Development of an in vitro diagnostic method to determine the genotypic sex ofXenopus laevis

Amin Eimanifar,Sean Gallagher,Henry Krueger,Suzanne Z Schneider,John Aufderheide

doi:10.7717/peerj.6886

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.

Highlights

The African clawed frog (Xenopus laevis) belongs to the Pipidae family of frogs, all of which have an aquatic lifestyle in adulthood (Franco et al, 2001)
African clawed frogs (X. laevis) offer a promising model to investigate the effect of potential endocrine disrupting chemicals (EDCs) within the framework of the United States Environmental Protection Agency’s (EPA’s), endocrine disruptor screening program (EDSP), larval amphibian growth and development assay (LAGDA) (United States Environmental Protection Agency (US EPA), 2015)
The plot indicates that the sample No 35 did generate a domain gene linked-W chromosome (DM-W) amplification curve by Rotor-Gene Q Real-time PCR, which occurred above the threshold line set by the software

Summary

Introduction

The African clawed frog (Xenopus laevis) belongs to the Pipidae family of frogs, all of which have an aquatic lifestyle in adulthood (Franco et al, 2001). Development of an in vitro diagnostic method to determine the genotypic sex of Xenopus laevis. African clawed frogs (X. laevis) offer a promising model to investigate the effect of potential endocrine disrupting chemicals (EDCs) within the framework of the United States Environmental Protection Agency’s (EPA’s), endocrine disruptor screening program (EDSP), larval amphibian growth and development assay (LAGDA) (United States Environmental Protection Agency (US EPA), 2015). Xenopus laevis has been used as a model to study sex chromosome evolution (Furman & Evans, 2016). Sex determination in X. laevis follows a ZW gametic system, where females are heterogametic (ZW) and males are homogametic (ZZ). Xenopus laevis is a sexually monomorphic species and the adult females are larger than males (Babošová et al, 2018). In X. laevis, a gene called DM domain gene linked-W chromosome (DM-W) is a master female sex determination gene (Yoshimoto et al, 2008). The DM-W gene appeared in an ancestor of X. laevis after divergence from the ancestor of X. tropicalis, and is present in many close relatives of X. laevis (Bewick, Anderson & Evans, 2011)

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