DODD, K.L.*; MURDOCK, C.A.; WIBBELS, T.; University of Alabama at Birmingham; University of Alabama at Birmingham; University of Alabama at Birmingham: Evaluation of aromatase mRNA levels in brain tissue during temperature-dependent sex determination in a turtle.

Some reptiles exhibit temperature-dependent sex determination (TSD), in which the incubation temperature of the egg determines the sex of the developing embryo. The physiology of TSD is not well understood, however, estrogen has been hypothesized to play a key role. One hypothesis suggests that that female incubation temperatures result in the increased production of mRNA for the enzyme cytochrome P450 aromatase that catalyzes the conversion of androgens to estrogens. Further, it has been suggested that the brain may be the source of aromatase activity prior to or during sex determination. In the current study, a quantitative competitive RT-PCR was validated for quantifying the aromatase mRNA levels in the brain tissue of Trachemys scripta, a turtle with TSD. Eggs were incubated at male and female temperatures and brain tissues were harvested at stages 13, 15, 17, 19, and 23. Total RNA was isolated from the tissues. A quantitative-competitive RT-PCR was preformed using a RNA competitor as an internal standard in each reaction tube. The competitor molecule was a fragment of the T. scripta aromatase mRNA that contained a deletion that allowed it to be differentiated from endogenous aromatase mRNA. A standard curve was run with each sample using five known dilutions of the competitor in order to quantify the amount of aromatase mRNA. The results indicate that aromatase mRNA is present during the thermosensitive period of sex determination in brain tissue. Further, the results provide a profile of aromatase mRNA levels in brain tissue at both male and female incubation temperatures during TSD.