Developmental changes in expression of pluripotent genes in early equine embryos.

Genes involved in maintaining pluripotency have potential use in establishing cell lines for regenerative medicine. However, the genes differ subtly between species, and are poorly described in the horse. In this study we examined changes in expression of pluripotency-associated genes in horse embryos during blastocyst formation. Twenty-one grade 1–2 embryos where recovered from mares by uterine lavage on Day 6–7 after ovulation. Embryos were classified by developmental stage (morula, early or expanded blastocyst: n = 5, 7 and 9, respectively) and their diameter measured by micrometer, before being snap frozen. Subsequently, mRNA from individual embryos was extracted, DNAse-treated and synthesized into cDNA using an AllPrep Mini Kit and Superscript III Reverse Transcriptase (Qiagen, Venlo, and Invitrogen, Breda respectively, the Netherlands). Equine-specific intron-spanning/overlapping primers were designed using PerlPrimer v1.1.14 by BLAST searching the NCBI horse genome for 5 genes associated with pluripotency in other species (octamer binding protein OCT4, transcription factor NANOG, developmental pluripotency-associated DPPA4, growth and differentiation factor GDF3 and telomerase reverse transcriptase TERT) and 2 reference genes (signal recognition particle SRP14 and phosphoglycerate kinase PGK1). Relative gene expression was then examined by quantitative PCR using an iQ5 RT PCR Detection System (BioRad, Veenendaal, the Netherlands). Relationships were tested by Pearson correlations and differences between developmental stages were tested by ANCOVA. Embryos ranged in diameter from 126 to 680 μm. As expected, absolute expression of all pluripotency markers increased with increasing embryo diameter (P = 0.000; R = 0.93, 0.92, 0.88, 0.86 and 0.76 for NANOG, DPPA4, GDF3, OCT4 and TERT, respectively). After normalization with SRP14 and PGK1, significant negative correlations with embryo diameter were apparent for OCT4, NANOG and DPPA4 (P < 0.001; R = –0.73, –0.69 and –0.53, respectively). Moreover, all 5 pluripotency genes were down-regulated as embryonic development progressed (P < 0.05), although the time-course differed between genes. The DPPA4 and OCT4 expression decreased significantly at both the morula-early blastocyst and early-expanded blastocyst transitions, whereas NANOG expression only decreased significantly between the early-expanded blastocyst stages and GDF3 and TERT expression only between the morula-early blastocyst stages. Down-regulation of pluripotent gene expression during early development is consistent with increased cohorts of cells differentiating into trophectoderm and primitive endoderm, leaving an ever decreasing proportion of pluripotent cells in the inner cell mass. Furthermore, the different time courses of down-regulation may reflect different roles of the examined genes in developmental processes. For example, early and continued down-regulation of OCT4 is consistent with a role in differentiation between ICM and trophectoderm, whereas the slightly later onset of NANOG down-regulation better fits a role in determining between pluripotent epiblast and primitive endoderm, as described in other species

Germ cells and the origins of mammalian pluripotent cells

Mammalian embryonic stem (ES) cells originate from preimplantation embryos and can be propagated indefinitely without loss of pluripotency; i.e. the potential to develop into any embryonic cell type. ES cells have been described for mouse, rhesus monkey, and human. There is considerable interest in human ES cells because of their potential role in future medicine to regenerate damaged tissues. Potential risks need to be investigated in adequate model species, before such therapies can be practiced in human. The pig is a good candidate model organism in terms of organ sizes, physiology, and physiological life-span. However, true ES-cell lines have not yet been described for livestock species. It has been hypothesized that the resistance of livestock species to ES-cell derivation is caused by the difference in embryonic development between species. An important finding that is described in the thesis is that key transciption factors that are important for pluripotency in the mouse, are differentially expressed in early pig, cow, and, mouse embryo development. This could have consequences for the mechanisms of generation of ES-cells in livestock species. Spermatogonial stem cells (SSC) can be another source of pluripotent cells, if cultured under the appropriate conditions. The aim of one study of the thesis was to isolate, culture, and characterize male porcine germ cells. In particular, the effect of four commonly used growth factors on the self-renewal of porcine SSCs was investigated. In primary cultures of male germ cells, a large effect was found of growth factor FGF, which had a negative influence on the expression of germ cell specific genes and a positive influence on the expression of genes specific for somatic cells. Little is known about the mechanism behind the transition of SSC to ES-like cells, but the tumor suppressors TRP53 and PTEN seem to be involved in inhibiting this process. To get more insight in the role of these tumor suppressors in SSC, their expression levels were independently knocked down through RNA interference in a murine SSC line. In either case, this resulted in upregulation of the pluripotency factor Nanog. Therefore, it is hypothesized that male germ cells are insulated from pluripotency through independent suppression of Nanog through TRP53 and PTEN. In mouse development, expression of the pluripotency factor NANOG is restricted to cells of the pluripotent epiblast and to primordial germ cells, which are both cell types that can give rise to pluripotent cells. Although recent reports demonstrate that the testis can also give rise to pluripotent cells and testis function is closely associated with pluripotency, expression of NANOG in the testis has not been described yet. In a comparative study, NANOG expression was detected in differentiating male germ cells of mouse, dog, pig, and human. The findings of this study indicate at a conserved role for NANOG in the meiotic and post-meiotic phase of spermatogenesis. It is hypothesized that NANOG is involved in the epigenetic events that occur before and after meiosis

