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In mammals, follistatin (FST) plays an important role in early ovarian differentiation, acting downstream of the Wnt pathway. In teleost fish, fst is implicated in folliculogenesis and oocyte maturation, and an early and specific expression during ovarian differentiation has been described in rainbow trout, Oncorhynchus mykiss. By in situ hybridization, we demonstrated that during rainbow trout gonadal differentiation, fst shares a similar expression pattern with cyp19a1a, the gene encoding ovarian aromatase, a key steroidogenic enzyme needed for ovarian differentiation in fish. Expression of fst and cyp19a1a was first detected in a few scattered cells in the embryonic ovary several days before hatching. Then, after histological differentiation, fst and cyp19a1a expression was localized in clusters of cells lining the future ovarian lamellae. As FST expression is known to be induced by the Wnt/β-catenin pathway in mammals, the Wnt pathway was inhibited in vivo with the IWR-1 molecule, and we analyzed by qPCR the effects of this treatment on fst expression. We found that IWR-1 decreased fst expression in female gonads, consistent with a regulation of fst expression by the Wnt pathway in rainbow trout. Furthermore, expression of cyp19a1a was also downregulated, suggesting an implication of the Wnt pathway in ovarian differentiation., (Copyright © 2013 S. Karger AG, Basel.)

The present review focuses on the roles of estrogens and aromatase (Cyp19a1a), the enzyme needed for their synthesis, in fish gonadal sex differentiation. Based on the recent literature, we extend the already well accepted hypothesis of an implication of estrogens and Cyp19a1a in ovarian differentiation to a broader hypothesis that would place estrogens and Cyp19a1a in a pivotal position to control not only ovarian, but also testicular differentiation, in both gonochoristic and hermaphrodite fish species. This working hypothesis states that cyp19a1a up-regulation is needed not only for triggering but also for maintaining ovarian differentiation and that cyp19a1a down-regulation is the only necessary step for inducing a testicular differentiation pathway. When considering arguments for and against, most of the information available for fish supports this hypothesis since either suppression of cyp19a1a gene expression, inhibition of Cyp19a1a enzymatic activity, or blockage of estrogen receptivity are invariably associated with masculinization. This is also consistent with reports on normal gonadal differentiation, and steroid-modulated masculinization with either androgens, aromatase inhibitors or estrogen receptor antagonists, temperature-induced masculinization and protogynous sex change in hermaphrodite species. Concerning the regulation of fish cyp19a1a during gonadal differentiation, the transcription factor foxl2 has been characterized as an ovarian specific upstream regulator of a cyp19a1a promoter that would co-activate cyp19a1a expression, along with some additional partners such as nr5a1 (sf1) or cAMP. In contrast, upstream factors potentially down-regulating cyp19a1a during testicular differentiation are still hypothetical, such as the dmrt1 gene, but their definitive characterization as testicular repressors of cyp19a1a would strongly strengthen the hypothesis that early testicular differentiation would need active repression of cyp19a1a expression., (Copyright 2009 Elsevier Inc. All rights reserved.)

A cDNA encoding for a novel rainbow trout SHBG was identified and characterized. Phylogenetic analysis showed that this novel SHBG, named SHBGb, was a highly divergent paralog of the classical SHBG (SHBGa) form previously known in vertebrates including zebrafish, seabass, and rainbow trout. Using all available sequences, no SHBGb-like sequence could be identified in any fish species besides Atlantic salmon. Rainbow trout SHBGa and SHBGb share only 26% sequence identity at the amino acid level and exhibit totally distinct tissue distribution, thus demonstrating a functional shift of SHBGb. Indeed, shbga mRNA was predominantly expressed in liver and spleen but could not be detected in the ovary, whereas shbgb had a predominant ovarian expression but could not be detected in liver. Despite its high divergence, rainbow trout SHBGb expressed in COS-7 cells could bind estradiol and testosterone with high affinity and specificity. Both rainbow trout shbgb mRNA and proteins were localized to the granulosa cells of vitellogenic ovarian follicles, whereas SHBGb immunoreactivity was also found in theca cells. Finally, shbgb ovarian mRNA expression exhibited a significant drop between late vitellogenesis and oocyte maturation at a time when ovarian aromatase (cyp19a) gene expression and estradiol circulating levels exhibited a dramatic decrease. Together, these observations show that SHBGb is a functional and highly divergent SHBG paralog probably arising from a salmonid-specific duplication of the shbg gene.

