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. 2014 Jul 17;9(7):e98531.
doi: 10.1371/journal.pone.0098531. eCollection 2014.

Intersex Occurrence in Rainbow Trout (Oncorhynchus Mykiss) Male Fry Chronically Exposed to Ethynylestradiol

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Free PMC article

Intersex Occurrence in Rainbow Trout (Oncorhynchus Mykiss) Male Fry Chronically Exposed to Ethynylestradiol

Sophie Depiereux et al. PLoS One. .
Free PMC article

Abstract

This study aimed to investigate the male-to-female morphological and physiological transdifferentiation process in rainbow trout (Oncorhynchus mykiss) exposed to exogenous estrogens. The first objective was to elucidate whether trout develop intersex gonads under exposure to low levels of estrogen. To this end, the gonads of an all-male population of fry exposed chronically (from 60 to 136 days post fertilization--dpf) to several doses (from environmentally relevant 0.01 µg/L to supra-environmental levels: 0.1, 1 and 10 µg/L) of the potent synthetic estrogen ethynylestradiol (EE2) were examined histologically. The morphological evaluations were underpinned by the analysis of gonad steroid (testosterone, estradiol and 11-ketotestosterone) levels and of brain and gonad gene expression, including estrogen-responsive genes and genes involved in sex differentiation in (gonads: cyp19a1a, ER isoforms, vtg, dmrt1, sox9a2; sdY; cyp11b; brain: cyp19a1b, ER isoforms). Intersex gonads were observed from the first concentration used (0.01 µg EE2/L) and sexual inversion could be detected from 0.1 µg EE2/L. This was accompanied by a linear decrease in 11-KT levels, whereas no effect on E2 and T levels was observed. Q-PCR results from the gonads showed downregulation of testicular markers (dmrt1, sox9a2; sdY; cyp11b) with increasing EE2 exposure concentrations, and upregulation of the female vtg gene. No evidence was found for a direct involvement of aromatase in the sex conversion process. The results from this study provide evidence that gonads of male trout respond to estrogen exposure by intersex formation and, with increasing concentration, by morphological and physiological conversion to phenotypic ovaries. However, supra-environmental estrogen concentrations are needed to induce these changes.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Testicular morphology.
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
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
Figure 3. Intersex morphology.
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
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
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
Figure 6. Vitellogenic follicles.
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
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
Figure 8. 11-ketosterone levels.
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
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
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.

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References

    1. Brennan J, Capel B (2004) One tissue, two fates: molecular genetic events that underlie testis versus ovary development. Nat Rev Genet 5: 509–521 Available: http://www.ncbi.nlm.nih.gov/pubmed/15211353 Accessed 12 April 2012 - PubMed
    1. Devlin RH, Nagahama Y (2002) Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences. Aquaculture 208: 191–364.
    1. Melamed P, Sherwood N, editors (2005) Hormones and their receptors in fish reproduction. Molecular aspects of fish and marine biology. Singapore.
    1. Pandian TJ, Sheela SG (1995) Hormonal induction of sex reversal in fish. Aquaculture 138: 1–22 Available: http://linkinghub.elsevier.com/retrieve/pii/0044848695010750
    1. Piferrer F (2001) Endocrine sex control strategies for the feminization of teleost fish. Aquaculture 197: 229–281 Available: http://linkinghub.elsevier.com/retrieve/pii/S0044848601005890

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Grant support

Fonds de la Recherche Scientifique – FNRS, Belgium, grant number: FC 83738 (http://www.frs-fnrs.be/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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