Effects of exposure to 17α-ethinylestradiol during early development on sexual differentiation and induction of vitellogenin in zebrafish (Danio rerio)

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Abstract

To determine the critical stage of zebrafish development where exposure to xenoestrogens can affect sex ratio and vitellogenin induction, zebrafish (Danio rerio) were exposed to 17α-ethinylestradiol (actual concentration 15.4±1.4 ng EE2/l) during early development: from fertilisation to hatch; hatch to 10 days post hatch (dph); 10–20 dph; 20–30 dph; 20–40 dph; 20–60 dph; fertilisation to 25 dph; or hatch to 60 dph. Vitellogenin was measured in whole body homogenate 30 dph by ELISA and sex ratio was determined 60 dph by histological examination of the gonads. All exposure periods significantly induced vitellogenin synthesis and affected the sex differentiation leading to development of ovo-testis or complete feminisation of the exposed fish depending on exposure period. Complete sex reversal was obtained in groups exposed from 20 to 60 dph and hatch to 60 dph. The half-life for degradation of vitellogenin was calculated. Juvenile zebrafish were exposed to 15.4±1.4 ng EE2/l (actual concentration) from fertilisation to 25 dph and transferred to clean water, after which weekly measurements of vitellogenin concentration in whole body homogenate were performed until day 46 post hatch. The half-life of vitellogenin was 2.4 days.

Introduction

During the last decades it has become more and more obvious that a number of organic pollutants cause adverse health effects to animals consequent to changes in endocrine functions (Gillesby and Zacharewski, 1998). Such chemicals are known as endocrine disrupters and most attention has been paid to compounds having estrogenic effects although compounds with anti-estrogenic, androgenic and anti-androgenic effects are known as well. Effects of endocrine disrupters in fish include reduced fertility (decreased sperm number and quality, or egg number), induction of the synthesis of the yolk precursor protein vitellogenin (VTG) in males and juveniles and effects on the development of the gonads (Billard et al., 1981, Andersen et al., 2001, Örn et al., 2000).

Endocrine disrupters are effective at sub-lethal concentrations (Stahlschmidt-Allner et al., 1997) and it is, therefore, not possible to detect effects of such compounds using existing standard tests with endpoints such as mortality. Great effort has, therefore, been put into this area to develop methods for testing endocrine disrupters containing new endpoints. At the 1st OECD Expert Consultation on Testing in Fish in 1998 gonadal development and VTG induction was, therefore, suggested as the key endpoints for in vivo evaluation of estrogenic compounds in fish (EDF2, 2000).

One of the fish suggested as a test animal was the zebrafish, Danio rerio. In this species, males pass through a stage of juvenile hermaphroditism. Approximately 10 days post hatch the differentiation of the gonads begins and all fish, irrespective of their definitive sex, develop ovaries. At approximately day 23 post hatch the ovaries of approximately half of the fish start to degenerate and are transformed into testes. This process is completed at approximately 40 days post hatch. In the remaining fish, the development and maturation of ovaries continue (Takahashi, 1977, Uchida et al., 2002).

Gonadogenesis in fish can be a very complex and plastic process. Although sex determination is under genetic control, the final differentiation of the gonads in fish also depends on endocrine signals, i.e. estrogens and androgens (Yamamoto, 1969, Arcand-Hoy and Benson, 1998, Campbell and Hutchinson, 1998). In most gonochoristic fish, the germ cells of the undifferentiated gonads are sexually bipotential (Kobayashi et al., 1991). During specific critical periods of early development, changes in sex hormone levels can, therefore, affect the final sex independently of the genetic sex (Donaldson and Hunter, 1982). Due to the lability of sex differentiation in fish, exposure to endocrine disrupters during certain critical periods of early development can lead to sex reversal.

To determine the critical stage where sex differentiation and induction of VTG in zebrafish can be affected by estrogenic compounds, zebrafish were exposed to 17α-ethinylestradiol at stages according to the observations on gonadal development by Takahashi (1977). If exposure to endocrine disrupters during this critical period is sufficient to manifest effects of endocrine disrupters, this may considerably lower the costs of future short-term methods for testing chemicals for an endocrine disrupting effect.

Section snippets

Test substance and artificial zebrafish medium

The test substance was 17α-ethinylestradiol (EE2) (ICN Biomedicals Inc., Ohio, USA) was dissolved in methanol. The final concentration of methanol did, in accordance with the OECD guideline no. 202 (1992), not exceed 100 μl methanol l−1 artificial zebrafish medium. Zebrafish were kept in artificial zebrafish medium, i.e. Millipore water filtrated through active charcoal and added salts. The medium was aerated until a stabile pH (pH 7.8±0.2) was reached (approx. 24 h). The approximate

Determination of water concentrations of EE2

The control group contained no EE2, while the water concentration of EE2 in the exposure group was 15.4±1.4 ng EE2/l.

Determination of the elimination, half-life, of VTG

The VTG measurements of this experiment evaluated the fate of VTG after termination of the exposure. At the end of the treatments, i.e. 25 dph, the VTG concentrations were significantly increased in fish exposed to 15.4±1.4 ng EE2/l when compared to the control fish (P<0.05). In the control group, no significant change in the VTG concentration from termination of exposure, 25–46

Determination of water concentrations of EE2

The actual concentration of EE2 in the exposure water was lower than the nominal concentration. Reduction of the nominal concentration is a problem when working with lipophilic compounds such as EE2. The fish were, therefore, kept in either glass jars or aquaria made of stainless steel to reduce the absorption of EE2 to the surfaces. Even though the aquaria were cleaned daily and the test solution was changed daily, microbial growth as well as absorption of EE2 to faeces and food leftovers

Acknowledgements

This project was financed by the Nordic Council of Ministers.

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