Cloning And Characterization Of Nuclear Estrogen Receptor And Nuclear Androgen Receptor Genes From Larimichthys Crocea

Large yellow croaker (Larimichthys crocea) is one of the most economically important marine fishspecies in China, distributed mainly in coastal regions of East Asia. Large yellow croaker is very popular dueto gold body colour, delicious, tender meat and abundant nutrition. There is an abundance of researchconcerning the morphology, breeding and gonadal development at the cellular level of the large yellowcroaker However, the molecular mechanisms of gonad development and sex differentiation remain poorlyunderstood. Therefore, cloning and characterization of gonad-related genes from large yellow crokar bymolecular biological techniques will provide insights into the molecular mechanisms of gonad developmentand differences in male and female.Based on established gonad cDNA library of large yellow croaker from our laboratory, partial sequenceof nAR was obtained. A partial nERα，nERβ1and nERβ2cDNA fragments were obtained by RT-PCR usingthe degenerate primers.. Three full-length cDNAs of nERα，nERβ1and nAR, and one cDNA fragment ofnERβ2were cloned by using SMART RACE technique. Real-time quantitative PCR (rtqPCR) was used toreveal their expression levels in different organs, in all stages of embryonic development. The results werereported as follows:1）The full-length cDNA of nERα is2989bp, consisting of a5’-terminal untranslated region (UTR) of508bp, a3’-terminal UTR (excluding the poly(A) tail) of494bp and an open reading frame (ORF) of1896bp.The deduced protein is composed of631amino acids (aa), with an estimated molecular weight (MW) of69.23kDa. RtqPCR revealed that nERα is ubiquitously expressed in all adult tissues examined. In liver andtestis of female and male fish, the expression was higher, and the expression level of nERα in the multiplecells, blastula and gastrula stage of embryonic development were higher than later stages of embryos.Expression levels declined substantially afterward during the course of embryonic development.2) The full-length cDNA of nERβ1is2538bp, consisting of a5’-terminal untranslated region (UTR) of572bp, a3’-terminal UTR (excluding the poly(A) tail) of288bp and an open reading frame (ORF) of1710bp.The deduced protein is composed of569amino acids (aa), with an estimated molecular weight (MW) of63.24kDa. RtqPCR revealed that nERβ1is ubiquitously expressed in all adult tissues examined. In gonad ofboth sexes, the expression was higher than other tissues, and the expression level of nERβ1in the early stagesof embryonic development was higher than later stages of embryos. Expression levels declined substantiallyafter blastula stage, and then AR mRNA was maintained at a low level.3) The full-length cDNA of nERβ2is2326bp, consisting of a5’-terminal untranslated region (UTR) of215bp, a3’-terminal UTR (excluding the poly(A) tail) of66bp and an open reading frame (ORF) of2025bp. The deduced protein is composed of674amino acids (aa), with an estimated molecular weight (MW) of74.97kDa. RtqPCR revealed a ubiquitous expression of nERβ2exists in all adult tissues examined. In liver ofboth sexes, and the expression level of nERβ2in female fish liver was higher than in male fish（P<0.05）.Expression levels of nERβ2in all stages of embryonic development maintained at a low level.4) The full-length cDNA of nAR is2840bp, consisting of a5’-terminal untranslated region (UTR) of240bp, a3’-terminal UTR (excluding the poly(A) tail) of382bp and an open reading frame (ORF) of2250bp.The deduced protein is composed of750amino acids (aa), with an estimated molecular weight (MW) of84.50kDa. RtqPCR revealed a ubiquitous expression of nAR is in all adult tissues examined. In gonad andliver of both sexes, the expression was higher than other tested tissues, and the expression level of nAR in theblastula stage of embryonic development was the highest. Expression levels were lower in all stages ofembryonic development except the multiple cells and blastula stages.We detected the expression and distribution of the above genes of large yellow croaker duringgametogenesis by using in situ hybridization with DIG antisense RNA probe. The results showed that:1) During spermatogenesis the strong hybridization signals of nERα were presented in nucleolus ofspermatogonium, meanwhile, the positive signals of nERα were detected in the cytoplasm of primaryspermatocyte, secondary spermatocyte and spermatid during spermatogenesis. During oogensis, the positivesignals of nERα were detected in the cytoplasm of phase I oocytes. Furthermore, the positive signals of nERαwere located in the follicle cell and the cytoplasm of phase II and III oocytes, lastly, the positive signals wereonly located in the follicle cells around the oocytes of phase IV and involuting phase oocytes.2) The stronger positive signals of nERβ1were detected in the spermatogonium during spermatogenesis.The signals become weaker in primary spermatocyte, and hybridization signal was also detected in secondaryspermatocyte, spermatid and spermatozoon. In oogensis, the positive signals of nERβ1were detected in thenucleolu of phase I oocytes. However, the positive signals of nERβ1were located in the cytoplasm in phase IIand III oocytes, and the positive signals were located in the follicle cells around occytes of the phase IV andinvoluting phase oocytes.3) During spermatogenesis, no signal was detected in spermatogonium, and weaker signals appeared inthe surrounding of primary spermatocyte, but there were stronger positive signals of nERβ2in the secondaryspermatocyte, spermatid and spermatozoon. During oogenesis, the positive signals of nERβ2were detected inthe cytoplasm of phase I oocytes and phase II oocytes. However, no positive signals of nERβ2were detectedin phaseIII, IV and involuting phase oocytes.4) The positive signals of nAR were detected in spermatogonium, primary spermatocyte, secondaryspermatocyte, spermatid and spermatozoon during spermatogenesis. Furthermore, the signals of nAR inspermatogonium and primary spermatocyte were significantly stronger than in secondary spermatocyte andspermatid. The positive signals of nAR were detected in the cytoplasm of phase I, phase II and phaseIIIoocytes. However, the positive signals of nAR were only detected in follicle cells and cytoplasm of oocytes phase IV and involuting phase oocytes.In summary, cloning, expression and characterization of nERα, nERβ1, nERβ2and nAR of large yellowcroaker suggested that these genes may play important roles in gonadal development, gametogenesis and earlystages of embryonic development and gametogenesis in present study. The results of this project will help usto elucidate the reproductive molecular mechanism of large yellow croaker.