Transcription Responses Of Er And Ar Genes And Estradiol Induces A Transcriptome Profile In Oryzias Curvinotus

Environmental estrogens?EEs?,one kind of natural or synthetic chemicals with estrogenic effects.EEs mediated by corresponding receptors and affect the transcriptional regulation of genes by simulating estrogen in vivo.Estrogen receptor and Andorgen receptor are group of the nuclear receptor superfamily,which mainly mediate sex hormone signaling in vivo and participate in physiological processes such as gonadal development and maturation.Oryzias curvinotus is the main species of native mangroves in Zhanjiang.It has the characteristics of strong fertility,short generation cycle and wide salt adaptability.It has the potential to develop into model organisms.In this study,we obtained the proper reference genes in RT-qPCR that is suiTab for different tissues and different stages of embryonic development and different exposure conditions by EEs.The er and ar were cloned by PCR and their characteristics analyzed.At the same time,in order to evaluate the potential hazard risks of EEs to organisms,the appropriate EEs biomarkers were screened.In this research,we analyzed transcriptome of estradiol?E₂?exposed for 24 hours?h?,and chose significant up-regulate genes,assessed their response to estrogen exposure and sensitivity to bisphenol A exposure.The main results are as follows:1.We have screened the internal reference genes suitable for RT-qPCR in different experimental condition.After RefFinder,geNorm,NormFinder and Bestkeeper analysis,the results showed that using eef1?and rps4x as two housekeeping genes for RT-qPCR internal reference genes in different tissues,different embryo development stages and different exposure EEs,a more accurate target gene quantification level was obtained.2.There are three subtypes of the er in the O.curvinotus.Also,with regards to ars,two subtypes exist in the O.curvinotus as well.In the detecter,6 tissues namely eye,muscle,brain,liver,gonad and gill from O.curvinotus were examined.All subtypes of er were mainly expressed in liver and gonad whereas the two kinds of ar were distributed in all tissues,ar?was highly expressed in eyes,liver and gonad while ar?is only expressed in gonad.During the 10 embryonic developmental stages,that is,1 cell stage,cleavage stage,blastocyst stage,gastrula,16 sarcolemma,eye stage,notochord cavitation,heart completion,spleen development,and hatch,the expression level of er and ar was different.3.Then,ers and ars have different expression characteristics by expose in different EEs.E₂ could up-regulate the expression of er?and er?1 in 2?g/L,20?g/L and 200?g/L,and down-regulate the expression of er?2.Each concentration?50 ng/L,500 ng/L,5?g/L,50?g/L?bisphenol A up-regulated er?expression,while er?2 was only up-regulated at higher concentrations?500 ng/L,5?g/L,50?g/L?.Bisphenol A decreased the expression of er?2 at each concentration.In addition,ar?was up-regulated at higher concentrations?50 ng/L,500 ng/L,5?g/L,50?g/L?and ar?was only up-regulated at 5?g/L.4.To analyze the transcriptome of E₂ exposed,it was found that 2?g/L of E₂would affect amino sugar and nucleotide sugar metabolism,myocardial function,phototransduction,spliceosome and renin-angiotensin system in post hatch.By selecting significantly up-regulated genes vtg and chg and assessing their sensitivity to EEs,the results showed that estradiol at different concentrations?2?g/L,20?g/L and200?g/L?up-regulated vtg and chg,however,there were differences in the response of bisphenol A at different concentrations?50 ng/L,500 ng/L,5?g/L,50?g/L?.ChgHm was significantly up-regulated under 50 ng/L bisphenol A exposure,while chgH and chgL were down-regulated under the influence of various concentrations of bisphenol A;vtg1 was up-regulated only at 500 ng/L,and vtg2 was down-regulated at higher concentrations?500 ng/L,5?g/L,50?g/L?.ChgHm is more suitable as an environmental biomarker for EEs than chgH,chgL,vtg1 and vtg2.The results of the study lays the foundation for the mechanisms of er and ar in O.curvinotus.At the same time,reference data were provided to assess the potential toxicity risks of EEs and the development of biomarkers for local mangrove EEs.