In mammalian and avian vertebrate groups, androgens act as controlling agents on male aggression and courtship behaviour by their conversion to oestrogens by cytochrome P450 aromatase in well-defined brain regions. Despite the fact that bony fishes have exceptionally high brain aromatase activity, little is known about it's possible regulatory effects on the reproductive behaviours of teleosts. In this study, Endler guppy males (Poecilia reticulata) were subjected to 26-29 days of 24-h exposure to two different concentrations (15 and 100 pg/L) of the aromatase inhibitor fadrozole in the water. Compared with the control males, two of three courtship activities in males exposed to the higher concentration were reduced when they were paired with receptive stimulus females. Reduction in brain aromatase activity was confirmed in both exposed groups with the use of the tritiated water assay.

Understanding the neuroendocrinological mechanisms behind reproductive behaviour is fundamental when studying endocrine disruption. Neuroestrogen production is a key step in the activation of reproductive behaviours among vertebrates. The actions of estrogens are transmitted through estrogen receptors (ERs) in distinct brain nuclei. Here we report alterations in reproductive behaviours in guppy males following 55-day food treatments with the antiestrogen fulvestrant (ICI) and the synthetic oestrogen 17α-ethinylestradiol (EE2). Male courtship and mating attempts were severely reduced in EE2 treated males after both 27-30 and 41-55 days of treatment when paired with females. ICI treatment gave a significant reduction in the frequency of sigmoid display behaviour after 27-30 days of treatment, and an almost 2.5-fold increase in gonopodium thrusting after 41-55 days of treatment. ICI treated males also decreased their frequency in successful mating attempts in comparison to the control males. The neurological effects of ICI were confirmed by Real Time-PCR analysis for brain aromatase and ERα gene expression. ICI treatment suppressed aromatase expression to 64% and stimulated ERα gene expression by over 300%. These results indicate that oestrogen action via ERs may play an important role for the complete display of male courtship and mating behaviour in the guppy. The results also suggest that local steroids are involved in regulating brain aromatase expression and that the negative effects of EE2 on sexual behaviour are linked via endocrine disruption of gonadal function.

Brain estrogen production, performed by the enzyme aromatase, can be disrupted/affected in teleost fish exposed to endocrine disruptors found in polluted aquatic environments. The guppy (Poecilia reticulata) was previously studied and confirmed to suffer negative effects on reproductive behaviors following inhibition of the brain aromatase reaction. Here adult guppies (Poecilia reticulata) of both genders were subjected to known endocrine disruptors: the androgen androstenedione (A), the synthetic estrogen 17 alpha-ethinylestradiol (EE2), and the estrogenic surfactant 4-nonylphenol (NP), at high (50 mu g/L) and at environmentally relevant concentrations (10 ng/L EE2, 5 mu g/L NP, and 0.7 mu g/L A) for 2 weeks followed by measurements of brain aromatase activity (bAA). In the adult males, bAA was stimulated by A and EE2 at 50 mu g/L. Female activity was also stimulated by the higher estrogenic treatment. At environmentally relevant concentrations only the EE2 treatment affected bAA, and only in males. The alkylphenolic substance NP produced no effect in either of the experiments, not on males nor females. The results indicate that short-term steroid treatments have stimulatory effects on guppy brain aromatase even at concentrations that can be found in the environment. We thus suggest bAA of adult guppies to be a suitable bioindicator of endocrine disruptors.

Oestrogens are biosynthesised by cytochrome p450-aromatase (Cyp19). Brain oestrogens serve several important functions of which nerve protection, cell proliferation, nerve development and behaviour control are a few. Teleost brain aromatase activity is exceptionally high (between 100-1000 times) compared to mammals and birds. We have successfully cloned and sequenced a 950 bp long partial fragment of the guppy CYP19B gene (PrCyp19b) derived from adult brain mRNA. Sequence alignment of translated amino acid sequence shows PrCYP19b having high sequence similarity to teleost brain aromatase. Anatomical distribution of PrCYP19b expression in adult guppy brains was studied using in situ-hybridisation with an antisense riboprobe synthesised from the cloned PrCYP19b gene. Expression of brain aromatase appeared at ventricular surfaces of the ventral telencephalic zones and the pre-optic area, in the hypothalamus, the pituitary gland, optic tectum and the cerebellum. This pattern is similar to previously reported distribution of brain aromatase in other teleosts. Measurement of brain aromatase activity in telencephalon, mesencephalon/diencephalon and rhombencephalon revealed female guppies producing the most oestrogens in mesencephalon/diencephalon, whereas males produces the most oestrogens in both telencephalon and mesencephalon/diencephalon. This indicates that brain oestrogen production is sexually dimorphic in the guppy and may serve different functions in the two genders.