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Sphingosine–sphingosine-1-phosphate pathway regulates trophoblast differentiation and syncytialization  Ambika T. Singh, Arunasalam Dharmarajan, Irving.

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Presentation on theme: "Sphingosine–sphingosine-1-phosphate pathway regulates trophoblast differentiation and syncytialization  Ambika T. Singh, Arunasalam Dharmarajan, Irving."— Presentation transcript:

1 Sphingosine–sphingosine-1-phosphate pathway regulates trophoblast differentiation and syncytialization  Ambika T. Singh, Arunasalam Dharmarajan, Irving L.M.H. Aye, Jeffrey A. Keelan  Reproductive BioMedicine Online  Volume 24, Issue 2, Pages (February 2012) DOI: /j.rbmo Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

2 Figure 1 Characterization of trophoblast differentiation markers. (A) Human chorionic gonadotrophin (HCG) production was measured by enzyme-linked immunosorbent assay in conditioned culture media over 5days in culture. Expression of the cytotrophoblast transcription factor glial cells missing homologue 1 (GCM1) was assessed by quantitative real-time PCR. Placental alkaline phosphatase (PLAP) activity was measured in cellular extracts from cultured trophoblasts. (B) Primary trophoblasts isolated from the human term placenta and visualized using Sigma fast 3,3-diaminobenzidine, detecting epithelial-specific marker cytokeratin 7 24h after isolation. (C) Islands of syncytiotrophoblast formed on day 5 after cytotrophoblast fusion. Values are percent of day-1 values as mean±SEM from three to five experiments. ∗P⩽0.05, ∗∗∗P⩽0.001, compared with day-1 concentrations by ANOVA. (B, C) Magnification=×40; bar=20μm. Reproductive BioMedicine Online  , DOI: ( /j.rbmo ) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

3 Figure 2 Concentrations of intracellular sphingosine kinase 1 (SPHK1), sphingosine (SPH) and secreted sphingosine-1-phosphate (S1P) during trophoblast differentiation in vitro. Western blotting was used to determine protein expression of SPHK1 enzyme (A) during 7days in culture during spontaneous villous trophoblast differentiation. Concentrations of intracellular SPH (B) and secreted S1P (C) were measured by LC-MS/MS. Values are percent of day 1 values as mean±SEM from three or four experiments. ∗P⩽0.05, ∗∗P⩽0.01, ∗∗∗P⩽0.001, compared with day 1 concentrations by ANOVA. Reproductive BioMedicine Online  , DOI: ( /j.rbmo ) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

4 Figure 3 Effects of sphingosine (SPH), sphingosine-1-phosphate (S1P) and sphingosine kinase 1 (SPHK1) inhibitor on trophoblast cell viability. Cells were treated with SPH (A), S1P (B) and SPHK1 inhibitor (C) for up to 72h and then viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Values are percentage of MTT reduction compared with control conditions at 24h for three or four experiments. ∗∗P⩽0.01 compared to control. Reproductive BioMedicine Online  , DOI: ( /j.rbmo ) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

5 Figure 4 Regulation of biochemical trophoblast differentiation by sphingolipid compounds. Effects of exogenous administration of sphingosine (SPH) (A,D,G), sphingosine-1-phosphate (S1P) (B,E,H) and sphingosine kinase 1 (SPHK1) inhibitor (C,F,I) on human chorionic gonadotrophin (HCG) secretion, glial cells missing homologue 1 (GCM1) expression and placental alkaline phosphatase (PLAP) activity compared with vehicle-treated cells during differentiation. Values are percent of 24h controls as mean±SEM from three to five experiments. ∗P⩽0.05, ∗∗P⩽0.01, ∗∗∗P⩽0.001, compared with controls for each treatment at respective time points (ANOVA). Reproductive BioMedicine Online  , DOI: ( /j.rbmo ) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

6 Figure 5 Changes in morphological differentiation. Immunocytochemistry (A–D) was used to determine the ratio of multinuclear cells (two or more nuclei per cell) to total number of nuclei, assessed by E-cadherin expression, in response to sphingosine (SPH), sphingosine-1-phosphate (S1P) and sphingosine kinase 1 (SPHK1) inhibitor (E) compared with untreated cells. Values are mean±SEM from three experiments. ∗P⩽0.05, compared with controls (ANOVA). Bar=20μm. Reproductive BioMedicine Online  , DOI: ( /j.rbmo ) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

7 Figure 6 Phosphorylation of Akt in response to sphingosine-1-phosphate (S1P) treatment. Immunoblots representing total and phosphorylated Akt (Ser473) after acute exposure to S1P for 3h compared with control. Values are mean±SEM from 3 experiments. ∗P⩽0.001, compared with control by two-tailed t-test. Reproductive BioMedicine Online  , DOI: ( /j.rbmo ) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

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