Forward and reverse primer sequences, product sizes and accession numbers for genes analyzed by qRT-PCR. bp: base pairs.

<p>Forward and reverse primer sequences, product sizes and accession numbers for genes analyzed by qRT-PCR. bp: base pairs.</p

Effect of direct fetal androgenization on molecular liver function in adult offspring.

<p>QRT-PCR showing the effect of prenatal fetal androgenization on hepatic <i>AR</i> (A), <i>ERα</i> (B), <i>GR</i> (C), <i>PEPCK</i> (D), <i>MAP2K4</i> (E), <i>UGCG</i> (F), <i>ACADM</i> (G), <i>IGF1</i> (H) and <i>IGFBP1</i> (I) mRNA expression. Values represent mean ± SEM and * = <i>P</i><0.05.</p

Effect of maternal prenatal androgenization on adult liver steroid receptor expression.

<p>A) There was no difference in adult plasma testosterone after maternal TP compared to controls. B–C) Immunohistochemistry showing androgen receptor (AR) localization in adult ovine liver sections (B, scale bar = 100 µm; arrow indicating staining in blood vessels and C, scale bar = 20 µm; arrow indicating negatively stained hepatocyte nucleus). Representative sections were selected from control animals. QRT-PCR showing the effect of maternal androgenization on hepatic <i>AR</i> (D), <i>ERα</i> (E) and <i>GR</i> (F) mRNA expression. Values represent mean ± SEM and * = <i>P</i><0.05.</p

Effect of maternal prenatal androgenization on hepatic metabolic and signaling pathways.

<p>A-B) Immunohistochemistry for PEPCK (brown) in adult liver sections. A) Hepatocytes are stained while supporting tissue, such as the capsule (arrow) are not immunostained (scale bar = 100 µm). B) Higher power section of the more intensely stained hepatocytes around the hepatic portal vessels (HPV). Arrow indicates no staining associated with the non-hepatocytes in this region (scale bar = 20 µm). Representative sections were selected from control animals. QRT-PCR showing the effect of maternal androgenization on hepatic <i>PEPCK</i> (C), <i>MAP2K4</i> (D), <i>UGCG</i> (E), <i>ACADM</i> (F), <i>IGF1</i> (G), <i>IGFBP1</i> (H) and <i>IGF1R</i> (I) mRNA expression. Values represent mean ± SEM and * = <i>P</i><0.05, ** = <i>P</i><0.01.</p

Summary of the effects of differing fetal programming models on hepatic gene expression in the adult ewe.

<p>Upward arrows (↑) represent an increase and downward arrows (↓) represent a decrease in mean gene expression in TP cohorts compared to controls. The fetal injection column is compared to the maternal controls. Asterisks (*) indicate significantly altered gene expression. Dashes (-) represent no change.</p

Effect of maternal prenatal androgenization on adult liver lipid content and other metabolic parameters.

<p>A) Representative oil red O histological stains for lipids in ovine adult liver sections illustrating negative (-), possible early (+/−) or positive (+) fatty liver (scale bars = 50 µm) and B) analysis of control (C; n = 5) and maternal TP (n = 9) showing increased fatty liver in the TP cohort. Maternal prenatal TP treatment had no effect on adult liver analytes including aspartate transaminase (AST; C) and gamma glutamyltransferase (GGT; D). There was no effect on body weight (E), weight of omental fat (F), plasma leptin (G) and cholesterol levels (J). H) Plasma glucose concentrations at basal levels and 15 min after i.v. glucose bolus showed no differences, however, after 15 min there were significantly higher plasma insulin concentrations in the TP group (I). Values are mean ± SEM and * = <i>P</i><0.05.</p

Expression of the <i>SLITs</i> and <i>ROBOs</i> in ovarian cancer.

<p><b>A</b>) Real-time quantitative PCR showing increased <i>SLIT2</i>, <i>SLIT3</i>, <i>ROBO1</i>, <i>ROBO2</i> and <i>ROBO4</i> in primary cultures of hOSE compared to malignant epithelial cells cultured from the ascites of ovarian cancer patients. <b>B</b>) RT-PCR showing that both the SKOV-3 and PEO-14 cell lines expressed <i>SLIT2</i>, <i>SLIT3</i>, <i>ROBO1</i> and <i>ROBO2</i>. <b>C</b>) Real-time quantitative PCR showing <i>SLIT2</i>, <i>SLIT3</i> and <i>ROBO2</i> transcripts were more abundant in the well differentiated PEO-14 cells compared to the poorly differentiated SKOV-3 cells. FB = Fetal Brain positive control; -RT = RT negative negative control; - = DNA negative negative control; * = <i>P</i><0.05; ** = <i>P</i><0.01; *** = <i>P</i><0.005.</p

The effect of manipulation of <i>GR</i> on <i>SLITs</i> and <i>ROBOs</i> in ovarian cancer cells.

<p><b>A</b>) RT-PCR showing that PEO-14 and SKOV-3 cells expressed <i>GR</i> and <i>MR</i> but not <i>PR</i>. The breast tumour cell line HTB-19 was used as a positive control and –RT was used as a negative control. <b>B</b>) Real-time quantitative PCR demonstrating that transfection with the <i>GR</i> siRNA reduced <i>GR</i> expression in both cell lines when compared to the scrambled (sc) siRNA control. <b>C</b>) Confirmation that <i>GR</i>siRNA did not affect <i>MR</i> expression in PEO-14 and SKOV-3 cells. <b>D</b>) Quantitative Real-time PCR showing an increase in <i>SLIT2</i>, <i>ROBO1</i> and <i>ROBO2</i> expression after <i>GR</i> knock down by <i>GR</i>siRNA in PEO-14 cells. <b>E</b>) Demonstration that <i>GR</i>siRNA transfected SKOV-3 cells also had increased <i>SLIT2</i> and <i>ROBO1</i> expression. * = <i>P</i><0.05.</p

The effect of cortisol on the SLIT/ROBO pathway.

<p><b>A</b>) Real-time quantitative PCR showing that Cortisol (1000 nM), compared to Ethanol carrier control, reduced <i>SLIT2</i>, <i>SLIT3</i>, <i>ROBO1</i>, <i>ROBO2</i> and <i>ROBO4</i> expression in primary cultures of hOSE. <b>B</b>) Cortisol did not alter the expression of <i>SLITs</i> and <i>ROBOs</i> in primary cultures of ovarian epithelial cancer cells. <b>C</b>) <i>SLIT2</i>, <i>SLIT3</i>, <i>ROBO1</i> and <i>ROBO2</i> expression was significantly reduced by Cortisol in more differentiated PEO-14 cells. <b>D</b>) However expression of these <i>SLITs</i> and <i>ROBOs</i> was not regulated by Cortisol in the poorly differentiated SKOV-3 cells. <b>E</b>) Cortisol reduced secreted levels of SLIT2 protein in the PEO-14 cells (control secretion is 0.5 ng/ml). <b>F</b>) Cortisol did not affect secreted levels of SLIT2 protein in the SKOV-3 cells (control secretion is 0.5 ng/ml). * = <i>P</i><0.05; ** = <i>P</i><0.01.</p

Primers used for qRT-PCR including primer sequence.

<p>All primers were pre-validated and supplied by Primer Design.</p><p>Primers used for qRT-PCR including primer sequence.</p