Study On Specification And Development Of The Primordial Germ Cells In Two Marine Fish Species By Vasa/dnd Gene

Primordial germ cells are the precursors of gametes responsible for genetictransmission to the next generation. The studies on specification and development ofprimordial germ cells are essential for genetic resource conservation and fishreproduction. However, little is known in the marine fish species. Turbot(Scophthalmus maximus) and red seabream (Pagrus major) are important marineaquaculture species in China. However, genetic resource reccession has brought downthe efficiency of aquaculture, due to rapid development of the industry. Therefore,conservation of genetic resource is increasingly demanded. In this study, we clonedthe vasa and dnd homologue gene from turbot and analyzed the expression profileduring germline development to reveal the origin and migration of turbot PGCs. Inaddition, we investigated the vasa expression during embryogenesis andgametogenesis in the red seabream. Furthermore, we isolated the vasa transcriptionalregulatory sequence from the red seabream and reported the functional analysis of theregulatory sequence in model fish. The results provide the basic knowledge of germcell specification and development in the two species, and will facilitate the germ cellmanipulation in those species. The main results are as follows:1) The full-length cDNA of vasa and dnd homologue were cloned from the turbot.Turbot vasa and dnd transcripts were exclusively detected in germ cell lineage byRT-PCR and in situ hybridization. Whole mount in situ hybridization showed thatthe specification mechanism of PGCs in turbot was similar to zebrafish. TurbotPGCs derived from the4cells inheriting the maternal germ plasm at32-cell stage.The PGCs underwent2-3mitoses and migrated to the presumptive genital ridge during embryogenesis.2) The turbot vasa transcripts were localized at the cleavage furrows during earlyembryogenesis, indicating that not all vasa lose the localization function inEuteleostei. After comparison of vasa expression patterns during oogenesis andearly cleavage stage in several fish species, we proposed that three modes of vasalocalization may exist in teleosts. The representative species were zebrafish, turbotand medaka, respectively.3) Sexually dimorphic expression of vasa and dnd was found during gonadaldevelopment in turbot by real-time PCR. The expression of vasa and dndincreased gradually during the testis development. This expression wassignificantly up-regulated when the spermatogonia proliferated and entered intomeiosis. The expression of vasa and dnd in ovary was significantly higher thanthat in testis before sex maturation. However, the expression in testis exceeded theovary during the spawning season.4) Whole mount in situ hybridization indicated that vasa transcripts were widelydistributed in all blastomeres during early embryogenesis in the red seabream.And vasa-positive cells couldn’t be detected until late gastrula. This expressionpattern resembled the medaka vasa during embryogenesis, suggesting that redseabream share a similar mechanism of PGCs specification with medaka.5) The transcriptional regulatory sequence was isolated by genome walking from thered seabream. It was showed that injection of GFP-Pmvas3’UTR mRNA was ableto label medaka PGCs during embryogeneis. Besides, we have constructedpPmvasGFP transgenic vector and established a stable transgenic medaka line,exhibiting GFP expression in germ cells including PGCs, mitotic and meioticgerm cells of both sexes under the control of Pmvas transcriptional regulatorysequences.