| 17?-ethinylestradiol(EE2),an important endocrine disrupting chemical(EDC),exists extensively in marine environment.EE2 possess endocrine disrupting effects and immunotoxicity,which have direct impact on the health and reproduction of fish.Innate immunity of fish plays an important role in resistance of pathogens infection.Therefore,it is of great important scientific significance in investigating the immunotoxicity effects of EDCs on fish.Previous studies mainly focus on the reproductive and immune toxicity effect of EE2 on fish,however,there is a few study on the immunotoxicity mechanism of EE2.In our study,a typical endocrine disrupting chemical EE2 was chosen as tested substance,while marine model animal Oryzias melastigma and commercially important cultured fish black porgy(Acanthopagrus schlegelii)as study objects.Embryos,larva fish and adult fish were exposed to EE2 with different concentrations and exposure time.Expression patterns of both gene and protein levels of endocrine related estrogen receptor(ERs)and various immunological parameters were accessed.By comparative evaluation of the results,we aimed to seek the relevance between ER genes and immune factors(such as antimicrobial peptide genes).Based on this research emphasis,this thesis was designed to illuminate toxicity effect of EE2 on marine fish and explore the possible immunotoxicity regulatory mechanism.This study contributes to intensively understanding mechanism of interaction between the toxic effects of environmental pollutants on marine fish and the response of fish innate immune factors,moreover provide important theoretical value and guiding significance.The main studies are as follows:1.cDNA sequence of three subtypes of estrogen receptor(ER)were cloned in O.melastigma and named as OM-ER?,OM-ER?1 and OM-ER?2.The cDNA length of OM-ER?(Genbank:JF972761)was 2762 bp,with an open reading frame(ORF)encoding 618 amino acids.OM-ER?1(Genbank:JF972762)cDNA length was 2835 bp,encoding 562 amino acids,while OM-ER?2(Genbank:JF972763)cDNA with a length of 2471 bp,encoding 631 amino acids.Phylogenetic analysis showed that the cloned three OM-ERs genes belonged to members of classic nuclear receptor family,containing five classic functional domains:A/B,C,D,E,F domain in the amino acid structures.Furthermore,polyclonal antibody of OM-ERa was acquired.2.Revealed the expression patterns of OM-ER?,OM-ER?1 and OM-ER?2 in different developmental stages and different tissues of normal male and female O.melastigma.Significantly high level of OM-ERa mRNA were observed in testis,ovary,spleen(both male and female)and liver(female),but relatively low expression in male liver.OM-ER?1 showed relatively high expression in liver(both male and female),testis and ovary,followed by intestine and stomach,moreover,expression in sexual gonads showed gender specificity which was significantly higher in male than that in female.OM-ER?2 was highly expressed in liver(both male and female),testis and ovary,and the expession in testis was higher than that in ovary.To sum up,OM-ERs were highly expressed in liver and sexual gonads,indicating their potential function in endocrine regulation.High expression of OM-ERa in immune organ(spleen)suggesting its function might relate to immune response.The expression of OM-ER?1 and OM-ER?2 continuously increased during the developmental period from 4 days-post-fertilization(dpf)to 30 days-post-hatching(dph),while the expression of OM-ERa stayed constant and low.3.Elucidated the expression patterns of OM-ERs genes under different dosages(5,50,500 ng/L)and various exposure time of EE2.The expression of OM-ERa was significantly induced in liver,testis and larva of O.melastigma exposure to EE2,moreover,the induction was amplified as the exposure time went on and exposure concentration increase,particularly in the liver of male adult individual.OM-ER?1 and OM-ER?2 were induced in liver at 500 ng/L but inhibited at 5 ng/L EE2 acute(96 h)exposure.Conversely,OM-ER?2 was upregulated in liver at 5 ng/L but downregulated at high dose EE2 chronic(21 d)exposure,which was similar to the expression patterns of larva acute exposed to EE2.The biomarker gene OM-vtg1 was remarkedly induced