Effects Of17α-ethinylestradiol On The Expression Of Gender-special Genes In Gobiocypris Rarus

17α-Ethinylestradiol (EE2), one of the most potent artificial estrogens of Endocrinedisruptors (EDCs), is widely used in birth control pills and many studies indicated that EE2has a significant impact on genes expression, especially the gender-specific genes expression.In environmental toxicology research, many studies found that Gobiocypris rarus (G. rarus) issensitive to exogenous hormones and can be used as an experimental animal for EDCs.Proteins encoded by Factor in the germLine alpha (figla), Forkhead box l2(foxl2),Synaptonemal complexes3(scp3), Sry-related high mobility group box9a (sox9a),Vitellogenins (vtg), Zona pellucida (zp2) and Zona pellucida (zp3) play important roles ingonad differentiation and reproduction. In the present study, firstly, full-length cDNAs of figla,scp3, sox9a and zp3were cloned and characterized by RT-PCR and RACE methods. Secondly,expression patterns of figla, scp3, sox9a and zp3in adult tissues were investigated byqRT-PCR. Thirdly, the gene expression profiles of figla, foxl2, scp3, sox9a, zp2, zp3and vtg inthe gonads and zp2, zp3and vtg in the liver of adult G. rarus were analyzed after exposed toEE2at1,5,25, and125ng/L for3and6days, respectively. Finally, the5′flanking regions offigla, foxl2, sox9a, zp2and zp3were isolated to better understand the regulation mechanismof gene expression of these genes following EE2exposure.The main results of this study are as follows:Full length cDNAs of G. rarus figla, scp3, sox9a and zp3are970,1005,1645and1489bp, respectively. The corresponding open reading frames (ORFs) of figla, scp3, sox9a and zp3are660,723,1404and1344bp encoding219,240,467and447amino acids, respectively.The expression patterns of figla, scp3, sox9a and zp3in adult tissues indicated that figla andzp3were predominantly expressed in the ovaries; scp3and sox9a were primarily expressed inthe testes. In addition, sox9a was also highly expressed in the brains of both genders.The most stable housekeeping gene in the G. rarus following EE2exposure is β-actinand it can be used as the reference gene. Three-day EE2treatment caused a significantincrease of the transcripts of figla, vtg and zp2in the testes, foxl2in the ovaries and vtg in thelivers of males. However, six-day EE2exposure repressed figla, scp3and zp2transcripts inthe testes and foxl2transcript in the ovaries. The results also shown that the vtg in the malesliver shows higher responsiveness than zp2in all EE2groups while the zp2in testes shows higher responsiveness than vtg in lower (1and5ng/L) EE2concentrations. Moreover, zp2ismore sensitive than zp3in the gonads of both genders in all treatment groups.The5′flanking regions of figla, foxl2, sox9a, zp2and zp3are1017,2351,2250,992and984bp, respectively. Many cis-elements are found in these5′flanking regions, such as thesex-determining region Y (SRY) binding site in the flanking regions of figla, foxl2, sox9a, zp2and zp3, the estrogen responsive element (ERE) in the flanking regions of figla and sox9a andthe oocyte-specific protein (Osp1) binding sites in the5′flanking regions of zp2and zp3.Furthermore, one CpG Island was found in the5′flanking regions of figla, foxl2and zp2.In conclusion, we can get the following conclusions. First, the most stable housekeepinggene in the G. rarus livers following EE2exposure is β-actin. Second, the testiculartranscripts of figla, scp3and zp2, the ovarian transcript of foxl2and the transcripts of vtg inthe G. rarus males′liver and both genders′gonads can be used as sensitive molecularbiomarkers for early warning to monitor EE2in fresh water environment. Third, the effectiveEE2dosage for feminization in male G. rarus might be at least25ng/L. Fourth, thecis-elements such as SRY, ERE and Osp1in the5′flanking regions of figla, foxl2, sox9a, zp2and zp3might involve in regulation of these genes following EE2exposure.