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

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.

Keywords: Genetic sex determination; OCSPP-LAGDA; TaqMan® real-time PCR; Xenopus laevis.

Figure 1. Linear amplification curves of DM-W gene for all specimens using the TaqMan-based assay.

All amplification curves crosses the solid threshold line denoted female’s genotype and the curves occurred below the threshold line are male genotypes. All curves indicate the amplification of DM-W gene generated by Rotor-Gene Q Real-time PCR with optimum cycling conditions, followed by 30 cycles. The threshold line was set automatically by the software.

Figure 2. Linear amplification curves of 18S rRNA gene for all specimens using the TaqMan-based assay.

All specimens showed stable expression curves occurred between 10 and 14 cycles. All curves indicate the amplification of 18S rRNA gene generated by Rotor-Gene Q Real-time PCR with optimum cycling conditions, followed by 30 cycles. The threshold line was set automatically by the software.

Figure 3. A representative example of linear amplification curve of a male specimen (100A-116-DNA-5) using the TaqMan-based assay.

The plot indicates that the sample No. 5 did not generate a DM-W amplification curve by Rotor-Gene Q Real-time PCR, which occurred below the threshold line set by the software.

Figure 4. A representative example of linear amplification curve of a female specimen (100A-116-DNA-35) using the TaqMan-based assay.

The plot indicates that the sample No. 35 did generate a DM-W amplification curve by Rotor-Gene Q Real-time PCR, which occurred above the threshold line set by the software.

Figure 5. A representative of micrographic image of oviduct stained with Hematoxylin and eosin (HE), magnified at 10×.

The characteristic structures for Oviduct (OV), Late Vitellogenic Oocyte (LVO) and Glandular Region (GR) are identified. The image produced by the histological sectioning of the specimen following the procedures published in OECD-LAGDA guideline, stained with Hematoxylin and eosin (HE). A female biological endpoint was confirmed by a certified pathologist.

Figure 6. A representative of micrographic image of oviduct stained with Hematoxylin and eosin (HE), magnified at 40×.

The characteristic structures for Oviduct (OV), Oviduct Ciliated Epithelium (OCE) and Glandular Region (GR) are identified. The image produced by the histological sectioning of the specimen following the procedures published in OECD-LAGDA guideline, stained with Hematoxylin and eosin (HE). A female biological endpoint was confirmed by a certified pathologist.

Figure 7. A representative of micrographic image of testis stained with Hematoxylin and eosin (HE), magnified at 20×.

The characteristic structures for Sperm (SR), Spermatogonia (SP), Primary Spermatocytes (PS), Secondary Spermatocytes (SS), Spermatids (SM), Interstitial Cells (IC) and Sertoli Cell (SC) are identified. The image produced by the histological sectioning of the specimen following the procedures published in OECD-LAGDA guideline, stained with Hematoxylin and eosin (HE). A male biological endpoint was confirmed by a certified pathologist.

Figure 8. Micrographic image of a male testis with presence of oocyte inside seminiferous tubules, stained with Hematoxylin and eosin (HE), magnified at 20×.

The characteristic structure for Oocyte (OO) is identified. The image produced by the histological sectioning of the specimen following the procedures published in OECD-LAGDA guideline, stained with Hematoxylin and eosin (HE). The presence of an oocyte inside seminiferous tubules is called “testis-ovum”.