Mechanisms Of Tebuconazole On Sex Differentiation In Zebrafish Mediated By CAMP/PKA Pathway

Tebuconazole is a triazole fungicide which has been widely used in agricultural production because of its broad-spectrum sterilization.Tebuconazole residues have also been detected in soil and water.However,the existing studies of tebuconazole mainly focus on its environmental behavior,detection methods and toxicity of mammals,and there are few studies on the toxicity of tebuconazole to aquatic organisms.In this paper,the effect of tebuconazole on sex differentiation of zebrafish was studied,and its possible molecular mechanism was discussed.(1)The article studied the most significant period of tebuconazole exposure on sex differentiation of zebrafish.Zebrafish at different life stages(0-5 dpf,5-19 dpf,0-19 dpf,19-60 dpf,5-60 dpf,0-60 dpf)were exposed to tebuconazole at the same concentration(0.5 mg/L).The results showed that tebuconazole exposure had developmental toxicity to zebrafish,including decreased survival rate and weight loss.However,exposure to tebuconazole before sex differentiation did not have a significant impact on it,and the 5-60 dpf exposure period was identified as the most significant and exposure stage for future research.(2)The article studied the effects of tebuconazole exposure on sex differentiation-related genes and sex hormone levels in zebrafish.The results showed that tebuconazole increased the transcriptional levels of male development related genes such as gata4,fzd2,fzd7a,and decreased the transcriptional levels of female development related genes such as rspo1,cyp19a1a,fox12 and sf-1,the transcriptional level of dmrt1 gene also decreased.Sex hormones was analyzed by ELISA Kits.After being exposed to tebuconazole,decreased level of estradiol and increased level of testosterone was detected.(3)The article studied the effects of tebuconazole exposure on the cAMP/PKA pathway.The results showed that tebuconazole inhibited the activity of the pathway,decreased the activity of PKA,hindered the phosphorylation of FOXL2 protein,inhibited the transcription of cvp19a,an aromatase regulated by FOXL2,and reduced the conversion efficiency of testosterone to estradiol,which led to the male bias sex ratio.(4)I used Label-free quantitative proteomics and phosphorylated Label-free quantitative proteomics sequencing to further identify the pathways affecting sex differentiation in zebrafish related with tebuconazole.The results showed that tebuconazole could affect the phosphorylation of protein.FOXO,a homologous protein of FOXL2,was enriched in KEGG pathway.Protein kinase B was also been significantly affected,which proves that our hypothesis that tebuconazole affects sex differentiation by interfering with phosphorylation.