Comparative Studies About The Expression Of Vg, Sox9, Sox9a2, Dmrt1 Gene In Diploid And Triploid Female Rainbow Trout (Oncorhynchus Mykiss)

Rainbow trout(Onchorynchus mykiss) are one of the most important cold-water fish in the world, in order to enhance the quality of the products, recently, cultivation of the artificial-induced triploid rainbow trout has become a tendency. Because of the polyploidy, the mechanisms of reproduction of triploid rainbow trout have changed, even lead to the results of the infertility of triploid female rainbow trout. Gonad is the foundation of the breeding of fish, even when we really know the principle of gonadal development and gamete production, could we make good control of the fish reproduction. Although there were some studies about the gonadal development of triploid rainbow trout, they nearly all concentrated on the histological, cytology, reproduction and endocrinology, especially, there were lack of studies at the level of molecule biology.When compared with the diploids, the ovaries of triploid female rainbow trout have underwent the unusual ways. The parth of development of ovaries of triploid female rainbow trouts was as follows: Oogoniums→Oocyte before the vittelogenices→the infertility oocyte+the cluster of oocyte→the cluster of oocyte+ Similar spermatocyte srimary→Similar spermatocyte srimary. The ovaries of triploid female have the trend of masculinization at the middle and end stage of development. We sampled the rainbow trout at 10 and 20 month old after fertilization, following the methods of flow cytometry to identify the ploidy of fish, fixed the tissue sampled with boens'fixtive and with the usual procedure of paraffin slicing to study the development of gonad. Ef1a gene was choosed as control gene, using the technology of real-time PCR to probe into the relative expression of the target gene of Vg, Sox9a2, Sox9, Dmrt1 in diploid and triploid female rainbow trout. All are aimed at finding out the reason for masculinization of triploid female rainbow trout.The results had showed that:1.When compared with diploid counterparts, the ovaries development of triploid female rainbow trout of 10 month old were not seen something unnormal. While the ovaries development of 20-month old was abnormal with the structure of cluster of Oogonium and Similar-spermatocyte-srimary co-existed together. And there were most of zone of illness of gonad because of the vacuole,dissoluttion,nacreous of the reproductive cells. 2.There were little autosynthes Vg in ovaries of triploid female rainbow trout in 10 and 20 month old. Duing to the de-feminazation of triploid female rainbow trout in 10 month old, the relative expression level of Vg were low and with the levels of 0.12 times lower than diploid conterparts, and the diversity was significant(p<0.05). While in 20 month old, because of the rapid speed of masculinzation of triploid female rainbow trout, the relative expression of Vg was rather low with 0.05 times lower than diploid, and the diversity was significant(p<0.05).3. It seemed that there were no direct relationship between sox9a2 gene and the gonadal differenciation of triploid female rainbow trout. The relative expression levels of sox9a2 in 10-month old triploid female rainbow trout were significantly lower than diploid conterparts(p<0.05) with 0.08 times lower than diploid. While in 20-month old, the relative expression of sox9a2 were still significantly lower than diploid(p<0.05) with 0.05 times lower than diploid. Obviously, there were no much changes of sox9a2 expression between 10 and 20 month old rainbow trout.4. Sox9 gene had some connections with gonadal differenciation of triploid female rainbow trout and it was insisted that it should play as a role of down-stream gene. The relative expression of sox9 gene in 10-month old triploid female rainbow trout were significantly(p<0.05) higher with the relative expression levels of 7.16 times than diploid couterpart. while in 20-month old rainbow trout, although the relative expression levels of sox9 were lower compared with 10-month old, still they were 1.84 times higher than diploid counerparts and the diversity was significant(p<0.05).5. The expression of Dmrt1 gene had some relationships with the masculinization of triploid female rainbow trout and it was insisited that it should play as a role of up-stream gene. When compared with the diploids in 10-month old, with the process of masculinzation iniciation, the relative expression of Dmrt1 gene started to grow with the levels of 1.14 times higer than diploid(p>0.05). Duing to the rapid process of masculinzation in triploid female rainbow trout, the expression levels of Dmrt1 were significantly(p<0.05) increased with the level of 14.5 times higher than diploid. It was inferred that Dmrt1 gene might play as a up-stream gene to activate the down-stream gene of sox9, leading to the masculinzation of triploid rainbow trout.6. Although it were not seen something unusual of triploid female rainbow trout in 10-month old, it was concluded both from the expression levels and the trend of Dmrt1 and sox9 gene that the masculinization of triploid female rainbow trout began at 10-month old.