Research On The Origination And Transplantation Technology Of Germ Cells In Marine Economic Fishes

Germ cells transmit genetic information to the next generation,which is very important for the continuation of race.In recent years,fish germ cell transplantation technology has been rapidly developed.Combined with cryopreservation technology,it has shown a broad space in the protection of endangered species and surrogate broodstock.However,most of the current researches on fish germ cell development and transplantation focus on models and freshwater fishes,and rarely involve in marine economic fishes.Turbot(Scophthalmus maximus),Summer flounder(Paralichthys dentatus),Japanese flounder(Paralichthys olivaceus)and black rockfish(Sebastes schlegelii)are important marine economic cultured species in China.With the long-term artificial breeding,the degradation of germplasm resources,the decrease of genetic diversity and the low fertility gradually appear.In this study,we studied the origination pattern and transplantation technology of germ cells in the Pleuronectiformes and Scorpaeniformes.To promote the understanding of the origination,differentiation and maturation mechanism of fish germ cells,and open up new ways for the preservation and efficient utilization of germplasm resources.The specific research results are as follows:1.Black rockfish germ cell origination and developmentThe expression of black rockfish vasa gene in germline was analyzed by in situ hybridization.The vasa RNA evenly distributed in blastomere during cleavage stage.Signal concentration appeared at early gastrulation stage,and the areas of embryonic shield seemed to be darker than the rest.Until the late gastrulation stage,some vasa-positive cells firstly appeared at germ ring of epiboly bottom,and the primordial germ cells(PGCs)formed.PGCs gathered on dorsal side of the narrow embryonic body at early neurula stage and randomly aligned along anterior of the body axes.Then,they moved posterior-ward and loosely lined along the anterior-posterior axis on both sides of the embryonic body at somite stage.PGCs formed two clusters and aligned bilaterally on the ventral side at heart beating stage.Finally,the PGCs localized at the dorsal sides of the gut,ventral side of notochord at hatching stage.After birth,PGCs were gradually surrounded by somatic cells and migrated along mesonephric duct.On the 10 th day,PGCs migrated to the primordial genital ridge and formed the primordial gonad.During the entire gonadal development process,vasa expressed in early germ cells.This study firstly revealed the origination and migration pattern of germ cells in marine viviparous fish,which was similar to medaka of Euteleostei without early localization function of germplasm(vasa RNA),but different from zebrafish of Ostariophysans with germplasm early localized at cleavage furrows.2.Function analysis of teleost vasa/nanos3 3’UTRThe 3’UTR of the maternal gene has localization function,fused with fluorescent protein,and can track fish PGCs in vivo.Constructed vasa/nanos3 3’UTR chimeric m RNA of several common teleost,including turbot and black rockfish,and injected1-2 cell fertilized eggs to realize the labeling of PGCs in turbot and other fishes in vivo.This simple,rapid,and non-gene pollution labeling method is suitable for commercial fishes.The conserved elements in vasa 3’UTR of several teleost were analyzed,and the key factors involved in localization were discussed.The zebrafish vasa 3’UTR with early localization was rich in localization elements,which could mark PGCs of various fishes.However,teleost that had lost their early localization,such as red seabream(Pagrus major)and marine medaka(Oryzias melastigma)vasa3’UTR could not label the PGCs of zebrafish and turbot.However,black rockfish vasa 3’UTR had three important conserved elements(M10,M12,M19)of zebrafish,which might be involved in marking zebrafish PGCs.3.Transplantation of spermatogonial stem cells within PleuronectiformesSpermatogonial stem cells(SSCs)were isolated and purified from the cryopreserved whole testes of Japanese flounder,summer flounder and turbot,then labeled with PKH-26,and intraperitoneally transplanted into triploid Japanese flounder larvae within 22 days post hatching,exploring the cell transplantation of genetic relationships from from near to far(intra-species,intra-genus,and inter-family).Fluorescence tracking revealed that donor cells almost all migrated to the primordial genital ridge at 14 days after transplantation(14dpt),and were randomly distributed and incorporated into the developing recipient gonads at 50 dpt;Histology showed that in intra-species and intra-genus transplantations,the recipients differentiated into male and female chimeras,while in in inter-family transplantation,the recipients differentiated into male and intersex chimeras;The In situ hybridization and immunohistochemistry identified the survival and proliferation of turbot germ cells in the triploid Japanese flounder recipient.It also proved that the intersex chimera included not only early oocytes and male germ cells of Japanese flounder,but also the spermatogonia and spermatocyte of turbot.Subsequently,the donor-derived spermatozoa and its function were further confirmed by scanning electron microscopy,PCR,and artificial insemination.Moreover,the intersex chimera could mature and produce functional turbot spermatozoa.Based on the above identification,the transplantation efficiency was turbot-Japanese flounder(33-50%),summer flounder-Japanese flounder(75-95%)and Japanese flounder-Japanese flounder(100%).This firstly realized inter-family transplantation in marine fish species and shortened the maturation time of turbot spermatozoa.4.Transplantation of spermatogonial stem cells within ScorpaeniformesThe SSCs were isolated from the cryopreserved whole testis of black rockfish,labeled with PKH-26,and then intraperitoneally transplanted into allogeneic hatched larvae at 5-10 days post hatching.Fluorescence observed that the donor cells migrated to the genital ridge at 20dpt(100%),and incorporated into the recipient gonad at90dpt(90%).Histology showed that the recipient gonad was normally differentiated,and the morphology was not significantly different from that of the control.One year after transplantation,29 recipients were identified by microsatellite,and the chimerism rate of intra-species transplantation reached 82.76%.At the same time,the SSCs of other species in Scorpaeniformes,such as Hwanghae Rockfish(Sebastes koreanus)and marbled rockfish(Sebastiscus marmoratus),were transplanted into black rockfish to explore the inter-species transplantation of viviparous fish.Through fluorescence tracking,it was found that the donor cells migrated to the genital ridge of the recipient at 15 dpt,with a migration efficiency of more than 60%.