| Primordial germ cells(PGC)are progenitor cells of germ cells capable of producing egg and sperm,which provides the basis for reproduction.Studies have shown that the number of PGCs in zebrafish,killifish,blackfish and crucian carp plays an important role in gender determination and differentiation.Meanwhile,the formation,migration,proliferation and differentiation of PGCs are determined by several conserved maternal germ plasm components,including dazl,dnd,nanos,piwi and vasa.In addition,PGCs isolation and transplantation provide effective methods for the restoration of endangered or valuable fish species.Therefore,the study of the development of PGCs has farreaching significance for reproductive development theory,conservation of germ plasm resources and breeding applications,and has become a hot research topic in the field of biological reproduction.Dnd is a highly conserved germ plasm component and plays a crucial role in PGCs development.Recently,medaka dnd has been defined as the first and the only PGCs specifier in fish,as its dosage has critical effects on PGCs number.Vasa gene plays important roles in regulating the formation and migration of PGCs.In fish,medaka vasa is necessary for PGCs migration but not for PGCs proliferation and survival,while zebrafish vasa is significant for PGCs maintenance and differentiation.Taken together,dnd and vasa directly affect the development of PGCs.Oryzias celebensis(O.celebensis)is a freshwater fish under the genus Oryzias,native to the rivers and lakes of Sulawesi(formerly known as Celebensis Island)and East Timor.The O.celebensis and Oryzias latipes belong to the same 14 species of genus,which have the characteristics of short growth cycle,transparent embryos and rapid reproduction.The adult O.celebensis can be up to 6 centimeters long,which about twice as large as medaka.So far,the studies of O.celebensis are limited to sex determination systems,sex chromosomes and sex determination sites.However,there are few studies on germ cell-specific genes.In this study,the cDNA sequences of the O.celebensis dnd and vasa were obtained by RACEs.The expression patterns of dnd and vasa in embryos and gonads were analyzed by RT-PCR,chemical in situ hybridization and dual color fluorescent in situ hybridization.The 3'UTR of dnd and vasa were fused with green fluorescent protein(GFP)to label PGCs.dnd knockdown and overexpression were used to study the specialization effect of PGCs.At the same time,medaka vasa transgenic fish was used as the donor for the early exploration of germ cell transplantation.Here,the cDNA of the O.celebensis dnd(Ocdnd)was obtained with a length of 1125 bp encoding 373 amino acids(including a stop codon).The cDNA of the O.celebensis vasa(Ocvasa)is 1848 bp in length and encodes 615 amino acids(including the stop codon).Amino acid homology alignments and phylogenetic tree results show that Ocdnd and Ocvasa are highly evolutionarily conserved and belong to the same family as medaka.RT-PCR analysis shows that Ocdnd and Ocvasa are specifically expressed in adult gonads,and Ocdnd RNA is appeared at high levels until the morula and apparently decreases thereafter during embryogenesis.In gonads,chemical sections in situ hybridization results show that both Ocdnd and Ocvasa have higher expression levels in early stage of germ cells,and are completely absent in mature sperm and egg.Dual color fluorescence section in situ hybridization results demonstrate that the expression patterns of Ocdnd and Ocvasa in both sexes are very similar and could not be effectively distinguished.In embryos,chemical section in situ hybridization reveals that the expression of Ocdnd and Ocvasa was significantly different in the early stage of embryonic development.By dual color fluorescent in situ hybridization of embryos,we find that Ocvasa has a wide expression or distribution before the gastrula stage,but Ocdnd is localized in the germ plasm particles from one cell;after the gastrula stage,Ocdnd and Ocvasa are simultaneously located in germ plasm particles.Moreover,it is found by co-injection of gfp-Ocdnd/Ocvasa 3'UTR with rfpDrnos1 3'UTR are able to transiently label PGCs of O.celebensis.In order to knockdown the Ocdnd gene we synthesize the Ocdnd Morpholino(MOOcdnd),use the Oryzias latipes dnd(Oldnd)recombinant expression vector pCSch Oldnd and transcribe the mRNA to overexpress the gene as well.Ocdnd knockdown by MOOcdnd injection leads to the loss of PGCs and this can be rescued by co-injection of MOOcdnd and Oldnd mRNA.Moreover,overexpression of Oldnd mRNA can boost PGCs number.It is worth noting that this experiment successfully transplanted spermatogonial cells from the vasa transgenic strain of medaka into the germ cell-deficient O.celebensis.Therefore,it is vital for understanding the development of O.celebensis to conduct researches on Ocdnd and Ocvasa expression patterns and functions,which also helps to explore the signaling pathways of Ocdnd and Ocdnd,and lay the foundation for further study of allogeneic germ cell transplantation mediated by O.celebensis. |
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