The Mechanism Of Origin, Proliferation And Migration Of Primordial Germ Cell And High Temperature Effect On Masculinization In Olive Flounder(Paralichthys Olivaceus)

Sex determination of fish is dependent on both genetic and environmental factors. In fish, temperature is one of the most important sex-determination factors. Previous studies about the mechanism of masculinization effect of high temperature on flatfishes mainly focused on the hypothalamic-pituitary-gonadal axis. Recently, it has been demonstrated that the primordial germ cells(PGCs) play import role in sex determination in model fish species. PGCs-ablated fishes develop as sterile males. Thus, questions associated with the effect of high temperature on germ cells need to answer, including: the effect of high temperature on migration and proliferation of PGCs in flatfishes; the changes in morphology, number and the gene expression of germ cells during sex differentiation under high temperature, the interaction between germ cells and somatic cells under high temperature, which results in the formation of testis. This study taken genetic female olive flounder(Paralichthys olivaceus)(XX) under high temperature treatment as the research object. 1) the migration and proliferation of PGCs; 2) dynamic changes in the number, morphology and gene expression; 3) endocrinological pathway, will be carried out to reveal the development process of germ cells and somatic cells and elucidate the mechanism of masculinization under high temperature. The results of the study will enrich the basic knowledge of fish sex differentiation, improve the regulation mechanism of reproduction and development in flatfishes. The main results are as follows:1) We cloned the full-length c DNA of dnd homologue and investigated its temporal and spatial expression pattern during embryogenesis and gametogenesis. A phylogenetic comparison of the peptide sequences of DND with other typical vertebrate showed that DND from olive flounder and other teleosts were grouped into a single lineage. It was demonstrated that expression of Podnd transcripts was exclusively restricted in germ cells but not in somatic cells by real-time quantitative PCR and in situ hybridization. In ovary, the dnd signal diffused evenly in the ooplasm in oogonia, stage II and III oocytes, with the strongest signal in stage II, and can’t be detected in stage IV and V; In testis, obvious signal was present in spermatogonium, weaker in spermatocyte, and undetectable in spermatid and spermatozoon. Furthermore, the highest expression of Podnd during embryonic development presented at the 2-cell stage, gradually decreased through subsequent development and reached the lowest at late gastrula stage till undectable. Generally, the Podnd transcrips localization and migration were similar to zebrafish. The detailed mechanism of dnd m RNA localization and PGCs origination and migration should be investigated in the future;2) The full-length c DNA of sdf-1 and cxcr4 were cloned from P. olivaceus. The analysis indicated these two genes were both hightly conserved. Sdf-1 and cxcr4 mainly expressed in gastrul stage and somites stage, in accordance with the PGCs migration characteristics, and under different temperature treatment, the expression of the above two factors have a significant change. The results indicated that they may involve in the regulation of the migration process;3) androgen(17α-methyltestosterone), high temperature(28 ℃) treated the larval at total length between 1.2-1.3 cm, both can induce masculinization:the control group(XX), the female ratio was 94.44%; androgen treatment group, the male ratio was 92.31%, at the same time significantly inhibit the growth of the juvenile fish; high temperature treatment group, the male ratio was 95.24%. There was no significant difference compared with the control group at the total length, that is to say, there was no necessary relationship between the total length and phenotypic sex during the sexual differentiation stage induced by high temperature;4) The number of germ cells and proliferation pattern presents dimorphism in the process of sexual differentiation: the quantity of germ cells toward the female differentiation group is significantly higher than the male. At the same time, the male apoptosis was significantly higher than that of females, apoptosis of male was strip type and the female was dots scattered type. The different features of apoptosis may participate in the differences in germ cell number between phenotypic sex;5) The gene of dnd and amh presented significantly sexual dimorphism during sex differentiation. The expression of dnd in female dominant population signigicantly higher than that in male dominant population while the expression of amh in female dominant population signigicantly lower than that in male dominant population. The result suggest that they may play an important role for the direction of the sexual differentiation.