19 research outputs found

    Differential Effects of Sodium Butyrate and Lithium Chloride on Rhesus Monkey Trophoblast Differentiation

    No full text
    <div><p>Trophoblast differentiation during early placental development is critical for successful pregnancy and aberrant differentiation causes preeclampsia and early pregnancy loss. During the first trimester, cytotrophoblasts are exposed to low oxygen tension (equivalent to~2%-3% O<sub>2</sub>) and differentiation proceeds along an extravillous pathway (giving rise to invasive extravillous cytotrophoblasts) and a villous pathway (giving rise to multinucleated syncytiotrophoblast). Interstitial extravillous cytotrophoblasts invade the decidua, while endovascular extravillous cytotrophoblasts are involved in re-modelling uterine spiral arteries. We tested the idea that sodium butyrate (an epigenetic modulator) induces trophoblast differentiation in early gestation rhesus monkey trophoblasts through activation of the Wnt/β-catenin pathway. The results show that syncytiotrophoblast formation was increased by butyrate, accompanied by nuclear accumulation of β-catenin, and increased expression of EnvV2 and galectin-1 (two factors thought to be involved in trophoblast fusion). Surprisingly, the expression of GCM1 and syncytin-2 was not affected by sodium butyrate. When trophoblasts were incubated with lithium chloride, a GSK3 inhibitor that mimics Wnt activation, nuclear accumulation of β-catenin also occurred but differentiation into syncytiotrophoblast was not observed. Instead the cells differentiated to mononucleated spindle-shaped cells and showed molecular and behavioral characteristics of endovascular trophoblasts. Another highly specific inhibitor of GSK3, CHIR99021, failed to induce endovascular trophoblast characteristics. These observations suggest that activation of the Wnt/β-catenin pathway correlates with both trophoblast differentiation pathways, but that additional factors determine specific cell fate decisions. Other experiments suggested that the differential effects of sodium butyrate and lithium chloride might be explained by their effects on TNFα production. The results provide valuable tools to manipulate trophoblast differentiation <i>in vitro</i> and to better understand the differentiation pathways that occur during early gestation.</p></div

    Effect of lithium chloride on metalloproteinase activity and cell invasion.

    No full text
    <p>(A) Cells were incubated in the presence of lithium chloride or sodium chloride (control) for 7 days after which MMP9 and MMP2 activities were measured by zymography as described in Methods. (B) To test for effects of lithium chloride on proliferation, equal numbers of trophoblasts were incubated in the presence of lithium chloride or sodium chloride for 7 days after which cell numbers were measured as described in Methods. (C) To assess invasive activity, trophoblasts were cultured in chamber inserts in the presence of lithium chloride or sodium chloride for 7 days after which invasion to the lower chamber was measured as described in Methods. Results are means ± SEM (n = 4).</p

    Expression of extravillous trophoblast marker proteins.

    No full text
    <p>Cells were cultured for 7 days (in the absence of sodium butyrate or lithium chloride) after which the expression of the selected adhesion molecules was detected by Western blotting as described in Methods. The graphs below the Western blot show densitometric quantitation of protein bands and values are shown as means ± SEM (n = 3). The asterisks indicate values that are significantly different (p<0.05) from the respective controls. ND, not detected.</p

    Primers used for qPCR.

    No full text
    <p>*, Sequence obtained from Esnault et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135089#pone.0135089.ref027" target="_blank">27</a>]</p><p>Primers used for qPCR.</p

    Comparison of the effects of CHIR99021 and lithium chloride on trophoblast adhesion molecule expression.

    No full text
    <p>Trophoblasts were incubated in the presence of 3 μM CHIR99021 or 20 mM lithium chloride for 7 days. (A) Phase contrast image of cells at 7 days. Note the absence of spindle-shaped cells in the presence of CHIR99021 and their presence when trophoblasts were incubated with lithium chloride. (B) Expression of adhesion molecules was assessed by Western blot as described in Methods. The graphs below the Western blot show densitometric quantitation of protein bands (n = 2). C, CHIR99021; L, lithium chloride.</p

    Characterization of trophoblasts isolated from early gestation rhesus monkey placenta.

    No full text
    <p>Trophoblasts were isolated as described in Methods. Fraction A represents the pool of tissue digests 1–3 and fraction B represents the pool of digests 4–8. The cells were cultured for 24 h and then fixed, permeabilized and stained using antibodies against cytokeratin 7 (green) and vimentin (red) as described in Methods. Nuclei were identified using DAPI (blue). The white bars represent 20 μm. The images are representative of cells isolated from 4 different placentas.</p

    Effect of lithium chloride on trophoblasts.

    No full text
    <p>The cells were incubated in the presence of 20 mM lithium chloride or 20 mM sodium chloride (control) for 7 days and then stained using an antibody against CK7 as described in Methods. The white bar represents 20 μm. The arrows point to spindle-shaped cells in the lithium chloride-treated cultures.</p

    Effect of sodium butyrate on the expression of selected trophoblast genes.

    No full text
    <p>Trophoblasts were incubated in the presence or absence of sodium butyrate for different times as indicated on the graphs. At each time point gene expression was measured by qPCR as described in the Methods section. The graphs on the left show relative expression as mean ΔCt values ± SEM for three experiments and higher values represent lower expression. The asterisks indicate that the values are significantly different from the respective control values (p<0.05, ANOVA with Bonferoni post-test). The graphs on the right represent fold-change relative to t = 0.</p

    Effect of Lithium chloride on the interaction of trophoblasts with endothelial cells.

    No full text
    <p>Trophoblasts were incubated in the presence of lithium chloride or sodium chloride (control) for 7 days as described in methods. The cells were then trypsinized and added on top of confluent monolayers of uterine microvascular endothelial cells. The cells were cocultured for 48 h. Other endothelial monolayers were incubated with trophoblast-conditioned medium (TCM) or trophoblast medium (TM) (A) Phase-contrast image of live cocultures. The arrows indicate trophoblast colonies. Note the large colonies seen in the presence of lithium chloride. (B) Immunofluorescence staining of cocultures and endothelial monolayers using antibodies against CK7 (green), VE-cadherin (red) and DAPI (blue). T, trophoblasts; EC, endothelial cells. The white horizontal bar represents 20μm.</p

    Effect of sodium butyrate on the expression of Galectin-1.

    No full text
    <p>Trophoblasts were incubated in the presence or absence of sodium butyrate for 7 days after which the expression of galectin-1 was measured by (A) qPCR and (B) Western blot as described in Methods. In A the graph to the left shows relative expression as mean ΔCt ± SEM for three experiments. The graph on the right show fold-change relative to t = 0. In B the graph to the right of the Western blot shows the results of densitometric quantitation (n = 3 in each case) and the asterisks indicate values that are significantly different (p<0.05) from the respective control values.</p
    corecore