Effects of 17a-ethinylestradiol on the expression of three estrogen-responsive genes and cellular ultrastructure of liver and testes in male zebrafish.

In order to monitor the influence of estrogenic compounds on the reproductive physiology of fish, molecular markers for zebrafish vitellogenin, estrogen receptor and ZP2 were developed. For this purpose, sequence information about the zebrafish estrogen receptor and vitellogenin had to be obtained. By means of RT-PCR, a sequence fragment of the zebrafish estrogen receptor alpha was cloned and sequenced. Continuous cDNAs of two zebrafish vitellogenin-like gene products (zfvg1 and zfvg3) were constructed by the help of expressed sequence tags of zebrafish and completely sequenced. The sequences of the estrogen receptor and of the vitellogenins showed significant similarities to corresponding cDNAs of other fish species. Expression of these gene products was measured following exposure to 17alpha-ethinylestradiol and compared with histological endpoints. RT-PCR was used as a semiquantitative technique to record gene expression in adult male zebrafish, which were exposed to 17alpha-ethinylestradiol in time-and dose-response experiments. As for time-dependent expression, all hepatic genes investigated were expressed at considerable amounts from 24 h after onset of exposure to 50 ng/l 17alpha-ethinylestradiol to the end of experiment (17 days). In testes, expression of the estrogen receptor- as well as ZP2-mRNA remained unchanged for the entire experiment, except for the individuals exposed for 17 days, which displayed elevated expression levels of ZP2. In the dose-response experiment, male zebrafish were exposed to 17alpha-ethinylestradiol in concentrations from 0.25-85 ng/l for 4 and 21 days. LOECs for vitellogenin as well as estrogen receptor alpha expression were found to be 2.5 ng/l already after 4 d of exposure. Extension of the exposure time to 21 days resulted in enhanced transcription of vitellogenin-mRNAs at 2.5 ng/l 17alpha-ethinylestradiol, whereas the detection limit could not be lowered. In contrast, in testes no induction of both ZP2 as well as estrogen receptor expression was detected at any concentration tested. To examine estrogen-caused alterations at the ultrastructural level, liver and testes of males exposed to 25 ng/l 17alpha-ethinylestradiol were analysed. Male livers responded with a feminisation reflected by the proliferation of rough endoplasmatic reticulum and Golgi apparatus typical of female hepatocytes during vitellogenesis. However, in testes no signs of feminisation were detectable; rather, destructive phenomena like phagocytosis of sperm cells by Sertoli cells were observed. Thus, in sexually differentiated males no reorganisation of the gonadal tissue towards an ovary could be definitely detected at any level investigated.