of a single embryo-secreted factor, interleukin-6 (IL-6), could improve the development of individually cultured embryos.MethodsMouse embryos were cultured individually in 2 µL of G1/G2 media in 5% oxygen and supplemented with a range of doses of recombinant mouse or human IL-6.ResultsMouse IL-6 increased hatching at doses of 0.01 and 10 ng/mL compared to the control (93% and 93% vs. 78%, p<0.05) and increased the total number of cells at a dose of 0.1 ng/mL compared to the control (101.95±3.36 vs. 91.31±3.33, p<0.05). In contrast, the highest dose of 100 ng/mL reduced the total number of cells (79.86±3.29, p<0.05). Supplementation with human IL-6 had a different effect, with no change in hatching or total cell numbers, but an increase in the percentage of inner cell mass per embryo at doses of 0.1, 1, and 100 ng/mL compared to the control (22.9%±1.1%, 23.3%±1.1%, and 23.1%±1.1% vs. 19.5%±1.0%, p<0.05).ConclusionThese data show that IL-6 improved mouse embryo development when cultured individually in complex media; however, an excess of IL-6 may be detrimental. Additionally, these data indicate that there is some cross-species benefit of human IL-6 for mouse embryos, but possibly through a different mechanism than for mouse IL-6.

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Global gene expression of the inner cell mass and trophectoderm of the bovine blastocyst.

Project description:BACKGROUND: The first distinct differentiation event in mammals occurs at the blastocyst stage when totipotent blastomeres differentiate into either pluripotent inner cell mass (ICM) or multipotent trophectoderm (TE). Here we determined, for the first time, global gene expression patterns in the ICM and TE isolated from bovine blastocysts. The ICM and TE were isolated from blastocysts harvested at day 8 after insemination by magnetic activated cell sorting, and cDNA sequenced using the SOLiD 4.0 system. RESULTS: A total of 870 genes were differentially expressed between ICM and TE. Several genes characteristic of ICM (for example, NANOG, SOX2, and STAT3) and TE (ELF5, GATA3, and KRT18) in mouse and human showed similar patterns in bovine. Other genes, however, showed differences in expression between ICM and TE that deviates from the expected based on mouse and human. CONCLUSION: Analysis of gene expression indicated that differentiation of blastomeres of the morula-stage embryo into the ICM and TE of the blastocyst is accompanied by differences between the two cell lineages in expression of genes controlling metabolic processes, endocytosis, hatching from the zona pellucida, paracrine and endocrine signaling with the mother, and genes supporting the changes in cellular architecture, stemness, and hematopoiesis necessary for development of the trophoblast.

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LIF-Dependent, Pluripotent Stem Cells Established from Inner Cell Mass of Porcine Embryos

Project description:The pig is important for agriculture and as an animal model in human and veterinary medicine, yet, despite over 20 years of effort, it has proved a difficult species from which to generate pluripotent stem cells analogous to those derived from mouse embryos. Here we report the production of LIF-dependent, so called naM-CM-/ve type, pluripotent stem cells from the inner cell mass of porcine blastocysts by up-regulating expression of KLF4 and POU5F1. These cells resemble mouse ES cells and are distinct from the FGF2-dependent, induced pluripotent cell type derived from porcine somatic cells. Transcript profiling utilized Affymetrix porcine microarrays were conducted to compare the gene expressions associated with pluripotency in the two LIF-dependent pESK lines (pESK I & II, passage 14 & 5, respectively) with a naM-CM-/ve phenotype and two porcine iPSC lines (ID4 & ID6, both passage 7, GSE15472) with a primed phenotype that re-programming of porcine fetal fibroblasts (EGFP-PFF, GSE15472). The pESK lines clustered separately from the porcine iPSC lines, EGFP-PFF and primary cultures established from explants of porine umbilical cord (PUC). Porcine pluripotent stem cells were derived from the inner cell mass with transduction with human KLF4 by lentiviral transduction.

2011-12-02 | E-GEOD-26369 | biostudies-arrayexpress

LIF-Dependent, Pluripotent Stem Cells Established from Inner Cell Mass of Porcine Embryos

Project description:The pig is important for agriculture and as an animal model in human and veterinary medicine, yet, despite over 20 years of effort, it has proved a difficult species from which to generate pluripotent stem cells analogous to those derived from mouse embryos. Here we report the production of LIF-dependent, so called naïve type, pluripotent stem cells from the inner cell mass of porcine blastocysts by up-regulating expression of KLF4 and POU5F1. These cells resemble mouse ES cells and are distinct from the FGF2-dependent, induced pluripotent cell type derived from porcine somatic cells. High throughput SNP chip genotyping was conducted on Illumina's Porcine SNP60 BeadChip (WG-410, a service provided by Geneseek, NE, http://www.neogen.com/GeneSeek/). The results exhibited that the two lines pluripotent stem cells from the inner cell mass of porcine blastocysts were porcine origin and genetically distinct. Porcine pluripotent stem cells were derived from the inner cell mass with transduction with human KLF4 by lentiviral transduction.

