Cytochrome P450 aromatase (CYP19) and sex differentiation in the Nile tilapia Oreochromis niloticus
Sex steroids are generally considered as natural sex inducers in fish. Aromatase (cytochrome P450 aromatase) catalyses androgens into oestrogens in the steroidogenic pathway. Three different approaches were taken to elucidate the action of aromatase in relation to sex differentiation in the Nile tilapia Oreochromis niloticus. The first was treatment with Fadrozole TM,a non-steroidal aromatase inhibitor (Al), by incorporating it in the diet or by immersing fish in a solution containing Al during the sex differentiation period. The Al treatment masculinised genetic females, indicating the importance of aromatase in sex differentiation. The result revealed that the most sensitive time to Al lies between 11-18 dpf (days post fertilisation). A partial brain type aromatase cDNA (1707bp) was identified from a brain cDNA library of O. niloticus. The amino acid sequence (that corresponds to exon 2-9) derived from this showed 63.7% identity to a previously reported ovarian aromatase gene of this species, and 96.7% identity to the brain type aromatase gene of a closely related species O. mossambicus. A semi-quantitative RT-PCR method was established to investigate expression of brain and ovarian aromatase genes during ontogeny. No sexually dimorphic expression of brain aromatase mRNA was detected. However, expression of ovarian aromatase was down-regulated from 15 to 23 dpf in genetic males but upregulated in genetic females. This period overlaps closely with the most sensitive period to Al. The pattern of temperature-dependent sex determination (TSD) was examined using three different genotypes (XX, XY, YY) at two temperatures (28 and 36°C). The results showed a bidirectional pattern of TSD. YY groups showed a significant percentage of feminisation at the higher temperature, which was suppressed by the Al treatment, implying that aromatisation is mechanistically associated with TSD in this species.All of these data consistently suggest that aromatase plays a crucial role in sex differentiation, and that the decisive aromatisation takes place between 13-25 dpf in this species. Considering the timing (26-30 dpf) of the first appearance of steroid producing cells in the gonadal area, the decisive aromatisation is not likely to take place there. The brain could be the primary aromatisation site in fish sex differentiation.