Alligator aromatase cDNA sequence and its expression in embryos at male and female incubation temperatures

In all species of crocodilians, sex is determined not by genetic mechanisms , but by the temperature at which the egg is incubated. In the American all igator (Alligator mississippiensis) the thermosensitive period (TSP) for se x determination is a 7- to 10-day window within stages 21-24 of development , around the middle third of the incubation period. Treating embryos with e strogen during the TSP produces female offspring, even at male incubation t emperatures. Conversely, blocking embryonic estrogen synthesis at female-in ducing temperature prevents development of the female phenotype. Therefore, it has been suggested that estrogen plays a role in determination of sex i n the alligator. Estrogen is produced from an androgen substrate by cytochr ome P450 aromatase (CYP19). If estrogen plays a critical role in sex determ ination, there should be differences in aromatase expression between embryo s at male- and female-producing temperatures during the TSP. Therefore, to address this question, we cloned and characterized the alligator CYP19 cDNA . Based on the sequence information, a quantitative kinetic reverse transcr iptase-polymerase chain reaction (TaqMan) assay was designed to measure exp ression of the alligator aromatase gene in RNA extracted from the gonadal a nd brain regions of alligator embryos incubated at male- or female-producin g temperatures from prior to the TSP through hatching. Aromatase expression was detected in the brain region from the earliest stage tested (stage 20) through hatching. The hypothalamus had significantly higher expression tha n the forebrain or hindbrain in both male and female embryos. Expression wa s not significantly different in the gonadal region between embryos at male and female temperatures until after the TSP, when there was a dramatic inc rease in expression at female temperature. These data indicate that aromata se expression and, thus, estrogen production, are not the initial trigger f or sex determination but play an essential role in ovarian differentiation in the alligator.