Category And Subcellular Localization Of Canine ERβ Isoforms In Hypothalamus-pituitary-gonad Axis

For the past few years,beagle dogs appeared many reproductive problems which influencethe production rate and quality of beagles severely and result in momentous economicloss,therefore, it is significant to carry out the research which is relate to canine reproductivemechanism in our country.Hypothalamus-pituitary-gonad axis is the most important regulation axis to mammalianreproductive action. With regard to female animals，estrogen plays a significant role inhypothalamus-pituitary-gonad axis. In addition, the physiological actions of estrogen is mediatedby ERs.The positive and negative regulation of estrogen is not only correlated to ERs which haveexpression difference, expression levels and expression period in different tissues,but also thisregulation is related to ER isoforms and its pattern of presence. Studies by some researchershave established that ERα gene konck out mouse are infertility in spite of female or male,endometrium cannot grow matured,and gland numbers are less than wild type mouse; ERβ geneknock out mouse developmental condition of endometrium is similar to wild type mouse andERβ-/-mouse can be pregnant,but less amount of birth.Afore-mentioned research resultsstrongly confirm that ERs are closely related to reproduction regulation of mammalian.Because ERs participate in the process of mammalian reproductive regulation,so we focuson ERβ and ERβ isoforms to study ERβ and ERβ isoforms in the canine hypothalamus-pituitary-gonad axis, and research its functions initially.The study results could supplytheoretical basis for the role of ERβ and ERβ isoforms in Beagles reproductive regulation andsolving problems encountered in the actual production furtherly.In this study, ERβ and its isoforms of canine hypothalamus-pituitary-gonad axis tissueswere cloned by nested PCR and the full-length CDs obtained by RACE-PCR.Later,we buildrecombinant plasmids，and transfected transiently into293cells. Cellular localization about ERβand its isoforms proteins were carried by subcellular localization experiments.At the meanwhile,we analyzed the expression of ERβ mRNA gene in tissues ofhypothalamus-pituitary-gonad axis on beagles during oestrus and dioestrus by Real-timequantitative PCR.Results：1.We obtained CDs sequences of two ERβ isoforms successfully. ERβ-1293coding regioncontains1293bp,lacks300bp that the fourth exon of the full-length ERβcompletely. ERβ-1257coding region contains1257bp,lacks181bp which the seventh exon of the full-length ERβcompletely.2. Real-time quantitative PCR show that the expression of ERβ mRNA in oestrushypothalamus、pituitary、ovary and uterus are all higher than dioestrus，and the expression levelof oestrus hypothalamus、pituitary、ovary and uterus is2.32times、2.26times、2.82times and5.73times than dioestrus，respectively.3.We constructed pEGFP-N1-ERβ、pEGFP-N1-ERβ1293and pEGFP-N1-ERβ1257expressionvectors,and transfected293cells successfully. We identified the expression of ERβ and ERβisoforms proteins by western blotting （WB）in vitro.WB results show that the fusion proteinexpressed successfully.4. Subcellular localization about ERβ and ERβ isoforms in293cells shows that bothERβ-1593and ERβ-1257locolize in nucleus,and ERβ-1293locolizes in cytoplasm.Research results indicate that there are ERβ isoforms in canine hypothalamus-pituitary-gonadaxis,and the subcellular localization of two ERβ isoforms have discrepancy.The study contributes to thefollowing research about the releation of beaches and oestrus regulation.