Expression Of Nup58 And Nop58 In Medaka And Their Potential Role In Sex Differentiation

Like mammals,the male of Oryzias latipes is determined by the XY sex system.Because of the advantages of fast life cycle,easy handling,small size of genome,etc.,Oryzias latipes is an animal model to study the mechanisms of sex determination and differentiation of vertebrates.Gonadal soma-derived factor（Gsdf）can regulate the male germ cels of Oryzias latipes and play a key role in switching on/off spermatogenesis before the last turn of mitotic proliferation.Targeted disruption of gsdf in medaka results in XY female sexual reversal,and leads to an expansive proliferation of spermatogonia,abnormal centrosome replication,reduction of pachytene cels and asynchronous cysts of germ cell development.This study is a supplement and improvement to the mechanism of gsdf regulating sex differentiation.The nuclear pore complex（NPCs）is the passage of substances into and out of the nucleoplasm/cytoplasm.Nucleopore can regulate the transport efficiency of RNA and protein in and out of the nuclear pore,thus regulating gene expression.Even if this regulation is only a subtle change,it may also have a dramatic impact on cell signal and cell fate.Nucleoporin 58（Nup58）is a member of the central channel of the NPC.It is very important for Drosophila male germ cels to enter meiosis,and may be necessary for human spermatogenesis.This experiment analyzed the expression pattern of nup58 gene in wild-type medaka,and found that nup58 gene was expressed in all stages and tissues of medaka,and the expression in testis was higher than that in ovary.The overall methylation level of nup58 gene promoter in female fish was 5.55%,and that in male fish was 2.96%,but this difference was not significant,indicating that the difference in expression of nup58 gene in male and female gonads of medaka was not regulated by promoter methylation.Analysis of the transcriptome data of gsdf^+/+and gsdf^-/-medaka testis showed that the expression of nup58 in the testis of gsdf deficiency was the most significant among all nuclear porin genes,and was confirmed by RT-q PCR.Meanwhile,the up-regulated differential gene KEGG pathway has enriched into the nucleocytoplasmic transport pathway,and the nup58 gene in this pathway suggests that the expression of nup58 may be regulated by Gsdf signal and the downstream Smad phosphorylation and dephosphorylation signal transduction pathway.The results of immunofluorescence experiment showed that nup58 was expressed in all germ cels at different stages of gonadal gametogenesis in gsdf^+/+and gsdf^-/-medaka,and the expression location in ovary would change with the change of nuclear membrane.Through transmission electron microscope observation of the testis of gsdf^+/+and gsdf^-/-medaka,it was found that the nuclear membrane of spermatocyte in pachytene stage was sunken and had the possibility of premature rupture,and this change might be caused by the increase of Nup58 content after gsdf knockout leading to the enlargement of the NPC on the nuclear membrane.Finally,the correct expression of PCMV-N-FLAG-Nup58recombinant vector in 293T cells will provide a good platform for cellular experiments.This study shows that Nup58,as a nucleoporin,can participate in the proliferation of male reproductive cels of medaka,and its high expression in the testis of gsdf signal deficiency is correlated with the abnormal cystic proliferation of germ cels.The role and dynamic changes of nucleocytoplasmic transport coordinated by Nup58 nucleoporin in sex differentiation of germ cels deserve further study.The nucleolus is mainly the site of ribosomal RNA production and ribosomal subunit assembly,and also the location of many meiotic genes.Nucleolar protein 58 is necessary for the stability or synthesis of sno RNA.The research on it focuses on tumor,but not on sex differentiation.The results of previous experiments showed that the expression of Nop58 protein in the gsdf-knockout ovary was significantly up-regulated,which showed that Nop58 had a potential role in fish sex differentiation and gametogenesis in addition to its role in tumorigenesis.The laboratory previously reported a number of potential interaction partners of Gsdf using His-tag"pull-down"experiment combined with shotgun LC-MS/MS,and found that Nop58 was one of the potential partners of Gsdf.This study first explored the expression pattern of nop58 gene in various tissues and stages of medaka through RT-q PCR experiment,and found that it can be expressed in different time and space of medaka,and its expression in testis will be significantly higher than that in ovary.The interaction between Gsdf and Nop58 was further confirmed by paired yeast two-hybrid experiment.To summarize,Nop58 may play a role in gametogenesis through its interaction with Gsdf,which provides new insights for nucleolus involving in the process of sex differentiation.