Germ cells and the origins of mammalian pluripotent cells

Mammalian embryonic stem (ES) cells originate from preimplantation embryos and can be propagated indefinitely without loss of pluripotency; i.e. the potential to develop into any embryonic cell type. ES cells have been described for mouse, rhesus monkey, and human. There is considerable interest in human ES cells because of their potential role in future medicine to regenerate damaged tissues. Potential risks need to be investigated in adequate model species, before such therapies can be practiced in human. The pig is a good candidate model organism in terms of organ sizes, physiology, and physiological life-span. However, true ES-cell lines have not yet been described for livestock species. It has been hypothesized that the resistance of livestock species to ES-cell derivation is caused by the difference in embryonic development between species. An important finding that is described in the thesis is that key transciption factors that are important for pluripotency in the mouse, are differentially expressed in early pig, cow, and, mouse embryo development. This could have consequences for the mechanisms of generation of ES-cells in livestock species. Spermatogonial stem cells (SSC) can be another source of pluripotent cells, if cultured under the appropriate conditions. The aim of one study of the thesis was to isolate, culture, and characterize male porcine germ cells. In particular, the effect of four commonly used growth factors on the self-renewal of porcine SSCs was investigated. In primary cultures of male germ cells, a large effect was found of growth factor FGF, which had a negative influence on the expression of germ cell specific genes and a positive influence on the expression of genes specific for somatic cells. Little is known about the mechanism behind the transition of SSC to ES-like cells, but the tumor suppressors TRP53 and PTEN seem to be involved in inhibiting this process. To get more insight in the role of these tumor suppressors in SSC, their expression levels were independently knocked down through RNA interference in a murine SSC line. In either case, this resulted in upregulation of the pluripotency factor Nanog. Therefore, it is hypothesized that male germ cells are insulated from pluripotency through independent suppression of Nanog through TRP53 and PTEN. In mouse development, expression of the pluripotency factor NANOG is restricted to cells of the pluripotent epiblast and to primordial germ cells, which are both cell types that can give rise to pluripotent cells. Although recent reports demonstrate that the testis can also give rise to pluripotent cells and testis function is closely associated with pluripotency, expression of NANOG in the testis has not been described yet. In a comparative study, NANOG expression was detected in differentiating male germ cells of mouse, dog, pig, and human. The findings of this study indicate at a conserved role for NANOG in the meiotic and post-meiotic phase of spermatogenesis. It is hypothesized that NANOG is involved in the epigenetic events that occur before and after meiosis

Developmental changes in expression of pluripotent genes in early equine embryos.

Genes involved in maintaining pluripotency have potential use in establishing cell lines for regenerative medicine. However, the genes differ subtly between species, and are poorly described in the horse. In this study we examined changes in expression of pluripotency-associated genes in horse embryos during blastocyst formation. Twenty-one grade 1–2 embryos where recovered from mares by uterine lavage on Day 6–7 after ovulation. Embryos were classified by developmental stage (morula, early or expanded blastocyst: n = 5, 7 and 9, respectively) and their diameter measured by micrometer, before being snap frozen. Subsequently, mRNA from individual embryos was extracted, DNAse-treated and synthesized into cDNA using an AllPrep Mini Kit and Superscript III Reverse Transcriptase (Qiagen, Venlo, and Invitrogen, Breda respectively, the Netherlands). Equine-specific intron-spanning/overlapping primers were designed using PerlPrimer v1.1.14 by BLAST searching the NCBI horse genome for 5 genes associated with pluripotency in other species (octamer binding protein OCT4, transcription factor NANOG, developmental pluripotency-associated DPPA4, growth and differentiation factor GDF3 and telomerase reverse transcriptase TERT) and 2 reference genes (signal recognition particle SRP14 and phosphoglycerate kinase PGK1). Relative gene expression was then examined by quantitative PCR using an iQ5 RT PCR Detection System (BioRad, Veenendaal, the Netherlands). Relationships were tested by Pearson correlations and differences between developmental stages were tested by ANCOVA. Embryos ranged in diameter from 126 to 680 μm. As expected, absolute expression of all pluripotency markers increased with increasing embryo diameter (P = 0.000; R = 0.93, 0.92, 0.88, 0.86 and 0.76 for NANOG, DPPA4, GDF3, OCT4 and TERT, respectively). After normalization with SRP14 and PGK1, significant negative correlations with embryo diameter were apparent for OCT4, NANOG and DPPA4 (P < 0.001; R = –0.73, –0.69 and –0.53, respectively). Moreover, all 5 pluripotency genes were down-regulated as embryonic development progressed (P < 0.05), although the time-course differed between genes. The DPPA4 and OCT4 expression decreased significantly at both the morula-early blastocyst and early-expanded blastocyst transitions, whereas NANOG expression only decreased significantly between the early-expanded blastocyst stages and GDF3 and TERT expression only between the morula-early blastocyst stages. Down-regulation of pluripotent gene expression during early development is consistent with increased cohorts of cells differentiating into trophectoderm and primitive endoderm, leaving an ever decreasing proportion of pluripotent cells in the inner cell mass. Furthermore, the different time courses of down-regulation may reflect different roles of the examined genes in developmental processes. For example, early and continued down-regulation of OCT4 is consistent with a role in differentiation between ICM and trophectoderm, whereas the slightly later onset of NANOG down-regulation better fits a role in determining between pluripotent epiblast and primitive endoderm, as described in other species

