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In fish, according to Yamamoto's model, androgens would drive testis differentiation and estrogens ovarian differentiation. In order to study the implication of steroid enzymes in rainbow trout gonadal differentiation, we examined the expression of some steroid enzyme genes during natural differentiation (cholesterol side chain cleavage = P450scc, 17-hydroxylase/lyase = P450c17, 3beta-hydroxysteroid dehydrogenase = 3betaHSD) and androgen-induced differentiation (P450scc, P450c17, 3betaHSD, aromatase = P450aro, and 11beta-hydroxylase = P45011beta). Expressions of P450scc, 3betaHSD, and P450c17 were all detected in male and female gonads at 55 days post-fertilization (dpf), i.e., two weeks before histological differentiation. There were no differences in their expression level respective to the sex. The androgen treatment was carried out by administration of 11beta-hydroxyandrostenedione (11betaOHDelta4) in genetic all-female populations and the resulting sex ratios were found to be 100% male even at a low dosage of 1 mg/kg of food. Following 11betaOHDelta4 treatment, only the expression of P450c17 was found to be sustained when compared with the female untreated control. In contrast, P450scc was clearly up-regulated and 3betaHSD and P450aro down-regulated by the androgen treatment. P45011beta gene expression remained low in gonads of androgen-treated females, as it did in control untreated females. These results together demonstrate that steroidogenesis in rainbow trout is potentially active in pre-differentiating gonads of both sexes, and that one of the masculinizing actions of androgens in the species may be to down-regulate the female-specific gonadal P450aro gene expression. However, in vivo androgen treatment in genetic females does not induce the same pattern of steroid gene expression as in genetic males. These data suggest that exogenous androgens might induce a male differentiation process with P450aro inhibition being one of the steps required. However, this process would not involve endogenously produced 11-oxygenated androgens.

In order to study the physiological implication of sex steroid hormones in gonadal sex differentiation in fish, we first investigated the potential role of estrogens using two fish models: the rainbow trout (Oncorhynchus mykiss) and a tilapia species (Oreochromis niloticus). All experiments were carried out on genetically all-male (XY) and all-female (XX) populations. In vivo treatments with an aromatase inhibitor (ATD, 1,4,6- androstatriene-3-17-dione) result in 100% masculinization of an all-female population in rainbow trout (dosage 50 mg/kg of food) and 75.3% in tilapia (dosage 150 mg/kg of food). In tilapia, the effectiveness of the aromatase inhibition by ATD is demonstrated by the marked decrease of the gonadal aromatase activity in treated animals versus control. No masculinization is obtained following treatment with an estrogen receptor antagonist (tamoxifen) in both species. Aromatase and estrogen receptor gene expression was studied in rainbow trout by semi-quantitative RT-PCR in gonads sampled before, during and after sex-differentiation. Aromatase mRNA is specifically detected in female gonads, 3 weeks before the first sign of histological sex-differentiation, i.e., first female meiosis. Aromatase expression in male gonads is at least a few hundred times less than in female gonads. Estrogen receptor gene is expressed in both male and female gonads at all stages with no dimorphic expression between sexes. Specific aromatase gene expression before ovarian differentiation was also demonstrated using virtual Northern blot, with no expression detected in male differentiating gonads. From these results it can be concluded that estrogen synthesis is crucial for ovarian differentiation, and transcription of the aromatase gene can be proposed as a key step in that process in fish.