Table 1.
Additional candidate genes studied using qPCR.
Figure 1.
Evaluation of trilostane treatment efficiency.
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).
Figure 2.
Expression of Fsh-responsive genes.
Heatmap representation of the hierarchical classification of the 102 clones differentially regulated in trout testis after an in vitro 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.
Figure 3.
Representation of the mean fold-change in each cluster.
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.
Figure 4.
Steroid-mediated action of Fsh on the steady-state level of mRNA transcripts measured by qPCR.
slc26a4, vt1, mmp19 and wisp1 and inha genes segregate in Cluster 1. The inhba gene belongs to Cluster 4. Three additional transcripts (fshr, lhcgr and igfbp6) previously demonstrated as up-regulated by gonadotropins are found to behave like genes in Cluster 1. Note that Fsh regulations of inha, wisp1 and igfbp6 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 rps15. Different letters indicate that treatments are significantly different as determined by non-parametric Mann & Whitney test.
Table 2.
Representative transcripts up-regulated by Fsh and sensitive to trilostane treatment.
Figure 5.
Real-time PCR measurement of candidate gene expression.
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 rps15. Different letters indicate that treatments are significantly different as determined by non-parametric Mann & Whitney test.
Table 3.
Representative transcripts regulated by Fsh independently of Δ4-steroid production.
Figure 6.
Steroid-independent action of Fsh on the steady-state level of mRNA transcripts measured by qPCR.
The mdka and hsd3b1 genes belong to Cluster 2 and the amh gene is segregated in Cluster 5. Four additional candidates previously demonstrated as being regulated by gonadotropins - ccnd1, fstl3, igf1b/igf3 and cyp11b2-2 - behave like genes of Cluster 2. Furthermore an increased response to Fsh in the presence of trilostane suggests an antagonism between Fsh and the Δ4- steroids. Bars represent mean ± SD of 5 to 6 replicates. Expression data were normalized to the reference gene rps15.
Figure 7.
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).
Figure 8.
Summary of the mechanisms underlying Fsh action on gene expression in rainbow trout testis.
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.