2011-01-05 | E-GEOD-26436 | biostudies-arrayexpress

Epigenetic disruptions of histone signatures for the trophectoderm and inner cell mass in mouse parthenogenetic embryos.

Project description:Epigenetic asymmetry has been shown to be associated with the first lineage allocation event in preimplantation development, that is, the formation of the trophectoderm (TE) and inner cell mass (ICM) lineages in the blastocyst. Since parthenogenesis causes aberrant segregation between the TE and ICM lineages, we examined several development-associated histone modifications in parthenotes, including those involved in (i) transcriptional activation [acetylated histone H3 lysine 9 (H3K9Ac) and lysine 14 (H3K14Ac), trimethylated histone H3 lysine 4 (H3K4Me3), and dimethylated histone H3 arginine 26 (H3R26Me2)] and (ii) transcriptional repression [trimethylated histone H3 lysine 9 (H3K9Me3) and lysine 27 (H3K27Me3), and mono-ubiquitinated histone H2A lysine 119 (H2AK119u1)]. Here, we report that in parthenotes, H3R26Me2 expression decreased from the morula stage, while expression patterns and levels of H3K9Ac, H3K27Me3, and H2AK119u1 were unchanged until the blastocyst stage; whereas H3K14Ac, H3K4Me3, and H3K9Me3 showed normal patterns and levels of expressions. Relative to the decrease of H3K9Ac in the ICM and increase in the TE of parthenotes, we detected reduced expression of TAT-interactive protein 60 acetyltransferase and histone deacetylase 1 deacetylase in the ICM and TE of parthenotes, respectively. Relative to the decrease of H3R26Me2, we also observed decreased expression of coactivator-associated arginine methyltransferase 1 methyltransferase and increased expression of the Wnt effector transcription factor 7L2 and miR-181c microRNA in parthenotes. Furthermore, relative to the decrease in H3K27Me3 and H2AK119u1, we found increased phosphorylation of Akt1 and enhancer of zeste homolog 2 in parthenogenetic TE. Therefore, our findings that histone signatures are impaired in parthenotes provide a mechanistic explanation for aberrant lineage segregation and TE defects.

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Leukemia inhibitory factor (LIF)-dependent, pluripotent stem cells established from inner cell mass of porcine embryos.

Project description:The pig is important for agriculture and as an animal model in human and veterinary medicine, yet despite over 20 years of effort, there has been a failure to generate pluripotent stem cells analogous to those derived from mouse embryos. Here we report the production of leukemia inhibitory factor-dependent, so-called naive type, pluripotent stem cells from the inner cell mass of porcine blastocysts by up-regulating expression of KLF4 and POU5F1. The alkaline phosphatase-positive colonies resulting from reprogramming resemble mouse embryonic stem cells in colony morphology, cell cycle interval, transcriptome profile, and expression of pluripotent markers, such as POU5F1, SOX2, and surface marker SSEA1. They are dependent on leukemia inhibitory factor signaling for maintenance of pluripotency, can be cultured over extended passage, and have the ability to form teratomas. These cells derived from the inner cell mass of pig blastocysts are clearly distinct from the FGF2-dependent "primed" induced pluripotent stem cells described recently from porcine mesenchymal cells. The data are consistent with the hypothesis that the up-regulation of KLF4, as well as POU5F1, is required to create and stabilize the naive pluripotent state and may explain why the derivation of embryonic stem cells from pigs and other ungulates has proved so difficult.

