COLEMAN, A.T.*; LEWIS, J.; DIZON, M.; COWAN, J.; WIBBELS, T.; Univ. of Alabama, Birmingham; Univ. of Alabama, Birmingham; Univ. of Alabama, Birmingham; Univ. of Alabama, Birmingham; Univ. of Alabama, Birmingham: Real-time PCR assay for quantifying mullerian-inhibiting substance (anti-mullerian hormone) in a turtle with TSD.

The physiology of temperature-dependant sex determination (TSD) has been the subject of many previous studies, but the temperature-sensitive element(s) in the sex determination cascade has not yet been identified. However, a variety of genes that were identified in mammals and birds appear to play important roles in the sexual differentiation of reptiles with TSD. Mullerian-inhibiting substance (MIS, also called anti-mullerian hormone or AMH) has been identified in reptiles, as well as other vertebrates. In mammals and birds, MIS is produced by the testis and stimulates the regression of the mullerian ducts in males. MIS is of particular interest in sex determination studies, because it is an early marker for testicular differentiation. In the current study, a real-time PCR assay for MIS was developed and evaluated for quantifying MIS from the red eared slider turtle, Trachemys scripta. A series of primers were utilized to amplify three different fragments from the 3� region of the open reading frame of T. scripta MIS cDNA. The three fragments (ranging in size from 162 to 825 bp) were used to generate and compare standard curves using a SYBR green PCR assay system. Dilution curves were generated from standards ranging from approximately 800 ng to 80 fg and compared. The 162 bp fragment proved to be optimal. Additionally, a fragment of beta-actin was cloned from T. scripta to use as a control for the real-time PCR assay. The development of a real-time PCR assay for quantifying MIS provides a new tool for further elucidating the physiology underlying sexual differentiation in a turtle with TSD.