The effect of agonists of estrogen receptors and xenoestrogen BPA on basal and hCG-stimulated testosterone production by Leydig cells from C57BL/6j and CBA/Lac mice.

<p>Leydig cells were incubated with the agonists alone (A) or co-treated with hCG for 17 h (B). Each experiment was performed independently five times with similar results. **P<0.01 compared to hCG treatment.</p

Comparative expression of steroidogenic genes in Leydig cells from CBA/Lac and C57BL/6j mice.

<p>Data are expressed as fold change ± S.E.M for four independent RNA preparations. *P<0.05 compared to CBA/Lac.</p

Basal and hCG-stimulated testosterone and estradiol production by Leydig cells from C57BL/6j and CBA/Lac mice as well as serum levels of LH.

<p>(A, B) Isolated Leydig cells were incubated with hCG or standard medium (control) for 17h, after which the concentrations of testosterone and estradiol were determined by RIA. Each experiment was performed four times independently obtaining similar results. (C). The data are expressed as means ± S.E.M (n = 23–27). *P<0.05, **P<0.01 compared to CBA/Lac; ♣P<0.05 compared to basal (untreated control).</p

Strain-related variations in sex hormone levels and their ratio in different mouse genotypes.

<p>The data are expressed as means ± S.E.M (n = 7–15). *P<0.05, **P<0.01, ***P<0.001compared to CBA/Lac; ♣♣P<0.01, ♣♣♣P<0.01 compared to C57BL/6j.</p

qPCR primer sequences and running conditions, bp-base pair.

<p>qPCR primer sequences and running conditions, bp-base pair.</p

Comparative expression of the family of estrogen receptor genes in Leydig cells from CBA/Lac and C57BL/6j mice.

<p>Data are expressed as fold change ± S.E.M for four independent RNA preparations. *P<0.05 compared to CBA/Lac.</p

Immunofluorescence detection of WT1 in HS207, HS360 and HS401 cells cultured in suspension.

<p>Nuclear expression of WT1 in cells was observed in all hES cell lines after spontaneous differentiation (A, C, and E) as well as after BMP7-stimulation (B, D and F) in cells mainly located on the edge of the spheres. Nuclear expression of WT1 was observed in Sertoli cells present in adult human testis (G; arrow heads). Negative controls exhibited no specific WT1 staining in either the nucleus or cytoplasm (H and small inserts in a-f). Red: WT1 staining. Blue: DAPI staining marking the nucleus. Scale bars: 100 μm in A to H, and 50 μm in the small inserts.</p

Expression of mRNA encoding the pluripotency markers <i>NANOG</i> and <i>POU5F1</i>, the germ cell marker <i>DDX4</i> and Sertoli cell markers <i>FSHR</i> and <i>VIM</i> by HS207, HS360 and HS401 cells cultured and differentiated in suspension.

<p>The relative level of mRNA was calculated by the ddCt procedure from the mean of triplicates and statistical analysis performed by way of One-way RM ANOVA. The error bars depict standard deviations. *p <0.05, **p <0.01, ***p <0.001 when comparing undifferentiated with differentiated cells. #p <0.05, ##p <0.01, ###p <0.001 when comparing BMP7-stimulated with unstimulated cells. For an explanation of the abbreviations, see the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144029#pone.0144029.g002" target="_blank">Fig 2</a>.</p

TaqMan Low-Density Array analysis of undifferentiated hES cells cultured on hFFs (hFF) or in suspension (sus).

<p>(A) Significantly lower and higher (p < 0.05) expression of genes in suspension culture relative to hFF culture. The expression values are dCt values relative to the mean expression of <i>ACTB</i>, <i>GAPDH</i> and <i>RAF1</i>, scaled by subtracting the dCt value from the lowest dCt+1 value. The value of dCt+1 was assigned to samples with no expression; thus blue (0) represents no expression, with increasing expression towards red. (B) Cell line-specific differences in gene expression between hFF and suspension culture conditions, with lower expression in suspension versus hFF cultures, and (C) higher expression in suspension versus hFF cultures (p < 0.05 for HS207 and HS360 cells). A list of gene names and abbreviations can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144029#pone.0144029.s011" target="_blank">S6 Table</a>.</p

Percentages of hES cell colonies exhibiting cells positive for DDX4.

<p>Cell colonies were divided into four groups according to their expression profile: no DDX4 expression, <25%, 25%–75% or >75% of DDX4-positive cells present per cell colony. A pool of three experiments (minimum 45 cell colonies) for each cell line and condition was evaluated to define DDX4 expression.</p><p>Percentages of hES cell colonies exhibiting cells positive for DDX4.</p