Stage specific changes in expression of OCT4, CDX2, NANOG and GATA6 mRNA during blastocyst formation in the horse

During blastocyst formation two consecutive lineage\ud segregation events give rise to the three primitive cell\ud types: trophectoderm, primitive endoderm, and pluripotent\ud epiblast. In the mouse, octamer binding protein 4\ud (OCT4) and caudal type homeobox 2 (CDX2) are essential\ud for inner cell mass (ICM) and trophectoderm formation\ud respectively, while differential expression of NANOG and\ud GATA binding protein 6 (GATA6) in cells of the ICM determines which will form epiblast or hypoblast (primitive\ud endoderm) respectively (Ralston and Rossant, 2005). However,\ud expression of OCT4 protein in the trophectoderm and\ud the absence of NANOG protein and gene expression during\ud blastocyst formation in large domestic species, suggest\ud that species-specific differences exist in the processes governing early lineage segregation (Kuijk et al., 2008).\ud Equine blastocyst formation occurs 6–7 days after ovulation\ud and is accompanied by an increase in cell numbers from around 150 to over 2900, where multiplication is\ud predominantly within the trophoblast and primitive endoderm\ud (Betteridge, 2007). Since little is known about the\ud roles of OCT4, CDX2, NANOG and GATA6 in early lineage\ud segregation events in the horse, we aimed to determine\ud whether expression of these genes is consistent with lineage\ud specification during equine blastocyst formatio

The roles of FGF and MAP kinase signaling in the segregation of the epiblast and hypoblast cell lineages in bovine and human embryos

At the blastocyst stage of mammalian pre-implantation development, three distinct cell lineages have formed: trophectoderm, hypoblast (primitive endoderm) and epiblast. The inability to derive embryonic stem (ES) cell lines in a variety of species suggests divergence between species in the cell signaling pathways involved in early lineage specification. In mouse, segregation of the primitive endoderm lineage from the pluripotent epiblast lineage depends on FGF/MAP kinase signaling, but it is unknown whether this is conserved between species. Here we examined segregation of the hypoblast and epiblast lineages in bovine and human embryos through modulation of FGF/MAP kinase signaling pathways in cultured embryos. Bovine embryos stimulated with FGF4 and heparin form inner cell masses (ICMs) composed entirely of hypoblast cells and no epiblast cells. Inhibition of MEK in bovine embryos results in ICMs with increased epiblast precursors and decreased hypoblast precursors. The hypoblast precursor population was not fully ablated upon MEK inhibition, indicating that other factors are involved in hypoblast differentiation. Surprisingly, inhibition of FGF signaling upstream of MEK had no effects on epiblast and hypoblast precursor numbers in bovine development, suggesting that GATA6 expression is not dependent on FGF signaling. By contrast, in human embryos, inhibition of MEK did not significantly alter epiblast or hypoblast precursor numbers despite the ability of the MEK inhibitor to potently inhibit ERK phosphorylation in human ES cells. These findings demonstrate intrinsic differences in early mammalian development in the role of the FGF/MAP kinase signaling pathways in governing hypoblast versus epiblast lineage choices

A Distinct Expression Pattern in Mammalian Testes Indicates a Conserved Role for NANOG in Spermatogenesis

Background: NANOG is a key player in pluripotency and its expression is restricted to pluripotent cells of the inner cell mass, the epiblast and to primordial germ cells. Spermatogenesis is closely associated with pluripotency, because through this process highly specialized sperm cells are produced that contribute to the formation of totipotent zygotes. Nevertheless, it is unknown if NANOG plays a role in this process. Methodology/Principal Findings: In the current study, NANOG expression was examined in testes of various mammals, including mouse and human. Nanog mRNA and NANOG protein were detected by RT-PCR, immunohistochemistry, and western blotting. Furthermore, eGFP expression was detected in the testis of a transgenic Nanog eGFP-reporter mouse. Surprisingly, although NANOG expression has previously been associated with undifferentiated cells with stem cell potential, expression in the testis was observed in pachytene spermatocytes and in the first steps of haploid germ cell maturation (spermiogenesis). Weak expression in type A spermatogonia was also observed. Conclusions: The findings of the current study strongly suggest a conserved role for NANOG in meiotic and post-meiotic stages of male germ cell development.Stem cells & developmental biolog

Validation of ACCESS: an automated tool to support self-management of COPD exacerbations

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