Shedding new light on early sex determination in zebrafish

Alex C King,Armin K Zenker,Michelle Gut

doi:10.1007/s00204-020-02915-y

Alex C King, Armin K Zenker + Show 1 more

Open Access

https://doi.org/10.1007/s00204-020-02915-y

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Abstract

In contrast to established zebrafish gene annotations, the question of sex determination has still not been conclusively clarified for developing zebrafish, Danio rerio, larvae, 28 dpf or earlier. Recent studies indicate polygenic sex determination (PSD), with the genes being distributed throughout the genome. Early genetic markers of sex in zebrafish help unravel co-founding sex-related differences to apply to human health and environmental toxicity studies. A qPCR-based method was developed for six genes: cytochrome P450, family 17, subfamily A, polypeptide 1 (cyp17a1); cytochrome P450, family 19, subfamily A, polypeptide 1a (cyp19a1a); cytochrome P450, family 19, subfamily A, polypeptides 1b (cyp19a1b); vitellogenin 1 (vtg1); nuclear receptor subfamily 0, group B, member 1 (nr0b1), sry (sex-determining region Y)-box 9b (sox9b) and actin, beta 1 (actb1), the reference gene. Sry-box 9a (Sox9a), insulin-like growth factor 3 (igf3) and double sex and mab-3 related transcription factor 1 (dmrt1), which are also known to be associated with sex determination, were used in gene expression tests. Additionally, Next-Generation-Sequencing (NGS) sequenced the genome of two adult female and male and two juveniles. PCR analysis of adult zebrafish revealed sex-specific expression of cyp17a1, cyp19a1a, vtg1, igf3 and dmrt1, the first four strongly expressed in female zebrafish and the last one highly expressed in male conspecifics. From NGS, nine female and four male-fated genes were selected as novel for assessing zebrafish sex, 28 dpf. Differences in transcriptomes allowed allocation of sex-specific genes also expressed in juvenile zebrafish.

Highlights

Fish embryos are an attractive model for the risk assessment of chemicals and for the investigation of effects of endocrine disrupting substances (Kazeto et al 2004; Yu et al 2018) and drug discovery (Vaz et al 2018)
Cycle threshold (Ct) values obtained from the dilution series were used to determine PCR efficiency of the seven tested genes so comparisons of gene expression could be made
The developed qPCR probes, as well as commercially available probes for the genes sox9a, dmrt1 and igf3 (Thermo Fisher Scientific, Switzerland) were used for the analysis of sex-specific gene expression in zebrafish in relation to age, with actb1 used as a housekeeping reference gene

Summary

Introduction

Fish embryos are an attractive model for the risk assessment of chemicals and for the investigation of effects of endocrine disrupting substances (Kazeto et al 2004; Yu et al 2018) and drug discovery (Vaz et al 2018). Toxicological studies which test acute toxicity by means of Fish Embryo Acute Test (FET) (OECD 2013) or Early-life Stage Toxicity Tests (ELS Test) (OECD 2010) often use zebrafish embryos as indicators of endpoints (Kazeto et al 2004; Yu et al 2018). At this early stage, the embryos cannot be morphologically. This highlights the need for identification of novel genes for sex identification at early stages in zebrafish to scrutinise sexspecific drug susceptibility and translate to human data

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