Fish gonadal phenotype is very sensitive to sex steroid and functional masculinizations can be obtained in most species using androgen treatments. To gain insight into the molecular effects of androgen-induced masculinization we characterized, in the rainbow trout, the gonadal expression profiles of 103 candidate genes involved in sex differentiation and early gametogenesis. The androgen treatment (11beta-hydroxyandrostenedione, 10 mg/kg of food for 3 months) was administered in a genetic all-female population. Gonads were sampled at different time points in genetic all-male and all-female control populations and in the androgen-treated group. Gene expression profiles were recorded by real-time RT-PCR and biological samples and gene expressions were compared using a global clustering analysis. This analysis revealed that masculinization with androgens acts firstly by repressing granulosa cell related genes, including genes involved in ovarian differentiation (foxl2a, fst, cyp19a1a), and subsequently by repressing genes important for early oogenesis (gdf9, bcl2lb, fancl, gcl, fshb, lhb, sox23, sox24, nup62 and vtgr). However, this masculinizing treatment did not induce a testicular differentiation similar to what was observed in the control male population. This was especially noticeable for many Leydig cell genes encoding proteins involved in steroidogenesis or its control (hsd3b1, star, cyp17a1, cyp11b2.1 and nr5a1b) that were down-regulated in the androgen-treated group. Concomitantly some Sertoli cells marker genes were up-regulated by the androgen treatment (sox9a.1, nr0b1, cldn11, dmrt1) whereas others were down-regulated (amh, sox9a.2), suggesting a partial differentiation of the Sertoli cell lineage. Overall, this suggests that the crucial step of this masculinization process is the de-differentiation of the granulosa cells.

Background: Fish gonadal sex differentiation is affected by sex steroids treatments providing an efficient strategy to control the sexual phenotype of fish for aquaculture purposes. However, the biological effects of such treatments are poorly understood. The aim of this study was to identify the main effects of an androgen masculinizing treatment (11beta-hydroxyandrostenedione, 11betaOHDelta4, 10 mg/kg of food for 3 months) on gonadal gene expression profiles of an all-female genetic population of trout. To characterize the most important molecular features of this process, we used a large scale gene expression profiling approach using rainbow trout DNA microarrays combined with a detailed gene ontology (GO) analysis., Results: 2,474 genes were characterized as up-regulated or down-regulated in trout female gonads masculinized by androgen in comparison with control male or female gonads from untreated all-male and all-female genetic populations. These genes were classified in 13 k-means clusters of temporally correlated expression profiles. Gene ontology (GO) data mining revealed that androgen treatment triggers a marked down-regulation of genes potentially involved in early oogenesis processes (GO 'mitotic cell cycle', 'nucleolus'), an up-regulation of the translation machinery (GO 'ribosome') along with a down-regulation of proteolysis (GO 'proteolysis', 'peptidase' and 'metallopeptidase activity'). Genes considered as muscle fibres markers (GO 'muscle contraction') and genes annotated as structural constituents of the extracellular matrix (GO 'extracellular matrix') or related to meiosis (GO 'chromosome' and 'meiosis') were found significantly enriched in the two clusters of genes specifically up-regulated in androgen-treated female gonads. GO annotations 'Sex differentiation' and 'steroid biosynthesis' were enriched in a cluster of genes with high expression levels only in control males. Interestingly none of these genes were stimulated by the masculinizing androgen treatment., Conclusion: This study provides evidence that androgen masculinization results in a marked dysregulation of early gene expression profiles when compared to natural testicular or ovarian differentiation. Based on these results we suggest that, in our experimental conditions, androgen masculinization proceeds mainly through an early inhibition of female development.