Fig. 1

Proliferation and transcriptomic response of zebrafish testis to Fsh. (A) Schematic representation of the experimental set-up used in this study. Testis tissue was collected from adult males exposed to 10 nM estradiol (E₂) in vivo for 21 days, to induce androgen insufficiency. Testicular explants were cultured for 48 h in the absence or presence of 100 ng/mL Fsh, co-incubated with 25 µg/mL trilostane to prevent the production of 3-keto,delta-4 steroids, and then used for different purposes. (B-C) Immunocytochemical detection of BrdU incorporation served to quantify germ cell proliferation activity. (D) Quantification of BrdU labeling indices of type A_und and type A_diff spermatogonia (n = 4). (E-F) Immunocytochemical detection of BrdU in Sertoli cells (E) and quantification of BrdU-positive Sertoli cells per section surface area (F) (n = 4). (G) Quantification of testicular androgen (11-KT) production to evaluate the effects of E₂-pretreatment and/or trilostane. Males were pre-exposed (white background) or not (grey background) to 10 nM E₂in vivo for 21 days and testes were then incubated in the absence or presence of 100 ng/mL Fsh with medium that did or did not contain 25 µg/mL of trilostane (n = 11-12). Incubation medium was collected after 48 h of culture and 11-KT levels determined by RIA. (H) Total numbers of up- (left) and down-regulated genes (right) identified by RNAseq. Numbers of differentially expressed genes (DEGs; 1-P_NOI < 0.05) are shown in two categories after fold change (FC) selection (FC > 2.0, dark grey background; or < 2.0, light grey). (I) KEGG pathways modulated in Fsh-treated testes. Each pathway shown is represented by at least 3 DEGs (1-P_NOI < 0.05) and has a ratio of regulated genes (up-/down-, or vice versa) higher than 3. DEGs are highlighted with red (up-) or green (down-regulated) background. In D, F and G, data are expressed as mean ± SEM and asterisks indicate significant differences between treated and control groups (*P < 0.05, **P < 0.01, ***P < 0.001). In G, bars with different letters are significantly different. A_und, type A undifferentiated spermatogonia; A_diff, type A differentiating spermatogonia; SB, swim bladder; SC, Sertoli cell; T, testis; Tril., trilostane. Scale bar represents 25 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)