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LIF-Dependent, Pluripotent Stem Cells Established from Inner Cell Mass of Porcine Embryos

Project description:The pig is important for agriculture and as an animal model in human and veterinary medicine, yet, despite over 20 years of effort, it has proved a difficult species from which to generate pluripotent stem cells analogous to those derived from mouse embryos. Here we report the production of LIF-dependent, so called naïve type, pluripotent stem cells from the inner cell mass of porcine blastocysts by up-regulating expression of KLF4 and POU5F1. These cells resemble mouse ES cells and are distinct from the FGF2-dependent, induced pluripotent cell type derived from porcine somatic cells. Transcript profiling utilized Affymetrix porcine microarrays were conducted to compare the gene expressions associated with pluripotency in the two LIF-dependent pESK lines (pESK I & II, passage 14 & 5, respectively) with a naïve phenotype and two porcine iPSC lines (ID4 & ID6, both passage 7, GSE15472) with a primed phenotype that re-programming of porcine fetal fibroblasts (EGFP-PFF, GSE15472). The pESK lines clustered separately from the porcine iPSC lines, EGFP-PFF and primary cultures established from explants of porine umbilical cord (PUC).

2011-12-02 | GSE26369 | GEO

LIF-Dependent, Pluripotent Stem Cells Established from Inner Cell Mass of Porcine Embryos

Project description:The pig is important for agriculture and as an animal model in human and veterinary medicine, yet, despite over 20 years of effort, it has proved a difficult species from which to generate pluripotent stem cells analogous to those derived from mouse embryos. Here we report the production of LIF-dependent, so called naïve type, pluripotent stem cells from the inner cell mass of porcine blastocysts by up-regulating expression of KLF4 and POU5F1. These cells resemble mouse ES cells and are distinct from the FGF2-dependent, induced pluripotent cell type derived from porcine somatic cells. High throughput SNP chip genotyping was conducted on Illumina's Porcine SNP60 BeadChip (WG-410, a service provided by Geneseek, NE, http://www.neogen.com/GeneSeek/). The results exhibited that the two lines pluripotent stem cells from the inner cell mass of porcine blastocysts were porcine origin and genetically distinct.

2011-01-05 | GSE26436 | GEO

Genome-wide histone modification profiling of inner cell mass and trophoectoderm of blastocyst stage bovine embryos by novel low input RAT-ChIP

Project description:Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) has revolutionized our understanding of chromatin related biological processes. The method, however, requires thousands of cells and has therefore limited applications in situations where cell numbers are limited such as highly pure cell populations or early developmental stages of mammalian preimplantation embryo where only few hundred cells might be available. Numerous attempts have been made to reduce the number of cells needed for successful ChIP-seq experiment, however, the developed methods are often complex, laborious or not sensitive enough. Here we describe a new simple method called Restrictase Assisted Tagmentation Chromatin Immunoprecipitation (RAT-ChIP) that enables to obtain high quality genome-wide histone modification profiles from as few as 100 cells. The method is simple, cost-effective, takes one day to complete and can potentially be automated. We subsequently use the novel method to derive the first genome-wide maps of histone H3K4me3 and H3K27me3 modifications of inner cell mass (ICM) and trophoectoderm (TE) of bovine blastocyst stage embryos.

2019-11-15 | GSE103734 | GEO

Transcriptome profiling of bovine inner cell mass and trophectoderm derived from in vivo generated blastocysts.

Project description:This study describes the generation and analysis of the transcriptional profile of bovine inner cell mass (ICM) and trophectoderm (TE), obtained from in vivo developed embryos by using a bovine-embryo specific array (EmbryoGENE) containing 37,238 probes.A total of 4,689 probes were differentially expressed between ICM and TE, among these, 2,380 and 2,309 probes were upregulated in ICM and TE tissues, respectively (P ? 0.01, FC ? 2.0, FDR: 2.0). Ontological classification of the genes predominantly expressed in ICM emerged a range of functional categories with a preponderance of genes involved in basal and developmental signaling pathways including P53, TGF?, IL8, mTOR, integrin, ILK, and ELF2 signalings. Cross-referencing of microarray data with two available in vitro studies indicated a marked reduction in ICM vs. TE transcriptional difference following in vitro culture of bovine embryos. Moreover, a great majority of genes that were found to be misregulated following in vitro culture of bovine embryos were known genes involved in epigenetic regulation of pluripotency and cell differentiation including DNMT1, GADD45, CARM1, ELF5 HDAC8, CCNB1, KDM6A, PRDM9, CDX2, ARID3A, IL6, GADD45A, FGFR2, PPP2R2B, and SMARCA2. Cross-species referencing of microarray data revealed substantial divergence between bovine and mouse and human in signaling pathways involved in early lineage specification.The transcriptional changes occur during ICM and TE lineages specification in bovine is greater than previously understood. Therefore, this array data establishes a standard to evaluate the in vitro imprint on the transcriptome and to hypothesize the cross-species differences that allow in vitro acquisition of pluripotent ICM in human and mice but hinder that process in bovine.

| S-EPMC4683974 | biostudies-literature

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