Expression Analysis Of Nanos2 During Gonadal Development Of Monopterus Albus

Ricefield eel(Monopterus albus)is a crucial aquatic economic fish in China.It is the first vertebrate that was found to be sequential hermaphroditic.However,the molecular mechanism of the sex reversal of Monopterus albus remains unclear.According to the previous results,the methylation level of nanos2 promoter region decreased during the process of sex change,which suggested that it may play an essential role in the gonadal development of Monopterus albus.In this study,the temporal and spatial expression characteristics of nanos2 gene in the gonadal development of Monopterus albus were studied by quantitative real-time PCR(q RTPCR),fluorescence in situ hybridization,and immunofluorescence.First of all,to determine gonadal developmental stages of Monopterus albus for subsequent study,histological analysis of gonads of Monopterus albus was carried out utilizing conventional paraffin section and HE staining method.According to features of the paraffin sections,the developmental stages of gonads were classified into ovaries at stage Ⅱ,Ⅲ and Ⅳ,intersexual gonad(early,middle,late),and testis at stage Ⅱ,Ⅲ,Ⅳ and Ⅴ,respectively.The NCBI search results showed that there were three nanos gene in Monopterus albus.The homology of the nanos gene family in Monopterus albus was analyzed by constructing a phylogenetic tree.The results showed that the nanos genes of Monopterus albus had some homology with other teleost.The temporal and spatial expression characteristics of three nanos genes(nanos1,nanos2,and nanos3)were analyzed in Monopterus albus by q RT-PCR.The quantitative results showed that nanos1 was highly expressed in the liver,followed by the brain and gonad;nanos2 and nanos3 were highly expressed in the gonad but weakly or not expressed in other tissues.nanos3 was mainly expressed in the gonads with late stage of oocyte development.And the expression level of nanos3 was low throughout the testicular development stage.In contrast,nanos2 was highly expressed in testis at stage Ⅳ and Ⅴ,and intersexual gonad at late stage,of which expression level was low through the developmental process of ovary.Due to many spermatids present in seminal vesicles of testis at stage Ⅳ and Ⅴ,and intersexual gonad at late stage,high expression level of nanos2 in these stages indicated that nanos2 may contribute to spermatogenesis.In order to further investigate the nanos2 gene,the whole sequence of Monopterus albus nanos2 was obtained by RACE-PCR.And the nanos2 m RNA fluorescence probe was synthesized for locating nanos2 m RNA in gonadal tissue.The recombinant Nanos2 polypeptide was prepared by prokaryotic expression,and was used to immunize rabbits for preparing antibodies.The full-length c DNA sequence of nanos2 is 1139 bp.The open reading frame contains 537 bp,which encodes 178 amino acids including conserved zinc finger structure.The results of fluorescence in situ hybridization and immunofluorescence indicated that nanos2 were mainly expressed in a type of somatic cells(similar to sertoli cell),spermatogonia,secondary spermatocytes,and primary spermatocytes.In addition,the nanos2 positive signal was also found in the ovary,in which a kind of cells was the same nucleus diameter(6～7 μm)with spermatogonia.Summarily,this work found that nanos2 was highly expressed in the gonads during spermatogenesis,and might play an essential role in spermatogenesis.In this study,the results of expression pattern of nanos2 would contribute to further understanding the function of nanos2 in the gonadal development of Monopterus albus.