The juvenile three-spined stickleback ( Gasterosteus aculeatus L.) as a model organism for endocrine disruption : I. Sexual differentiation

Abstract

Juvenile three-spined stickleback ( Gasterosteus aculeatus L.) is introduced as a unique model organism for both androgenic and oestrogenic endocrine action. Intersex is often used as an indicator for disruption of sexual differentiation in fish exposed to different kinds of effluents from human activities. In wild fish it has exclusively been reported in terms of feminisation due to xenoestrogens in the environment. The assumption that the intersex individuals are feminised genetic males can only be proven by genetic sex identification of the intersexual individuals. Intersex and gonadal sex reversal were induced in three-spined sticklebacks by treatment with natural and synthetic steroid hormones. Juvenile sticklebacks were exposed to three nominal concentrations of 17β-oestradiol (E2); i.e. 0.01, 1.0 and 10.0 μg/L; which were administered to the water either continuously from hatching to the end of the experiment (39–58 days post hatch), during the first 2 weeks after hatching only, from 14 days after hatching onwards, or during the chorionated embryo stage until hatching. Other groups were exposed to 17α-ethinylestradiol (EE2) at 0.05 μg/L and 17α-methyltestosterone (MT) at 1.0 μg/L (nominal concentrations). MT was applied continuously, during the first 2 weeks post hatch only, or from 14 days after hatching onwards. Gonad histology was examined and the genetic sex was identified with male sex-linked PCR markers. Treatment with oestrogens caused feminisation at the two highest E2 concentrations and with EE2. Exposure to E2 before hatching had no effect. Intersexual individuals from oestrogen treatments were genetic males. The genetic sex marker identified apparent total reversal of the gonad type of genetic males. Treatment with MT did not reveal a clear picture, since intersex was observed in both genetic females and males. MT also caused severe testis abnormalities, mainly the development of large branched cavities with unidentified origin. The process of sex differentiation is most sensitive to the influence of external steroids during the first 2 weeks after hatching. A lower incidence of intersex could also be induced in sticklebacks exposed from 14 days after hatching by E2 treatment, but not with MT. The combination of gonad histopathology with genetic sex identification in juvenile sticklebacks is suggested as a tool for detecting endocrine disruption in laboratory studies, and might become very useful in field surveys.