in liver of adult individual and larva in a time-and dose-dependent manner,however,it showed no obvious change in embryos,which imply adult fish and larva were more sensitive than embryo to EE2 exposure.All these suggested that O.melastigma of different developmental stages showed different sensitivity to EE2 exposure,and the response mode of OM-ERs also showed differences.4.Illustrated the expression patterns of immune-related genes in liver of male adult O.melastigma under EE2 exposure.The expression of OM-hepl was inhibited under both acute(12-96 h)and chronic(7-21 d)EE2 exposure.The expression of JAK/STAT immune signal pathway genes were inhibited by acute and chronic EE2 expsoure.As a stimulus,EE2 acute exposure for 3-12 h activated the MyD88/NF-?B signal pathway,as exposure time went on,EE2 exposure inhibited MyD88/NF-?B signal pathway,affected the liver immune function.Metabolic gene CYP1A1,physiological index ACP and AKP in liver was significantly inhibited by 500 ng/L EE2exposure for 14 and 21 d,showing decreasing metabolic ability of posion and subsequent liver damage.All the results above suggested that EE2 exposure had significantly inhibitory effect on immune response and immune activities in O.melastigma and A.schlegelii.5.Revealed the key transcriptional regulatory element in the promoter of OM-hep1.By genome walking,we cloned the 5' flanking sequence of O.melastigma antimicrobial peptide OM-hep1 with a length of 2558 bp.Using the dual-luciferase reporter gene system and cultured cell lines(Carp EPC,HepG2),various recombinant plasmids with deletions and mutations were constructed,furthermore,a key transcriptional regulatory elementlocated in-315 bp to-289 bp of OM-hep1 promoter wassuccessfully characterized,while-315 bp to-293 bp was a predicted ERE-like binding site.Using EPC and HepG2 cell lines,we found that EE2 stimulation significantly suppressed the activity of OM-hep1 promoter;co-transfected pCMV-HA-ER? with dual-luciferase reporter gene system into HepG2,over-expression of OM-ERa also inhibited the promoter activity of OM-hep1.Results above indicating OM-ERa might function as a transcriptional regulation factor mediated the down-regulation of OM-hepl gene by EE2 exposure.In vivo experiment then confirmed this assumption.When O.melastigma exposed to EE2,OM-ER?expression in liver was remarkably induced while OM-hepl was inhibited;conversely,intraperitoneal injection of ER inhibitor ICI 182780 into O.melastigma followed with EE2 exposure,the inhibiting effect of EE2 on OM-hepl was eliminated.In vivo assay verified the relationship between OM-hepl and OM-ERa.6.Through EMSA and SPR assays,we further confirmed the key transcriptional regulatory element HepERE in OM-hep1 promoter.Synthetic probe with biotin labeled(HepERE-biotin)was acquired.pCMV-HA-ERa was transfected into 293T cells,then nuclear protein was extracted for EMSA assay.A supershift band was detected by EMSA suggesting OM-ERa could bind to HepERE-biotin.DNA binding domain(DBD)of estrogen receptor was highly conserved among different species,so human ERa was applied to determined the bonding strength with HepERE-biotin.In this case,BiacoreTM T200 was applied to detect surface plasmon resonance,results showed that the affinity between human ERa,and HepERE-biotin was KD = 8.6 nmol/L,which could be defined as potent combination.In summary,EE2 exposure exhibited immunotoxicity on marine fish,affected their immune response and immune activities.Furthermore,we first revealed HepERE(including the ERE binding site)in OM-hep1 promoter was the key transcriptional regulatory element in the immunotoxicity mechanism exposure to EE2.Meanwhile,we revealed a new signal pathway involved in fish hepcidin transcriptional regulation.In addition,the newly revealed EE2-mediated ERa/HepERE pathway for hepcidin transcriptional expression significantly enriched our knowledge of the regulatory mechanism of mammal and fish hepcidin expression in correspondance with multiple stimulants.The work we done can lay the foundation for further research on the relationship between immunotoxicity of marine environmental endocrine disrupters and marine fish healthy and survival. |
PDF Full Text Request