Figure 1.
A) Concentration of endogenous cortisol in larvae reared at masculinizing (MPT, 29°C), mixed-sex-producing (MixPT, 24°C), and feminizing (FPT, 17°C) temperatures between 0 and 7 wah. Larvae reared at the MPT and MixPT showed higher cortisol values than those at the FPT between 3 and 5 weeks, a period considered as critical for gonadal sex determination in pejerrey. Different letters inside and above the bars indicate statistically significant differences between weeks for the same temperature and between treatments on the same week, respectively (One-way ANOVA followed by the Tukey test; P≤0.05; GraphPad Prism v.4.00). B) Relation of rearing temperature and the concentration of endogenous cortisol in individual larvae between 1 and 5 weeks after hatching. C) Relation of the mean concentration of endogenous cortisol between 1 and 5 weeks after hatching and the percentage of males.
Figure 2.
Abundance of mRNA transcripts of the sex differentiation genes amh and cyp19a1a in cortisol-treated (0.8 mg/g food) larvae at 4 and 6 wah.
Typical values obtained at masculinizing (MPT; 7 wah) and feminizing (FPT; 6 wah) temperatures are shown for comparison. Symbols represent individual values for 10 larvae each in control and cortisol-treated groups and 5 larvae each in the MPT and FPT. Values were normalized by the respective values of β-actin. Horizontal dotted lines represent threshold values (mean±2SD) for cyp19a1a (0.00258) and amh (0.00247) that were calculated using values from the MPT and FPT, respectively. Cortisol-treated animals showed consistent cyp19a1a downregulation and amh upregulation comparable to the typical molecular signature of masculinization of animals at the MPT whereas controls showed a bimodal distribution of mRNA transcripts that agreed with the sex ratio of 69.2% males (that is, 6–7 and 3–4 out of 10 individuals resembled those at the MPT and FPT, respectively).
Figure 3.
Incidence of gonadal apoptosis (TUNEL assay) in cortisol-treated (0.8 mg/g food) larvae between 4 and 6 wah.
Apoptosis occurrence is defined as the percentage of individuals showing each of the categories of apoptosis indicated in the left side of the top panels (red: abundant, yellow: few, grey: none; the right side shows DAPI nuclear staining for visualization of cells). Scale bars represent 10 µm; numbers within parenthesis indicate sample number. Cortisol-treated larvae showed markedly higher incidence of gonadal apoptosis compared to control larvae at 6 wah.
Figure 4.
Percentage of males in groups treated with cortisol and the synthetic glucocorticoid receptor (GR) agonist Dexamethasone.
All groups were reared at an intermediate temperature (24°C) during the trials. Groups treated with cortisol and Dexamethasone had increased proportions of males compared to the respective controls. Numbers within parenthesis indicate the sample number; asterisks indicate statistically significant differences between groups in the same trial (χ2; P≤0.05; GraphPad Prism v.4.00).
Figure 5.
Concentrations of 11-ketotestosterone (11-KT) and testosterone (T) in cortisol-treated larvae reared at MixPT (24°C) and in untreated larvae reared at MPT (29°C), MixPT (24°C) and FPT (17°C) between 0 and 6 wah.
Different letters inside and above the bars indicate statistically significant differences between weeks for the same temperature and between treatments on the same week, respectively (One-way ANOVA followed by the Tukey test; P≤0.05; GraphPad Prism v.4.00). Larvae fed cortisol showed higher titres of 11-KT than the corresponding control (MixPT) between 2 and 4 wah. Both 11-KT and T were significantly higher in the MPT compared to the FPT on the same weeks.