Changes Of Vitellogenin And Transcriptomic Analysis Of Gonads Of Misgurnu Anguillicaudatus Under PHE Stress

The loach（Misgurnus anguillicaudatus）is a benthic fish,belonging to the genus Misgurnus in the family Cypriniformes,which is distributed throughout the country in natural freshwater waters and is highly adaptable to the environment.Phenanthrene（PHE）is a typical polycyclic aromatic hydrocarbon pollutant,which is highly toxic to aquatic organisms and may cause long-term pollution to the water environment.However,there are few studies on the mechanism of toxic effects of PHE on loach,especially on the response process of gonadal development.In this study,we performed q PCR and ELISA on the changes of vitellogenin（VTG）in liver and gonadal tissues of loach under PHE exposure,and explored the toxicological response mechanism of gonads to PHE exposure by using transcriptomic sequencing to screen for genes related to gonadal development with significant differences.It is worth mentioning to explore the effects of PHE exposure on aquatic organisms,provide basic data and ideas to further investigate the expression patterns of genes related to exogenous estrogen interference with gonadal development in fish and to further explore its reproductive toxicity in fish.The main findings are as follows.1.The loaches（Misgurnus anguillicaudatus）were exposed to 1.33,1.77,2.36 and3.13 mg·L^-1 for 28 days.During the PHE treatment,the liver and gonad tissues of loach were collected on 7,14 and 28 days.The hepatosomatic index（HSI）and gonadosomatic index（GSI）were measured by weighing method,the expression of vitellogenin 1（vtg1）and vitellogenin c（vtgc）in the liver and gonad of M.anguillicaudatus was detected by q RT-PCR,and the content of vitellogenin（VTG）was determined by Enzyme Linked Immunosorbent Assay（ELISA）.The results showed that:（1）PHE stress significantly increased HSI and decreased GSI of male,and significantly increased HSI and GSI of female.（2）PHE stress caused significant increase of vtg1 and vtgc expression in M.anguillicaudatus,and the expression of vtg1 and vtgc in male M.anguillicaudatus showed a trend of low concentration induction and high concentration inhibition.（3）PHE exposure significantly increased VTG content in liver and gonad of M.anguillicaudatus,especially in the group with higher concentration of PHE.It is suggested that certain concentration of PHE could induce abnormal development of liver and gonad of M.anguillicaudatus.Additionally,the lower concentration of PHE could induce expression of vtg1 and vtgc in liver and gonad,while the higher concentration of PHE could inhibit the expression of those two genes,and the expression of vtg1 and vtgc were different under PHE stress.Moreover,PHE could induce the production of VTG in liver and gonad of loach.2.The loaches were exposed at concentrations of 0.30、1.00、3.00 mg·L^-1for 60 days.The effects of PHE on the testis development were evaluated by calculating the survival rate,observing the structure of testis and analyzing transcriptome.To begin with,PHE markedly decreased the survival rate in a dose-dependent manner.Then,the number and density of spermatogonia,primary spermatocytes,secondary spermatocytes and spermatids were substantially reduced under PHE exposure.The space in the seminiferous tubule obviously increased in the high PHE concentration group.Meanwhile,transcriptome comparative analysis identified 5329 differentially expressed genes（DEGs）including 2928 up-regulated and 2401 down-regulated in the testis of Misgurnus anguillicaudatus exposed PHE for 60 days.Meiotic cell cycle,arganelle fission,ATPase activity and adenylate nucleotide binding were significantly differences by GO enrichment.KEGG pathway analysis revealed that TNF signaling pathway,CAMs,cytochrome P450 and lipid metabolism were markedly regulated.In addition,eight DEGs were randomly selected from the testis transcriptomics results for q PCR verification,the results were consistent with RNA-Seq.Finally,related genes（piwil2,dmc1,vasa,ubr2,dnd,rnf17）of testis development were further confirmed and they were differentially regulated after PHE exposure.3.The transcriptome library of PHE-treated female loach ovaries were constructed using Illumina platform,and the results showed that a total of 278 DEGs were screened,of which 168 were up-regulated expressed genes,mainly immune response and cytokine receptor interaction-related genes,and 110 were down-regulated genes,mainly protein binding and fatty acid metabolism-related genes.These DEGs were enriched to 1638categories in GO database,mainly involved in adhesion,molecular binding,signaling and other processes.KEGG analysis enriched to 27 pathways,including PI3K-Akt signaling pathway,RAS signaling pathway,HIF-1 signaling pathway.Among the screened DEGs,representative genes were selected for q PCR analysis,and the expression levels of il-6 and pcbp2 were found to be significantly up-regulated,and the expression levels of vcam1,atp6,opn1b and itga2 were significantly down-regulated.These differential genes may be involved in the response of loach ovarian tissue to PHE by activating the innate immune response of the organism and interfering with oxidative phosphorylation and protein synthesis.In conclusion,PHE exposure induced an increase in VTG content and gene expression in loach.Gonadal transcriptome analysis revealed significant changes in immune and inflammatory responses,gonadal development-related genes and metabolic pathways in loach under PHE exposure,which to some extent indicates the response mechanism of loach under PHE exposure.