Figure 1.
Structure of the male gonopodium.
In adult swordtail males the anal fin forms a gonopodium (bracket, A). A naturally developed gonopodium with typical terminal structures that form distal lepidotrich segments (B) and a testosterone-induced gonopodium (24 days of testosterone treatment) (C); caudal is to the top and ventral to the left (B, C). Arrows indicate terminal structures. Abbreviations: C, claw; RA, ramus; TB, terminal blade; S, spine; TH, terminal hook. Scale bars: 500 µm.
Table 1.
Gene specific primers used for qPCR.
Figure 2.
Phylogenetic reconstruction of aldh1a and rar sequences.
Phylogenetic analysis of chordate aldh1a enzymes (A) and retinoic acid receptors (B) using PhyML (upper values) and MrBayes (lower values). For the analysis the coding regions of aldh1a and rar cDNAs were used. The position of the X. hellerii orthologs of aldh1a2, rar-ga and rar-gb within the two phylogenies is highlighted (grey box).
Figure 3.
Phylogenetic reconstruction of androgen receptor sequences.
Phylogenetic analysis of vertebrate androgen receptors using PhyML (upper values) and MrBayes (lower values). For analysis the coding regions of androgen receptor (ar) cDNAs were used. The position of the X. hellerii ara and arb orthologs within the phylogeny is highlighted (grey box).
Figure 4.
Expression of aldh1a2, rar-ga and rar-gb during gonopodium development.
aldh1a2 and both rarg paralogs are expressed in developing gonopodia of X. hellerii. At 5 (A), 7 (D) and 18 (J) days of testosterone treatment (dt) aldh1a2 is expressed the distal-most mesenchyme of the main gonopodial rays and extends into rays 6 and 7 (G). aldh1a2 expression was not detected under identical conditions in control fins (M). rar-ga and rar-gb are expressed in an overlapping pattern. At 5 (B, C), 7 (E, F), and 18 dt (K, L) strong expression of both genes could be detected in the gonopodial rays 3–5 and also partly in ray 6 and 7. At 18 dt both genes appeared slightly down-regulated. The expression domains of both genes include the distal-most and more proximo-lateral mesenchymal cells (H, I). No up-regulation was detected in the control fins (N, O). White arrowheads highlight gene expression. (n = 6 for 5 and 7dt, n = 4 for 18 dt, and n = 5 for controls for every probe); scale bars: A-F and J-O: 200 µm; G-I: 100 µm.
Figure 5.
Expression of androgen receptor b in the developing gonopodium.
androgen receptor b(arb) is expressed in developing gonopodia of X. hellerii. At 5 days of testosterone treatment (dt) arbis strongly up-regulated in distal tip of the gonopodial rays 3, 4 and 5 and clearly weaker in the rays 6–7 (A). Expression could also be detected in the inter-ray tissue (black arrowhead). This expression pattern persists at 7 (B) and 18 dt (D). Longitudinal sections of anal fins after 7 dt revealed arb to be expressed both in the distal and proximal mesenchyme (C, C'). In control fins arb is expressed at basal levels (E). White arrowheads indicate gene expression. (n>10 for every stage and probe; scale bars: A, B, D–F and H, I: 200 µm; C, C', G: 100 µm).
Figure 6.
Increase in aldh1a2 expression in testosterone-induced gonopodia.
Fold-changes of aldh1a2 expression in testosterone-induced gonopodia after 7 days of treatment (A) and in naturally developing gonopodia (B). Anal fins of females (n = 5) were used as control group and developing (n = 4) and complete (n = 8) gonopodia from males were used (B). Asterisks indicate statistically significant differences compared to the control (p<0.05, ANOVA). Figure is plotted as means ± SE.
Figure 7.
Segment numbers of rays 3, 4, 5, 6, 7 and 8 in testosterone-induced gonopodia at day 7 during treatment.
The number of segments of each ray in different concentrations of 17-α-methyltestosterone was measured (n = 7 to 8). Asterisks indicate statistically significant differences (*p<0.05, **p<0.01, rank by Tukey). Figure is plotted as means ± SE.
Figure 8.
Increased activation of RA signaling in testosterone-induced gonopodia affects growth and development.
The number of new segments (A) and the length (in μm) of the tissue added by growth (B) in artificially induced gonopodia were compared in control and RA-injected fish after 7 days of treatment (n = 4 for control and n = 7 for RA). For these measurements, we initially set a reference point, which is the distal most segmental border of the last formed segment at day 0 (before treatment) (E). The distance from the reference point to the tip of developing gonopodia at day 0 was excluded from the same distance at day 7 (B). The total distance at day 7 in each ray was significantly increased in RA-injected fish (B). The length of new segments (from the reference point to the distal boundary of the last segment at day 7) (C) and the length of tissue of tip of gonopodia (from the distal border of the last segment to the tip of gonopodia at day 7) (D) were measured separately. Both lengths (C, D) were contributed to the increased total length of outgrowth of gonopodia (B). *indicates statistically significant differences compared to the control and § indicates marginal significance (*p<0.05, **p<0.01, 0.05<§p<0.08). Figure is plotted as means ± SE.