The Cloning And Expression Patterns Analysis Of Gcnf In Medaka

Germ cell nuclear factor(GCNF)is a ligand-activated transcription factor of the nuclear receptor(NR)gene family,including a DNA binding domain(DBD)characterized by NRs structure and a highly conserved ligand binding domain(LBD).GCNF plays key roles in the growth and reproduction of vertebrates and in the differentiation and division of embryonic stem cells(ESCs).Studies have shown that during embryonic development,by regulating pluripotency-related genes,GCNF can directly or indirectly affect embryo pluripotency,cell division and differentiation,neurodevelopment,disease occurrence,etc.In the process of gonad development,GCNF has an important regulatory effect on germ cell development,reproductive hormone secretion,and gonadal maturation.Although GCNF has been cloned and studied in many species,it has rarely been reported in fish,and its expression pattern varies among different chordates.Medaka(Oryzias latipes)is a good model species for studying sex determination,vertebrate development and embryonic stem cells.A series of advantages make it become a good model organism,including small size,rapid reproduction,easy survival,small genome and complete gene sequences,and it provides a broad research platform for many scholars to study cell biology and developmental biology.we currently have many varieties of transgenic fish,whose germ cells are specifically labeled.In order to lay the foundation for the research of vertebrate reproductive development and related genes function,we have long-term exploration and verification of germline development and sex-controlling for gonadal genes in medaka,such as boule and igf1.Medaka boule affects the testis development,and igf1 influences the estrogen production of medaka ovary.In addition,knock down of piwi or vasa in medaka affected primordial germ cells(PGCs)migration.Meanwhile,depletion of dazl or dnd led to the defect of PGCs specification.Therefore,medaka is a good model organismfor studying gonadal development and germ genes.At present,the research about GCNF in medaka has not been reported,but GCNF has been widely studied in mouse.Studies have shown that mouse gcnf RNA is localized in the chromatoid body(CB)around the nucleus of germ cell during the spermatid differentiation.The CB belongs to germ plasm,which has a perinuclear cytoplasmic cloud-like structure,and it is a germ-cell-specific centre for RNA storage and processing.In addition,studies have found that some germ genes are components of CB,such as piwi and vasa,and vasa is a germ cell marker gene that regulates medaka gonadal development.Therefore,we identify the localization of CB by cloning and expression analysis of medaka gcnf,and further analyze the potential role of gcnf in gonad development.In this study,the full-length coding sequences of gcnfa and gcnfb,two gene subtypes of medaka gcnf,were obtained.Gcnfa and gcnfb encodes 484 amino acids and451 amino acids,respectively,and their expression were analyzed between gene level and transcription level.Gcnf is highly conserved in evolution and contains typical DNA binding regions and ligand binding regions,is about 60% identical to that of several species.By molecular cloning techniques,both gcnfa and gcnfb were found to associate with the development of adult tissues and embryo stages.In adult tissues,gcnfa RNA was restricted in the adult gonad.Except for the expression of gcnfb RNA in the gonad,its signal was also present in the eye,brain,and weaker in the kidney.In developing embryos,gcnfa RNA persisted throughout embryogenesis,whereas gcnfb RNA was not expressed until the neurula stage.In situ hybridization analysis showed that,in the medaka ovary,gcnf RNA was present in early stages of oocytes,and had no detectable signal in mature oocytes,oogonia and in the somatic cells of ovary.In the medaka testis,gcnf RNA was expressed in the round spermatids and sperm during spermiogenesis,and had no detectable signal in spermatocytes and spermatogonia.In addition,the signal occurred around the peripheral nucleus of round spermatids in the early stage,distributed dynamically,and finally was concentrated in the acrosome cap region and the flagellar manchette of sperm.By compared with the germ marker gene,vasa,gcnf RNA accumulated in the CB during spermatid differentiation,is a component of CB.In addition,it changes dynamically during spermatogenesis.The CB is dispersed aroundthe cytoplasm of round spermatids and condensed to form dense particles after cell deformation,which eventually becomes one of essential components for sperm development.Therefore,we identify the role of GCNF in the gonadal development and reproductive development in medaka,and our results will pave the way for future studies of GCNF in the regulation mechanism of gonad and reproductive development.