Identification And Expression Of Cbx2 And Its Function In Medaka Gonad Development

For sexually reproducing organisms,gonadal development and its molecular mechanism is one of the most fundamental biological problems.It is also a complex and essential process.Although new genes related to gonadal development are constantly being discovered and identified,the regulatory mechanism of gonadal development has not been fully elucidated.Fish is the most diverse group in vertebrates,and for many economically fish,their growth rate has obvious sex differences,and the monosex cultivation has higher economic value.Therefore,the gonadal development researches have become the hotspot in aquatic zoology.CBX2(Chromobox homolog2)is one of the key members of Polycomb group(Pc G)family.As an important developmental-related transcription factor,it plays a key role in gonadal development in mammal.In humans and mice,lacking cbx2 will lead to sex reversal.Bai et al indicate that cbx2 may affect the gonadal development by regulating the expression of sry and Nr5a1(the key transcription factor)in mammals.However,most studies have focused on humans and mice,and that in fish is still not clear.In this study,we selected medaka(Oryzias latipes)to identify cbx2 c DNA by using PCR cloning and sequencing.The result showed that cbx2 c DNA was 1443 bp in length and encoded 480 amino acids.Then we used various softwares to perform bioinformatics analysis.The SMART and Swiss model predicted that the secondary structures of CBX2 contained a Chromo domain,four low-complexity domains,and a Pfam domain.In the tertiary structures,the content of ?-helix is 13.125%,?-sheet is 7.08%,and the random coils accounted for 79.795%.The Ex PASy-Prot Param tool found that among the amino acids,serine had the highest proportion,accounted for 14.0%.The phylogenetic tree showed the CBX2 evolved more conservatively and had strong homology from fish to humans.All fish in phylogenetic tree grouped into one branch,while the rest clustered into another.The multiple sequence alignment suggested that the Chromo domain and the Pfam domain were more conservative and the middle part was less conservative.In our study,the cbx2 expression profile of medaka embryos and gonadal development was established by using real-time PCR,immunohistochemistry and in situ hybridization.Real-time PCR showed that during the embryonic development,cbx2 m RNA was expressed steadily lower from unfertilized to cleavage stage.The expression level began to increase from the blastocyst stage,and it reached to peak in gastrula stage.Cbx2 still kept to be highly expressed during the neurula and organogenesis stage and the expression level decreased during later development,indicating that the cbx2 might be involved in the formation and development of fish organs,and also related to the formation of primary germ cell(PGC).Compared to other tissues,the cbx2 m RNA expression was higher in gonads and kidney,and especially in testis,suggesting that cbx2 might be related to the testis development.The results from immunohistochemistry and in situ hybridization were consistent.In testis,both cbx2 RNA and protein were mainly expressed in spermatogonia and spermatocytes.In ovary,they all had a higher expression in oocytes at stage I,II,and III.In addition,cbx2 and vasa(a marker gene)were co-localized in gonads by fluorescent in situ hybridization(FISH),indicating that their expression patterns were consistent.These suggested that cbx2 was a gene expressed in germ cells,and it might be related to the spermatogenesis and oogenesis.In order to further study the cbx2 function,we injected si RNA to inhibit its expression level in 1-cell stage embryos and gonads.The microinjection results suggested that the expression of cbx2 was significantly suppressed after injecting si RNA.The male sex-related gene,sox9(sry-related HMG box 9)was inhibited;and the expression of foxl2(Forkhead transcriptional factor 2),was up-regulated.The results of living injection were similar with microinjection results,the expression of cbx2 was significantly inhibited in gonads both testis and ovary,the expression of sox9 was decreased in testis and the expression of foxl2 was increased in ovary,which indicated that cbx2 might also participated in gonadal development by regulating the expression of genes including sox9 and foxl2 in medaka.In addition,in order to verify the cbx2 role in meiosis,the expression changes of meiosis-related genes,SCP3(Synaptonemal complex protein 3)and Exol(Exonuclease 1)were identified.The results suggested that both SCP3 and Exol expression decreased along with the cbx2,indicating that cbx2 might be also involved in meiosis.