Effects of bisphenol A and fadrozole exposures on cyp19a1 expression in the Murray rainbowfish, Melanotaenia fluviatilis.

Several endocrine-disrupting chemicals (EDCs) have been attributed to the alteration of reproduction in fish through disrupting endogenous sex steroidogenic pathways including aromatisation of androgens to oestrogen by CYP19 aromatase. Here we investigate this hypothesis in adult male and female Melanotaenia fluviatilis by examining the mRNA expression of cyp19a1 isoforms after exposure for ≤96 h to two EDCs with contrasting modes of action: one a weak oestrogen mimic, bisphenol A [BPA (100 or 500 µg/L)], and the other a nonsteroidal aromatase inhibitor, fadrozole [FAD (10 or 50 µg/L)]. The results suggest that BPA did not affect cyp19a1a expression significantly at both concentrations, whereas 50 µg/L of FAD significantly upregulated its expression in ovary. In contrast, BPA exposures increased expression of cyp19a1b in brain of both males and females, whilst FAD had contrasting effects in brain: It increased in males but decreased in females. Similar contrasting responses of cyp19a1b were induced by BPA in gonads: upregulation in ovary and downregulation in testis. FAD did not have a significant effect on gonadal expression of cyp19a1b. Collectively, the results suggest that BPA and FAD can disrupt cyp19a1b activity more readily than can cyp19a1a, albeit with contrasting effects in either a tissue- or sex-specific context that is conceivably consistent with their (BPA and FAD) opposing modes of action. Enhanced spatial and temporal sensitivity of cyp19a1b compared with cyp19a1a suggests that brain sex of fish is more susceptible to disruption by environmental pollutants such as BPA and FAD. Therefore, we propose that the response of cyp19a1b in brain tissue of M. fluviatilis is a more suitable indicator of oestrogenic pollution in the aquatic environment.

Exposure to estrogenic chemicals induces ectopic expression of vtg in the testis of rainbowfish, Melanotaenia fluviatilis.

Vitellogenin (Vtg) is the major egg-yolk precursor protein in oviparous organisms normally synthesised only in mature females. In males and juveniles, the vtg gene, although present, is silent, but its hepatic expression may be activated by xenoestrogens. Surprisingly, its induction and potential consequences in non-hepatic tissues remain unexplored. Here we test the hepatic and testicular response of vtg expression in adult male rainbowfish Melanotaenia fluviatilis exposed to either 1, 3, 5 µg/L 17ß-estradiol or 100, 500 µg/L 4-n-nonylphenol for 24-96 h. Significant increase in the expression level of vtg mRNA in the liver and testes of exposed males was observed. The early (24 h), sensitive and reliable detection of the vtg induction using qPCR demonstrates the assay's robustness to monitor xenobiotic exposure particularly in smaller fish like rainbowfish, an emerging indicator species. Whilst, the ectopic induction of vtg mRNA in testes suggests a more complex Vtg pathway.

Effects of exposure to oestrogenic compounds on aromatase gene expression are gender dependent in the rainbowfish, Melanotaenia fluviatilis.

This study investigated the influence of two endocrine disrupting chemicals (EDCs)-an exogenous oestrogen 17ß-estradiol (E2) and the oestrogen mimic 4-n-nonylphenol (NP) on the expression of aromatase transcripts in both sexes of adult Murray river rainbowfish. Reproductively active mature fish were exposed to 1, 3, and 5 µg/L E2 or 100 and 500µg/L NP for 24, 48, 72 and 96 h. The results show a significant reduction in the expression of cyp19a1a isoform in ovarian tissues with complete inhibition at the higher concentrations (3 and 5 µg/L E2; 500µg/L NP between 24 and 72 h) and at all concentrations after 96 h. There was no expression of the cyp19a1a isoform in female brain, male brain or testes in any treatment. E2 significantly increased expression of cyp19a1b in female brain except at 5 µg/L after 24h exposure. In male brain tissue E2 exposure decreased cyp19a1b expression except at 1 and 5 µg/L at 24h. NP significantly upregulated cyp19a1b in the female brain (except with 500 µg/L at 72 h) and in testes tissues. NP downregulated expression of cyp19a1b in the male brain tissue. Collectively, these observations support the hypothesis that the expression of cyp19a1b is regulated via both positive and negative feedback mechanisms, with differential modulation based on the type and concentration of the exposed oestrogens, duration of exposure, fish tissue and gender of the fish. The results also imply that exogenous oestrogens can have a disruptive effect on the steroidogenic pathway and may lead to effects on sex differentiation, sexual behaviour and reproductive cycles in this fish.

Ontogenic and sexually dimorphic expression of cyp19 isoforms in the rainbowfish, Melanotaenia fluviatilis (Castelnau 1878).

To investigate the role of cytochrome P450 aromatase, we isolated cyp19 isoforms in the Murray River rainbowfish, M. fluviatilis. The cloned cDNA for cyp19a1a and cyp19a1b had an open reading frame (ORF) of 492 and 499 amino acid residues, with shared identity of up to 83% and 87% with the corresponding homologues of other teleosts respectively. In contrast, the cyp19a1a and cyp19a1b of the Murray River rainbowfish had a shared identity of only 61%. Not surprisingly, the phylogenetic analysis clustered the M. fluviatilis cyp19 isoforms with the corresponding isoforms of other teleosts, suggesting a shared evolutionary ancestry of the respective isoforms. We also studied the expression of cyp19 isoforms during ontogeny and in adult fish using quantitative Real-Time PCR (qPCR). Results suggest that uniquely only cyp19a1b transcripts are maternally inherited, suggesting its role in early development and growth in the species. In contrast to reports in many teleosts, the cyp19a1a was exclusively expressed in the ovarian tissue and completely absent in other tissues examined, including testis. The cyp19a1b like in most teleosts was predominantly expressed in the brain of both males and females with low level of expression in other tissues including gonads of both sexes.