Abstract
A genotypic sex determination assay provides accurate gender information of individuals with well-developed phenotypic characters as well as those with poorly developed or absent of phenotypic characters. Determination of genetic sex for Xenopus laevis can be used to validate the outcomes of Tier 2 amphibian assays, and is a requirement for conducting the larval amphibian growth and development assay (LAGDA), in the endocrine disruptor screening program (EDSP), test guidelines. The assay we developed uses a dual-labeled TaqMan probe-based real-time polymerase chain reaction (real-time PCR) method to determine the genotypic sex. The reliability of the assay was tested on 37 adult specimens of X. laevis collected from in-house cultures in Eurofins EAG Agroscience, Easton. The newly designed X. laevis-specific primer pair and probe targets the DM domain gene linked-chromosome W as a master female-determining gene. Accuracy of the molecular method was assessed by comparing with phenotypic sex, determined by necropsy and histological examination of gonads for all examined specimens. Genotypic sex assignments were strongly concordant with observed phenotypic sex, confirming that the 19 specimens were male and 18 were female. The results indicate that the TaqMan® assay could be practically used to determine the genetic sex of animals with poorly developed or no phenotypic sex characteristics with 100% precision. Therefore, the TaqMan® assay is confirmed as an efficient and feasible method, providing a diagnostic molecular sex determination approach to be used in the amphibian endocrine disrupting screening programs conducted by regulatory industries. The strength of an EDSP is dependent on a reliable method to determine genetic sex in order to identify reversals of phenotypic sex in animals exposed to endocrine active compounds.