Table 1.
Primer sequences used for Q-PCR analysis.
Table 2.
Fish size, weight and mortality.
Figure 1.
A–B: Rainbow trout testis at 136 days post fertilization (dpf), control fish (1360 degree-days). This picture shows the differentiation status of a control testis, with a dominance of gonocytes (Go, future spermatogonia) surrounded by supporting cell (S, future sertoli cell) organized in cords (C, future tubules). HES. Scale bar A = 100 µm, B = 50 µm. C–D: Rainbow trout testis at 136 dpf, fish exposed chronically to 0.01 µg/L of EE2 for 76 days. HES. Go: gonocyte; S: supporting cell. Scale bar C = 100 µm, D = 50 µm. E–F: Normal ovarian morphology. Rainbow trout early stage ovarian morphology, control fish (1350 degree-days). PG: primary growth oocyte; L: lamellae; ST: stroma. Scale bar E = 100 µm, F = 50 µm.
Figure 2.
Altered testicular morphology.
All-male rainbow trout testis at 136 dpf exposed chronically to 1 µg/L EE2. HES. Scale bar = 50 µm. This picture illustrates the degeneration of the testis observed in several fish exposed to EE2, such as a loss of tubular arrangement, loss of germ cell number (Go) and differentiation, presence of lacuna (La). There is no recognizable differentiation of the gonad into male or female phenotype.
Figure 3.
A–B: Ovotestis 1. All-male rainbow trout testis at 136 dpf exposed chronically to 0.01 µg/L and 0.1 µg/L for 76 days. HES. L: lamellae; C: cords of gonocytes (Go); CN: cell nest in different meiosis stages: Le: leptotene; Zy: zygotene; Pa: pachytene. Scale bar = 50 µm. C–D: Ovotestis 2. All-male rainbow trout testis at 136 days post fertilization (dpf) exposed chronically to 0.01 µg/L EE2 for 76 days. HES. Scale bar C = 50 µm, insert in C = 25 µm, D = 50 µm. Figure C shows oocytes appearance in an altered testis structure. The insert in Figure C shows the normal testis structure, with gonocytes (Go) organized in cords (C) observed in other areas of this gonad. Figure D shows oocyte appearance in a normal testis structure. PG: primary growth oocytes; TE: altered testicular tissue; FC: follicular cells. E–F: Ovotestis 3. All-male rainbow trout testis at 136 dpf exposed chronically to 0.01 µg/L for 76 days. HES. Inserts show the altered testis structure at higher magnification. Dotted line represents the enlarged tissue section. OM: ovarian-like morphology; PG: primary growth oocytes; TE: altered testicular tissue; Sg: spermatogonia, La: lacune; Td: tubule disorganization; Va: vacuolation. Scale bar E = 300 µm, F = 100 µm, inserts = 50 µm.
Figure 4.
Ovarian-like morphology (sex-reversed fish).
All-male rainbow trout testis at 136 dpf exposed chronically to 1 µg/L EE2 for 76 days. HES. FC: follicular cell; PG: primary growth oocyte; L: lamellae; ST: stroma. Scale bar = 100 µm.
Figure 5.
Proliferative oviduct-like epithelioid structure.
All-male rainbow trout testis at 136 dpf exposed chronically to 1 and 10 µg/L EE2 for 76 days. HES. Ci: cils; E: columnar epithelial cells; BM: basement membrane; Ep: epithelioid; OM: ovarian-like morphology. Scale bars A = 50 µm; B = 500 µm.
Figure 6.
A–B: Vitellogenic follicles found in gonads of all-male rainbow trout testis at 136 dpf exposed chronically to 0.01 and 10 µg/L EE2 for 76 days. HES. Zp: Zona pellucida; FC: follicular cells (granulosa); TC: thecal cells; CA: cortical alveoli; P: protrusions probably originated from the follicular cells. C–D: Masson’s staining of a vitellogenic follicle. Scale bars 50 µm.
Figure 7.
Relative frequencies of the gonad mophological forms.
This graph represents the percentage of fish belonging to each morphological category, per concentration of EE2 used in the chronic experiment. n refers to the number of fish analyzed per experimental concentration.
Figure 8.
This graph show the linear relationship between [11-ketosterone] and LOG[EE2] in rainbow trout fry gonads submitted chronically to increasing concentrations of EE2. For each group, data represents the mean ± 2 SEM from 6 replicates measured independantly. Each replicate consisted of a pool of 10 pairs of gonads.
Figure 9.
Genes expression profiles in the testis.
Relationship between fold change (expressed as mRNA relative expression ratio with control group) of differentially expressed genes and LOG[EE2] in the testis of rainbow trout fry exposed chronically to increasing concentrations of EE2. For each group, data represents the mean ± 2 SEM from 6 replicates measured independantly. Each replicate consisted of a pool of 5 pairs of gonads. The letters a, b, c summarize the post hoc comparisons (p<0.05), the groups with the same letter being not significantly different. When the lack of fit to linear regression is not significant (p>0.05) the linear regression and associated R2 are shown.
Figure 10.
Genes expression profiles in the brain.
Relationship between fold change (expressed as mRNA relative expression ratio with control group) of differentially expressed genes and LOG[EE2] in brains of juvenile male rainbow trout gonads exposed chronically to increasing concentrations of EE2. For each group, data represents the mean ± 2 SEM from 9 replicates measured independantly. Each replicate consisted of a pool of 3 brains. The letters a, b, c summarize the post hoc comparisons (p<0.05), the groups with the same letter being not significantly different. When the lack of fit to linear regression is not significant (p>0.05) the linear regression and associated R2 are shown.