Study On Sex Related Genes Sox3, Sox9and Dmrt1in Black Rockfish, Sebastes Schlegeli

The black rockfish, Sebastes schlegeli, is an important cultured fish in North Chinawhich has the advantages of strong disease resistance, larger individual, growing fastand ovoviviparous. However, little is known about its molecular mechanism of sexdetermination or sexual development. Studying on sex-related gene of S.schlegeli maycontribute to future studies of their breeding work. Moreover, selecting ovoviviparousS.schlegeli as a model may help us to further understand the molecular mechanisms ofsex determination in fish. In this study, based on the homologous cloning and RACEtechniques, the full-length cDNA of the Sox3, Sox9and Dmrt1genes were obtained. Theresult identified the DNA structure, promoter sequence and temporal expression of thesethree genes in S. schlegeli. The methylation patterns of the Sox3gene promoter CpGisland was also studied in this paper. Meanwhile, S.schlegeli GnRH-I (SsGnRH-I) fusionprotein was obtained, and the expression patterns of these three genes were analyzed ingonads by SsGnRH-I injection (in vivo). Addtionally, the stabilities and reliabilities ofthe commonly used reference genes were evaluated for promoting analysis of geneexpression in different tissues and during larvae developmental stage from S.schlegeli.In the present study, mRNA transcription profiles of eight putative reference genes(18S rRNA, ACTB, GAPDH, TUBA, RPL17, EF1A, HPRT, and B2M) were examinedusing quantitative real-time PCR (qRT-PCR) in different tissues and larvaedevelopmental stage of S.schlegeli. Three common statistical algorithms (geNorm,NormFinder, and Bestkeeper) were used to assess expression stability and RPL17orEF1A were selected as the optimal reference genes.The Sox3full-length cDNA of S. schlegeli had a length of1386bp and contained a906bp ORF, a197bp5’UTR and a283bp3’UTR. The predicted SsSox3protein of301amino acids (aa) contained a highly conserved HMG domain. Throughbioinformatic analysis by a series of online prediction softwares, a number of putative binding sites for transcription factors including AP-1, GATA-3, Oct-1, FOXD3, PBX1,HNF4, HNF3β, C/EBP, GATA-1, GATA-3, GATAx, NF-1, Sp1, USF and thesex-related protein SRY, Sox5, Sox9. Additionally, CpG methylation pattern of theSsSox3gene in different tissues was analysed. The result showed that CpG methylationlevel of this gene was not consistent with its expression quantities. qRT-PCR analysisindicated that the expression of SsSox3was detected in all of the inspected larvaldevelopmental stages of1,5,10,15,25and35days after birth (DAB), and exhibitted adownward trend. The expression of the SsSox3gene in adult gonads was sexuallydimorphic, with a higher level in ovary than testis. Cellular localization analysis showedthat SsSox3was mainly located in the oocytes and follicular cells of the ovary, and aweaker signal was also detected in the germ cell of the testis.The Sox9full-length cDNA of S. schlegeli had a length of2039bp, comprising of a1461bp open reading frame (ORF), a336bp5’UTR and a242bp3’UTR. Thepredicted SsSox9protein of486aa contained a highly conserved HMG domain.Bioinformatic analysis revealed a lot of common binding sites for transcription factorwere found between Sox3and Sox9promoters, including AP-1, GATA-3, Oct-1,FOXD3, and the sex-related protein SRY, Sox5, Sox9. qRT-PCR was performed for thelarval development stages of S. schlegeli. During early developmental days, analysisexhibited that the expression of SsSox9were detected in all stages of1,5,10,15,25and35DAB. Moreover, the expression of SsSox9was higher at the sensitive period ofgonadal differentiation (15-25DAB). The expression of the SsSox9gene in adult gonadswas sexually dimorphic, with a higher level in testis than ovary. In situ hybridization(ISH) to gonadal sections was performed and ISH signals was detected in the germ cellsand Sertoli cells in the testis, oocytes and follicular cells in the ovary.The full-length cDNA of S. schlegeli Dmrt1was1587bp and contained a189bp5’UTR, a489bp3’UTR and a909bp open reading frame (ORF), which encoded302amino acids with a conserved DM domain and an MSM domain. Several transcriptionalfactor binding sites in the5’promoter were identified that might regulate SsDmrt1expression, including AP-1, GATA-1, GATA-2, GATA-3, Oct-1, FOXD3, STATx,C/EBPa and GATAx. Besides, sex related protein binding sites for SRY, Sox5, Sox9 were also predicted in the Dmrt1promoter. qRT-PCR analysis indicated that SsDmrt1was expressed in all of the inspected larval developmental stages from1to35DAB andthat the level of expression gradually decreased. The expression of SsDmrt1in adultgonads was sexually dimorphic with extremely high expression in the testis but very lowexpression in the ovary. Moreover, SsDmrt1was specifically expressed in the germ cellsof both the testis and the ovary by in situ hybridization analysis.The expression of Sox3, Sox9and Dmrt1mRNA were examined after SsGnRH-Itreatment in immature S. schlegeli. The expression of Sox9and Dmrt1mRNA wassignificantly increased at24h after SsGnRH-I treatment (in vivo experiment) inimmature testis, but not in the ovary. The expression of Sox3mRNA was increased at24h after SsGnRH-I injection both in ovary and testis. These expression pattern revealedthat GnRH may affect the gonadal development by regulating the expression of the Sox3,Sox9and Dmrt1genes.