Representative transcripts regulated by Fsh independently of Δ4-steroid production.

<p>Up-regulated genes mostly segregated in Cluster 2 whereas down-regulated genes segregated in Cluster 5. The response to Fsh was further identified as insensitive to trilostane in pairwise comparisons (Limma statistical test, p≤5%). When the information was available, we indicated the response to Lh (<b>¹</b>) Sambroni et al., 2013), the testicular expression profile as well as the <i>in vivo</i> regulation by androgens ((<b>²</b>)Rolland et al., 2009 and (<b>³</b>) Rolland et al., 2013). -: not determined.</p

Steroid-mediated action of Fsh on the steady-state level of mRNA transcripts measured by qPCR.

<p><i>slc26a4</i>, <i>vt1</i>, <i>mmp19</i> and <i>wisp1</i> and <i>inha</i> genes segregate in Cluster 1. The <i>inhba</i> gene belongs to Cluster 4. Three additional transcripts (<i>fshr</i>, <i>lhcgr</i> and <i>igfbp6</i>) previously demonstrated as up-regulated by gonadotropins are found to behave like genes in Cluster 1. Note that Fsh regulations of <i>inha</i>, <i>wisp1</i> and <i>igfbp6</i> are only partially lost in the presence of Tri. Bars represent mean ± SD of 5 to 6 replicates. Expression data were normalized to the reference gene <i>rps15</i>. Different letters indicate that treatments are significantly different as determined by non-parametric Mann & Whitney test.</p

Representative transcripts up-regulated by Fsh and sensitive to trilostane treatment.

<p>The corresponding genes segregated in Clusters 1or 2 and the response to Fsh was further identified as highly or moderately sensitive to trilostane in pairwise comparisons (Limma statistical test, p≤5%). When the information was available, we indicated the response to Lh (<b>¹</b>) Sambroni et al., 2013), the testicular expression profile as well as the <i>in vivo</i> regulation by androgens ((²)Rolland et al., 2009 and (³) Rolland et al., 2013). -: not determined.</p

Summary of the mechanisms underlying Fsh action on gene expression in rainbow trout testis.

<p>1- The primary action of Fsh is to stimulate steroidogenic cells to produce steroids which in turn regulate gene expression. 2- Fsh exerts specific regulatory effects independently of steroid mediation. 2 bis- In some cases steroids could have either an antagonistic or a redundant effect on gene expression. Plain lines with an arrow head indicate stimulatory effects whereas dotted lines illustrate inhibitory effects.</p

Meta-analysis: heatmap representation of the unsupervised hierarchical classification of the 58 clones found regulated by Fsh in the present study and in our previous study where Fsh and Lh effects on testicular transcriptome were measured (Sambroni et al., 2013).

<p>Meta-analysis: heatmap representation of the unsupervised hierarchical classification of the 58 clones found regulated by Fsh in the present study and in our previous study where Fsh and Lh effects on testicular transcriptome were measured (Sambroni et al., 2013).</p

Evaluation of trilostane treatment efficiency.

<p>11KT production in culture media after 48(500 ng/mL) alone or in combination with trilostane (10 µg/mL). Culture media were replaced after 48 h. Each bar represents the mean ± SD of 6 replicates. Different letters for each incubation duration indicate that treatments have significantly different effects as determined by non-parametric Mann & Whitney test (p<0.01).</p

Expression of Fsh-responsive genes.

<p>Heatmap representation of the hierarchical classification of the 102 clones differentially regulated in trout testis after an <i>in vitro</i> 4-day incubation without any substance (Ctrl) or with Fsh alone at 500 ng/mL (Fsh), trilostane alone at 10 µg/mL (Tri) or with both Fsh and trilostane (Fsh+Tri). Trilostane was added 1 h before Fsh addition in the medium. Media and hormones were renewed after 2 days. Clones segregated into 5 main groups, corresponding to genes which were up- or down-regulated by Fsh and sensitive or not to trilostane. Normalized expression values are shown according to the scale bar. Each line represents a clone and each column is a sample.</p

Representation of the mean fold-change in each cluster.

<p>Fold changes are relative to the control group. All the 102 clones included were individually found significantly affected by Fsh. Bars represent mean ± SEM. Y-axis is log-2 scaled and value 1 represents the control level. For convenience, note that the clusters are not shown in numerically ascending order.</p

Real-time PCR measurement of candidate gene expression.

<p>Relative expression of selected mRNA transcripts in testicular explants cultured during 4 days in the absence (Ctrl) or presence of either Fsh (500 ng/mL), 11-ketotestosterone (11KT) or 17α-methyltestosterone (MT) at 300 ng/mL (∼10⁻⁶ M). Bars represent mean ± SD of 5 to 6 replicates. Expression data were normalized to the reference gene <i>rps15</i>. Different letters indicate that treatments are significantly different as determined by non-parametric Mann & Whitney test.</p

Results obtained for the pathways enrichment analysis approach.

<p>Significantly enriched pathways (EASE score < 0.05) retrieved by the DAVID web tool for the “Total intersection” group in this analysis (4,160 gene IDs restricted to a set of 2,159 well annotated differentially expressed genes recognized by ENSDARG identifiers in DAVID). This group represents the common genes differentially expressed following chronic steroid modulation of immature gonads in rainbow trout at 0.01 and 0.1 μgEE2/L. They were retrieved from the intersection between two contrasts (CT1 and CT2) represented by a majority of fish displaying intersex gonads.</p