Exploration Of Gonadal Development, Cloning Of Vasa And Its Expression In Pristella Maxillaris

The body of Pristella maxillaris is transparent,and its bones,muscles and gonads and other internal organs can be observed clearly,therefore,Pristella maxillaris can be used as animal model to study gonadal development.In this study,the basic time nodes of gonadal development and sex differentiation of Pristella maxillaris were investigated by through intergation several methods in morphology,anatomy and histology.Meanwhile,the full-length cDNA of gonad-associated gene Vasa was cloned using homologous cloning and RACE-PCR,and its expression was also studied by semi-quantitative RT-PCR and in situ hybridization.The results are shown as follows:1 The sexual characteristics of Pristella maxillaris and Histological changes of Larval during sex DifferentiationThe appearance of the yellow granular structure or uplifted structure in abdomen can be used to judge the sex of transparent fish with age of over 4 months or silver-peritoneum fish older than 5 months.Four different phages of oocyte can be observed in ovary from 6-month-old fish,including primary growth stage,cortical alveolus stage,vitellogenesis stage andoocyte maturation stage,through histological H.E.staining.A large number of sperm filled in small sperm duct can also be observed in testis in fish with the same age.When hook-shaped teeth appeared in anal fins from fish with over 100 days,which can be used to judge the fish as male,but such structure was absent in fishes less than 2 months.Furthermore,the gonadal development of juvenile between 5 to 50 days old were also explored by using parffin section.No differentiated gonads could not be observed on both sides of dorsal abdominal cavity in 10 dpf fish.In 15 dpf fish,germline cyst was observed in the abdominal cavity,and gonad tissue only appeared on one side.The overy-like structure?‘juvenile ovary'?symmetrically appeared in 25 dpf fish.And baculiform testis appeared in 30 dpf fish,which shows that sex differentiation begins around 30days.2 Cloning and Expression Analysis of Vasa Gene in Pristella maxillarisThe full-length cDNA sequence of Vasa was cloned usingby homologous cloning and RACE-PCR,and its consists 2734 bp,named as Pm-Vasa.The length of Vasa ORF was 2013 bp,which codes 671 amino acids,its 5'and 3'untranslated regions were 143 bp and 578 bp respectively,and the 3'UTR contained a typical AATAAA polyA signal and polyA tail structure with 43 bp.The analysis of Vasa amino acid sequence showed that its molecular formula was C₃₁₂₂H₄₈₈₆N₉₁₄O₁₀₀6_S21 with a molecular weight of 71993.92 Da,and the isoelectric point was 5.53.The amino acid sequence contains all nine conserved domains common to the DEAD-box family of proteins,including GYVKPTPVQ,AQTGSGKT,PTREL,TPGR,DEAD,SAT,LVFVE,ARGLD and HRIGRTGR.There are glycine-rich regions at the N-terminus,including 9 RGG repeats and 8 RG repeats.In addition,ARKF frames were found,and Vasa-specific GG structures were present between the domains Ia and Ib.The amino terminus and the carboxy terminus are conserved with tryptophan?W?residues,aspartic acid?D?residues and amino acid?E?acidic residues.Semi-quantitative RT-PCR analysis showed that Vasa mRNA was mainly expressed in gonadal gland,and weak signal was also checked in liver and pancreas.The expression of Vasa mRNA decreased gradually after fertilization to 10 dpf embryos,continued to decrease with development of larvae?10 dpf to 70 dpf?.Finally,with the sexual maturation?the ovarian development of the testis?and body maturation,level of Vasa mRNA showed a trend of increase,which indicates that Vasa is a maternal gene.gonads in situ hybridization showed that Pm-Vasa mRNA in the original cells express strong,then gradually weakened,at all levels of secondary sexual mother cell to verify the Vasa gene location in gonads of Pristella maxillaris.All these results provide a theoretical basis for further study of sex differentiation,gonadal development and mechanism regulating them in Pristella maxillaris.