Molecular Cloning And Expression Of Several Germ Cell Markers Of The Nile Tilapia, Oreochromis Niloticus And Southern Catfish, Silurus Meridionalis

Sex determination and differentiation have always been the focus in the researches of biology, physiology and endocrinology.In mammals,sex was controlled by genetic factors,especially the sex determining gene,SRY/Sry.While in lower vertebrates,including fish,sex is also influenced by environmental factors,such as temperature,photoperiod and exogenous sex steroids.These factors might even cause sex reversal in many species.Sex differentiation includes the differentiation of both somatic cells and germ cells.In the past,much attention has been drawn to the differentiation of somatic cells,genes closely related to the sex determination and differentiation have been cloned and their functions further investigated.Recently,researches on the differentiation of germ cells have gradually become a new hotspot based on the better understanding of sex differentiation and requirement of fishery industry.To date,researches of germ cells have been mostly restricted in mammals,while in lower vertebrates,especially in fish,the germ cells were poorly investigated.One of the most important reasons responsible for this is due to the lack of germ cell markers. Investigations on the germ cell marker genes in fish will lay foundations for the researches on the mechanisms of germ cell differentiation and development,and expanding the understanding of sex differentiation in fish.It also has practical implications for aquaculture.The Nile tilapia(Oreochromis niloticus),because of its fast growth rate and short reproductive cycle,is developed as one of the most important species for aquaculture and experiment globally.As the growth of male is much faster than female by 50%,males are always favored.Because the masculinization methods generally utilized was of much disadvantages,new techniques by modern molecular biology is highly required in growing monosex progeny.As a result,investigations on the sex differentiation,especially on the differentiatioin and development of germ cells,are of decisive theoretical significance and practical implications.In the present study,several germ cell markers were cloned and their expressions in various adult tissues and ontogenic expressions in gonad were investigated.Sex reversals were successfully obatained by treatment with aromatase inhitor(fadrozole) and estrogen(17-βestrodial).The expressions of these marker genes in the gonads of sex reversed fish were also examined.First of all,two cDNAs of germ cell markers(Oct4 and Figla) responsible for early development of tilapia gonad were isolated by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends(RACE).The length of Figla cDNA is 1254bp, containing an open reading frame(ORF) of 594bp,which encode a 198aa protein.The length of Oct4 cDNA is 1787bp,containing an ORF of 1380bp,which encode a 460aa protein.The expression of Figla mRNA initiated from the 5 days after hatching(dab) and showed a sexual dimorphic pattern from 10 dah onwards.The expression level of Figla mRNA in testis remained low till 70 dah,while increased significant when sexually matured detected by both RT-PCR and quantitative RT-PCR(qRT-PCR).High expression of ntOct4 was detected during early gonadal development in both sexes,indicating its decisive role in this period.In situ hybridization(ISH) results revealed that ntOct4 showed a sexual dimorphic expression pattern, expressing higher in ovary than in testis from 1 month after hatching,after which,its expression level decreased significantly and became undetectable in the adult male.cDNAs of Spo11,one of the earliest marker related to recombination process in meiosis and Scp3 encoding the structural protein of synaptonemal complex during meiosis were also isolated by RT-PCR and RACE methods in the Nile tilapia.Both of them are markers closely related to the meiosis of germ cells.Full length cDNAs of Scp3 and Spo11 were 1070 and 1468 bp,which contain ORFs of 717 and 1173 bp,encoding proteins of 239 and 390 aa,respectively.Expressions of both markers were restricted to the gonads of the tilapia,with higher level in testis for Scp3 and higher level in ovary for Spo11.Tissue distribution analyse showed that the the expression of Scp3 was much higher in testis than in ovary.Ontogeny analysis by RT-PCR also revealed this pattern basically.It was also revealed that Scp3 was expressed mainly in theⅠ,ⅡandⅢphase oocytes and its expression level decreased in oocytes ofⅣphase,in which mainly concentrating on the edge.Spo11 expression also showed sexual dimorphic in tissue distribution analysis,being higher in ovary than in testis.Ontogeny analysis by qRT-PCR revealed the same pattern in 10 dah,30 dah,40 dah and 5 months fish and adults.Vasa protein,a member of the DEAD(Asp-Glu-Alu-Asp) box protein family of ATP-dependent RNA helicases,plays key roles in germ cell formation in higher metazoans.The Vasa gene,found exclusively expressed in germline cells of many metazoans,has been extensively characterized as germ cell specific marker in many vertebrate and invertebrate species.Two isoforms of Vasa cDNA (scVasa and it's short form scVasa-s) were isolated and characterized in a teleost fish,southern catfish by RT-PCR and RACE methods.Analysis of the nucleotide sequences revealed that the full length cDNA of scVasa comprises 2,522 bp with an ORF of 1,989 bp encoding 662 amino acids, while that of scVasa-s comprises 2,475 bp with an ORF of 1,926 bp encoding 641 amino acids. scVasa-s lacks a part of the N-terminal region found in the scVasa.Both of the two deduced amino acid sequences contain all of the known characteristics belonging to the DEAD-box protein family, four arginine-glycine-glycine(RGG) motifs and other eight conserved motifs.They show high similarity to Vasa homolog of Giebel carp(75.2%and 73.8%).Tissue distribution analysis by RT-PCR revealed that these two isoforms were exclusively expressed in the gonads of both female and male.In adult fish,scVasa was found to be mainly expressed in the primary oocytes atⅠandⅡphases in the ovary while in the spermatogonia and primary spermatocytes in the testis by in situ hybridization.Semi-quantitative RT-PCR analysis showed that the expressions of both scVasa isoforms were much higher in ovarian recrudescent stage(mainly with phaseⅡoocytes) than in vitellogenic stage(mainly with phaseⅢandⅣoocytes).This is the first time to clone so many germ cell markers in teleosts,the Nile tilapia and Southern catfish,both of which are important for aquaculture.Although investigations on these markers have been taken by reseachers all over the world extensively,most studies were focused only on a few model species,such as mouse,zebrafish and medaka,while little attentions have been paid to the important aquacultural speices.In order to lay foundations for growing monosex progeny and improving the growth rate of the aquacultural fishes by biologcial techeniques,expression patterns and changes during sex reversal by drug treatment of these germ cells and meiosis markers were investigated in the Nile tilapia and Southern catfish,two important aquacultural fish species in our country.The results of the study are important not only for researches on the mechanisms of sex differentiation,meiosis regulation and the cross talk between germ cells and somatic cells,but also important for practical implications in future aquaculture.