| Letrozole is a synthetic benzotriazole derivative that inhibits the activity of cytochrome P450 aromatase,which leads to a decrease in estrogen levels in the body and is generally used for chemotherapy and radiotherapy of breast cancer.Atrazine,also known as atrazine,is a three-benzene herbicide,mainly used for corn,sorghum,sugar cane,fruit trees and woodland,etc.,which may be washed into the low-lying areas by rain,resulting in a certain injury to the paddy field environment.In recent years,there have been lots of reports of the presence of letrozole and atrazine in urban sewage,surface runoff,drinking water,rivers,lakes and even rainwater.Because of the poor degradation of letrozole and atrazine,the persistence of long residues and the role of endocrine disrupting,it poses a potential threat to the aquatic environment and human health.However,how they act as endocrine disruptors to affect fish reproduction and sex differentiation is still unclear.In order to further understand their mechanism of endocrine disruption in fish,we used histological and molecular biology methods to study the effects of different concentrations of letrozole(25?g/L,125?g/L,625?g/L)and atrazine(2680 ?g/L,268?g/L,26.8?g/L)to expose female and male loach for a certain period of time(7 days,14 days,21 days),and investigate the changes of gonadal histological changes and sex related genes.The expression of miRNAs in the testis and ovarian of normal loach and exposed to letrozole and atrazine were studied by high-throughput sequencing,then investigating the effects of miRNAs on the gonad differentiation and development of loach by the treatment of the external environmental substances.The results showed that the number of primary oocytes treated with 625 ?g/L of letrozole in females was significantly lower than that of the control group;males showed spermatogenic tubule lobes increased,lumen expansion and increased sperm count.The expression levels of six genes related to gonad development and sex differentiation,including sox9a(sry-related HMG box-9a),sox9b(sry-related HMG box-9b),sf1(steroidogenic factor-1),dmrt1(double sex and mab 3)and amh(anti-Mullerian hormone)were raised in the testis or ovary,but did not change or decrease in the brains of both sexes,except that ar(androgen receptor)showed the opposite expression pattern.The expression of other four genes cyp19a(cytochrome P450 19a),foxl2(winged helix/forkhead transcription factor2),er?(estrogen receptor ?)and er? receptor(estrogen receptor ?)in the brain and gonads showed a concentration-dependent manner with gender and organizational specificity.In atrazine treatment experiments,we first identified the acute toxicity of atrazine to the loach,then on the basis of acute toxicity experiment,we can accurately estimate the environmental toxicity of atrazine and the effects on aquatic organisms reproductive development.The results showed that the half lethal concentration(LC50)of atrazine on loach was 31.60mg/L,26.82mg/L,18.98mg/L,the safe mass concentration(SC)was 5.8mg /L.With the increase of atrazine concentration and the prolongation of treatment time,the mortality of loach was significantly increased.Through the histological study,it was found that high concentration of atrazine(2680?g/L)could inhibit the occurrence of spermatozoa and promote the development of ovarian cells.At the same time,qRT-PCR results showed that different doses of atrazine could significantly promote the expression of cyp19 a,foxl2,er?,sf1 and other genes except er?showed an opposite expression pattern.Sox9 b,sox9b,dmrt1 and amh had showed significant inhibitory effects.The testis and the ovaries of the mature loach were established by high-throughput sequencing.The sequencing results showed that the majority of the short sequences of the loach gonads were small RNA,the length is concentrated in 27-28 bp,suggesting that there are a lot of piRNA in the gonad of loach.Twoknown and two new miRNAs(miR-138-x,mi R-335-x,novel-m0694-5p,novel-m0301-3p;miR-130-x,miR-205-x,novel-m0279-5p,novel-m0333-5p)with significant different expression levels in the two selected groups(DC-S24 hAC and DX-S24 h LX)were identified by fluorescence quantitative PCR.The results are consistent with the sequencing results,indicating that miRNA expression profiles obtained by high-throughput sequencing are reliable.A total of 14,359 target genes were predicted to target 6415895 target gene locus by using three target gene prediction software to predict all 1442 miRNAs.In the analysis of target gene predictions,we predicted several genes related to sex(foxl2,er?,er?,ar,dmrt3,cyp17,etc.).We found that some miRNAs(miR-181-y,let-7-x,miR-132-y,etc.)may play a certain role in the Gonads of fish by analyzing the miRNAs corresponding to each gene and combining the data of differentially expressed miRNAs between the samples.The target gene of differentially expressed miRNAs was found to be consistent with the target gene in each group by GO analysis,which involved cell formation,signal transduction,cell membrane and cell membrane composition,molecular binding,catalytic and transcription processes. |
PDF Full Text Request