Induction of Female-to-Male Sex Change in Adult Zebrafish by Aromatase Inhibitor Treatment

In fish, the sex reversal of reproductive organs can be induced by treatment with sex steroids during sexual differentiation in juveniles. This experimentally induced sex change was first described in medaka1, and artificial sex change has been intensively studied since2. Although these experiments demonstrated that gonadal cells and somatic cells in the gonads possess cell type plasticity, it was not possible to induce sex change using sex steroids following sex differentiation. Thus, it was thought that sexual plasticity is lost after sex differentiation. Female-to-male sex change is associated with a decrease in estrogen levels, followed by an increase in androgen levels3. Estrogens are produced by the conversion of aromatizable androgens by cytochrome P450 aromatase (P450arom), and the actions of P450arom are essential for sex differentiation and ovarian development in fish and other vertebrates4,5. Recently, it has become possible to reduce estrogen synthesis by inhibiting this aromatase activity using non-steroidal aromatase inhibitors (AIs), such as fadrozole hydrochloride, which is a reversible competitive inhibitor6. Further studies showed that sex change could be induced in many types of fish by aromatase inhibitor (AI) treatment during sex differentiation. In Japanese flounder (Paralichthys olivaceus) and tilapia (Oreochromis niloticus), brief treatment with AI during sex differentiation causes a sex reversal in which genetic females develop into phenotypically normal males7,8. Undifferentiated ovary-like gonads are initially developed during gonadal development in juvenile zebrafish, regardless of genotypic sex9. In genotypic male zebrafish, all oocytes disappear from the gonad by 30 days post-hatching, and spermatocytes develop concomitant with testicular differentiation10. In contrast, oocytes in the female ovaries continue to grow to maturation. The phenomenon of the presence of undifferentiated ovary-like gonads during the juvenile period is known as juvenile hermaphroditism. Recently, it has been reported that the massive, male-specific disappearance of oocytes from the gonad during the transition from ovary-like tissue to testis tissue is caused by apoptosis11. Therefore, it was suggested that oocyte apoptosis plays a significant role in the testicular differentiation of juvenile zebrafish. In zebrafish, the gonadal masculinization of genetic juvenile females can be induced by the dietary administration of an AI (fadrozole)12. Recently, two independent studies provided evidence for a population of undifferentiated stem cells in the ovary of adult zebrafish that can produce both types of gametes13,14. In this study, we examined whether AI (fadrozole) treatment induces a sex change in adult zebrafish. Our results support the hypothesis that sexual plasticity persists in adult zebrafish following sex differentiation, indicating that undifferentiated stem cells are maintained in adult fish that do not undergo sex change under natural conditions.