| Vitellogenin(Vtg)is the precursor of vitellogen,and it is produced by estrogen receptors activation mediated through the hypothalamic-pituitary-gonadal(HPG)axis [1].Egg of vertebrate can store a lot of yolk,which is composed mainly of vitellogenin also named the female serum specific protein.Vtg is a key substance during the occurrence of yolk,and it is a reproductive protein,which plays an important role in the process of reproduction development.The study of the synthesis mechanism of Vtg will improve the basic research of fish physiology and developmental biology,and provide the theoretical basis for fish reproduction as well.In order to understand the synthesis mechanism of Vtg and analyze the effect of exogenous estrogen on the synthesis of Vtg in fish,Scatophagus argus was used as the research object.Vtg synthesis mechanism was analyzed through cloacal aperture injection of EE2(17?-ethynylestradiol)in vivo,as well as EE2 exposure to primary cells of liver in vitro,by the method of gene cloning,fluorescence quantitative PCR and H.E.staining.We cloned the full-length cDNA sequence of three types of vtg using reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends procedures.The length of S.argus vtgAa cDNA sequence was 5360 bp(GenBank no.: KY676847),encoding a protein consisted of 1696 amino acids.The protein was 186.07 ku and isoelectric point was 9.16.The first 15 amino acids in N-terminal were signal peptide(Fig.1-3).The length of S.argus vtg Ab cDNA sequence was 5346 bp(GenBank no.:KY654346),and it encoded a protein consisted of 1699 amino acids.The protein was 186.37 ku and isoelectric point was 9.24.The first 15 amino acids in N-terminal were signal peptide(Fig.1-3).The length of S.argus vtgC cDNA sequence was 4244 bp(GenBank no.:KY676848),encoding a protein consisted of 1275 amino acids.The protein was 142.87 ku and isoelectric point was 6.43.The first 15 amino acids in N-terminal were signal peptide(Fig.1-3).According to the results of H.E.staining,all of the experimental fish were in the immature stage of gonadal development,which was accord with the prerequisite of EE2 injection experiment.Therefore,it was speculated that the Vtg synthesized after injection were induced by EE2 injection experiment.In this study,the S.argus primary cells of liver were isolated using perfusion method[2](Fig.3-1),and the isolated hepatocytes were treated with EE2 after cultured to the third generation.According to the results of RT-qPCR,the expression of liver vtgAb gene after EE2 cloacal aperture injection was higher than that of the other two subtype genes under both different sampling time and different doses of EE2.Therefore,vtg Ab was selected as the major gene to analyzfe the mRNA expression level(Fig.2-5).According to the RT-qPCR results of vtg Ab after the injection of EE2,the expression level of vtgAb was higher at EE2 concentration of 1.0 ?g/g and 10.0 ?g/g,while lower at concentration of 0.01 ?g/g and 0.1 ?g/g(Fig.2-6).At 24 h after EE2 injection,the expression level of vtgAb was low,and increased gradually to the highest one at 72 h.Then,the expression level of vtgAb was decreased at 96 h.The expression level of S.argus vtgAb gene in primary cells of liver was higher at EE2 exposure concentration of 10-8 mol/L,10-7 mol/L,10-6 mol/L and 10-5 mol/L.However,the expression level of vtgAb at EE2 exposure concentration of 10-5 mol/L was lower than that of 10-6 mol/L(Fig.3-2).The expression level of vtgAb was reached the lowest at which EE2 exposure concentration was 10-11 mol/L.vtgAb gene was initially expressed at 24 h after exposure,and increased gradually from 48 h,and reached to the highest one at 72 h.Then,the expression level of vtgAb decreased at 96 h,which was lower than that at 48 h,expressions of Vtg homologs could be induced by EE2. |
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