| Orange-spotted grouper,a protogynous hermaphroditic fish,is an excellent model for studying the mechanisms of gonadal differentiation,sex determination and sexual reversal,and is also a cultured marine fish with high nutritional value.In vertebrates,androgen plays key roles in regulating the gonad differentiation and development via the nuclear androgen receptor(AR).Forkhead box L 3(foxl3),one copy of foxl2,is important to the testis development in teleost fish.Rec8 and fbxo47 are involved in meiosis,regulating spermatogenesis.In grouper,androgens can promote gonadal sex reversal,as well as testis development and spermatogenesis,but the mechanism of androgen regulating testis development is unclear.In this study,using real-time quantitative PCR,the expression patterns of rec8 and fbxo47 in different tissues were detected,and the expression profiles of foxl3,rec8,and fbxo47 during different developmental stages in the gonad of the orange-spotted grouper were also investigated.Subsequently,AR regulating the activity of foxl3 promoter,and Foxl3 regulating the activity of rec8/fbxo47 promoter were studied by using the dual-luciferase report gene system.In addition,the subcellular localizations of foxl3,rec8,and fbxo47 in GS(Grouper spleen)cells was also determined.The experimental results are as follows:1.Fbxo47 has an open reading frame(ORF)of 1425 bp,encoding 414 amino acids.rec8 open reading frame is 1752 bp in length,encoding 513 amino acids.Tissue distribution of rec8 and fbxo47 m RNA showed that they are expressed in all tissues examined,with the highest expression in the gonad.According to the references,gonadal tissues in different development stages were collected.During the gonadal development,the expression levels of fox13,rec8,and fbxo47 increased gradually from O2 gonad to testis,with the highest expression in the testis,followed by intersex gonad.The expression levels of foxl3,rec8,and fbxo47 in the intersex gonad and testis were 7-10 times,29-188 times,and 11-320 times higher than that in the O4 vary,separately,suggesting that these genes may involve in the regulation of testis development in range-spotted grouper.2.The subcellular localization analysis in GS cells showed that foxl3 signal was observed in the nucleus,fbxo47 was expressed in the nuclear membrane,and rec8 was mainly distributed in the cytoplasm.3.The luciferase reporter assay showed that AR could enhance the activity of the foxl3 promoter,and Foxl3 could up-regulate the activities of rec8 and fbxo47 promoters,with a maner that the activities increased at the beginning and then declined.The highest luciferase activities in the groups of AR-PGL4.10-foxl3 and Foxl3-PGL4.10-rec8 were observed at the concentration of 20 ng of transfected plasmid,while Foxl3-PGL4.10-fbxo47 group showed significant luciferase activities at 50 ng of transfected plasmid.Different dose of β-testosterone treatment(10,50,100,200 n M)could enhance the activities of the fox13,rec8 and fbxo47 promoters in a dose-dependent manner.The luciferase activities is most clearly observable in the groups of AR-PGL4.10-foxl3 and AR-PGL4.10-rec8 after 50 n M β-testosterone treatment,and in the group of AR-PGL4.10-fbxo47 after 100 n M β-testosterone treatment(p <0.001).Taken together,the expression patterns of foxl3,rec8,and fbxo47 in the gonad of orange-spotted are highly suggestive of their involvement in the regulation of testis development.Moreover,our results indicate that there is a AR-Fox13-Rec8/Fbxo47 axis in grouper.The present study clarified the regulatory relationships among foxl3,rec8,and fbxo47,providing theoretical guidance for further research on the molecular biology and aquaculture production of grouper. |
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