FLETCHER, J.; WIBBELS, T.*; University of Alabama at Birmingham; University of Alabama at Birmingham: Development of a quantitative-competitive PCR for anti-mullerian hormone in the red-eared slider turtle.

The physiology underlying temperature-dependant sex determination (TSD) and sexual differentiation in reptiles is not well understood. However, several genes that were initially identified in other vertebrates may play important roles in the sexual differentiation of reptiles with TSD. Anti-m�llerian hormone (AMH, also called m�llerian inhibiting substance or MIS) has been identified in reptiles, as well as other vertebrates. In mammals and bird, AMH is produced by the testis and stimulates the regression of the mullerian ducts in males. In the current study, a 900 bp fragment of AMH from the red-eared slider turtle, Trachemys scripta, was cloned and then used to develop a quantitative-competitive PCR (QC-PCR). A PCR-based strategy was used to generate an AMH competitor molecule that had a 152 bp deletion. The deletion allowed the competitor to be distinguished from the original 900 bp fragment by agarose gel electrophoresis. The competitor could be amplified with the same primers used to amplify the original 900 bp fragment of AMH. The competitor and the original 900 bp fragment were shown to amplify at approximately the same efficiency. The competitor will be used as an internal standard for a QC-PCR for examining the expression of AMH during sex determination and sexual differentiation in T. scripta. This information will further elucidate the physiology underlying sexual differentiation in reptiles with TSD.