Sexual differentiation in zebrafish occurs after hatching and is a labile process. During early life, gonads are undifferentiated and become either testes or ovaries during sexual differentiation. The balance between circulating levels of male (androgen) and female (oestrogen) hormones is crucial to the process of sexual differentiation, determining whether a fish becomes male or female. Therefore, zebrafish are particularly susceptible to factors in the environment that may modify or interfere with this process.
Endocrine-disrupting chemicals are natural or man-made compounds that can mimic endogenous hormones and cause physiological disturbances. Current research has focused mainly on chemicals that mimic the effect of natural oestrogens, but endocrine disruptors can also interfere with the synthesis of endogenous hormones. So, can endocrine disruptors influence the sexual differentiation of zebrafish?
The enzyme aromatase converts androgens to oestrogens, playing a vital role in maintaining a balance between male and female hormones. Two forms of the aromatase-encoding gene have previously been identified and are mainly expressed in the gonad (CYP19A) and brain (CYP19B). The important nature of this enzyme made it an appealing focus for this study. Exposing fish to aromatase-inhibiting chemicals, Martina Fenske and Helmut Segner measured both the formation of gonads and the expression of the aromatase genes to determine the effects of these chemicals on fish sexual differentiation. The team chose to work with a compound called fadrozole, as it is a competitive inhibitor of aromatase activity. Taking two sets of fish,the team kept one lot in water to monitor normal development, while they exposed the other zebrafish to fadrozole during the period of sexual differentiation. After fadrozole exposure, they sampled some fish directly,while others were returned to control water and raised to adulthood to see if exposure-related effects were reversible.
In control fish, 44% developed testes and 56% ovaries, and aromatase gene expression varied depending on the developmental stage. During sexual differentiation there was no detectable difference in aromatase gene expression between males and females, but in reproductively active adult zebrafish CYP19A expression was higher in females than in males.
Amazingly, complete gonadal `masculinisation' occurred in all the fish exposed to fadrozole, and the effects persisted through to adulthood. Fadrozole also reduced gonadal CYP19A expression during sexual differentiation. Interestingly, while these fish possessed male gonads, 36% of fadrozole-treated adult fish showed female-like expression of CYP19A,whereas 64% had male-like expression. So while 100% of fish would be identified as males based on their gonad morphology, sexing of fish based upon expression of CYP19A might suggest that some of the fish were female.
This study highlights that zebrafish sexual differentiation is susceptible to interference by chemicals. Manipulation of the aromatase system completely and irreversibly altered the formation of gonads. Despite this, gene expression still displayed a dimorphic expression in adult fish, suggesting a partial recovery from fadrozole treatment. However, the genetic gender of these fish could not be identified due to the lack of sex-linked markers for zebrafish. The likelihood of masculinisation of female organisms by aromatase inhibitors is likely to depend on species, developmental stage and exposure concentrations. Nevertheless, it appears that endocrine-disrupting chemicals provide potential for the environment to decide: male or female?
