Exposure to an anti-androgenic herbicide negatively impacts reproductive physiology and fertility in Xenopus tropicalis

F Orton,A Eriksson,B Verbruggen,T Mcmillan,C R Tyler,T Uren Webster,E Jansson,J Fick,Y Carlsson,M Leishman,C Berg,T Economou,M Säfholm

doi:10.1038/s41598-018-27161-2

Abstract

Amphibians are threatened on a global scale and pollutants may be contributing to population declines, but how chemicals impact on their reproduction is poorly understood. We conducted a life cycle analysis to investigate the impacts of early life exposure to two anti-androgens (exposure until completion of metamorphosis;stage 66): flutamide, (50 µg/L)/linuron (9 and 45 µg/L)) on sexual development and breeding competence in Xenopus tropicalis. Our analyses included: mRNA levels of dmrt1, cyp17, amh, cyp19, foxl2 and ar (tadpoles/metamorphs), gonadal histomorphology (metamorphs/adults), mRNA levels of ar/gr (adult male brain/gonad/forelimb), testosterone/corticosterone levels (adult males), secondary sexual characteristics (forelimb width/nuptial pad: adult males) and breeding competence (amplexus/fertility: adult males). Compared to controls, feminised sex ratios and increased number of spermatogonia (adults) were observed after exposure to flutamide and the lower linuron concentration. Exposure to the lower linuron concentration also resulted in demasculinisation of secondary sexual characteristics and reduced male fertility. Flutamide exposure resulted in masculinisation of the nuptial pad and elevated mRNA levels of dmrt1, cyp17, amh and foxl2 in brains (metamorphs). Testosterone levels were higher in all treatment groups, however, overall few effects were observed in response to the higher linuron concentration. Our findings advance understanding of reproductive biology of X. tropicalis and illustrate negative effects of linuron on reproductive processes at a concentration measured in freshwater environments.

Highlights

Amphibian populations are disappearing at a rate faster than for any other vertebrate group[1]
We conducted a life cycle analysis with X. tropicalis exposed to the anti-androgens linuron and flutamide to assess for effects on sexual development, secondary sex characters, and subsequent gametogenesis, fertility and breeding behaviour in adult male and female frogs
Metamorphs (NF 66) not required for this grow out phase were sacrificed, the gonad-mesonephros complex (GMC) collected and placed in neutral buffered formalin (NBF) for analysis of gonadal histomorphology and the brains dissected and placed in RNAlater for analysis of gene expression

Summary

Introduction

Amphibian populations are disappearing at a rate faster than for any other vertebrate group[1]. Many pesticides, including herbicides, possess anti-androgenic activity[27,28] and high level application of agrochemicals occurs most often during the spring, coinciding with amphibian breeding and the period of sexual development during early life. We conducted a life cycle analysis with X. tropicalis exposed to the anti-androgens linuron and flutamide to assess for effects on sexual development (including gonadal differentiation and maturation), secondary sex characters, and subsequent gametogenesis, fertility and breeding behaviour in adult male and female frogs. We hypothesised that anti-androgenic chemicals would have feminising effects on physiological features, expression of gene related to sex in the brain and gonad (dmrt[1], amh, cyp[17], ar, foxl[2] and cyp19) and on behavioural endpoints in male frogs. The sensitivity of gonadal differentiation to estrogenic endocrine disruption in X. tropicalis is comparable with that of a temperate, terrestrial frog species[41]

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Conclusion