| Anti- müllerian hormone(AMH) is a dimeric glycoprotein belonging to the transforming growth factor-?(TGF-?) superfamily. In mammals, the anti- müllerian hormone is responsible for the regression of the müllerian ducts during male gonadal differentiation period, and preventing the development of female reproductive organs. As an important factor of sex differentiation, AMH can regulate the development and differentiation of germ cells. Anti-müllerian hormone type II receptor(AMHRII) also belongs to members of TGF-? superfamily. AMHRII can play a role through the intracellular serine / threonine protein kinase catalytic domain when it binding to the AMH. In this study, amh and amhrII expressions were analyzed in kidney, brain, heart,spleen, liver, testis and ovary of mature Takifugu rubripes, and were analyzed at individual level during different times(23, 30, 40, 60, 80 days of age(da), and 2, 3 years of age(Y)) in T. rubripes by flurescence quantitative Real-time Polymerase Chain Reaction(qRT-PCR).The results showed the significantly higher expression of amh in spleen, kidney and liver, than that in other organs(P<0.01). The amh expression level of testis was significantly higher(P<0.01), than that in ovary and brain. And the expression of amh in spleen was the highest, and the lowest was brain. During the different periods(23, 30 days of age(da), and 2, 3 years of age(Y)) of T. rubripes, the male expression level of amh was higher than that in female individuals significantly(P<0.01). The female expression level of amh was higher than that in male individuals significantly(P<0.01) in 60 da and 80 da. No statistically significant difference was observed of expression level between males and females in 40 da. The expression of amh in 3Y was significantly higher than that in 2Y(P<0.01). The amh expression level in males increased from 23 da to 30 da and decreased in later stages. In females, the amh expression level increased from 23 da to 80 da and subsequently decreased in later stages. Amh gene may play an important role in gonadal development and sex differentiation of T. rubripes.AmhrII expression in gonads were significantly higher than other tissues(P <0.01), and the highest expression of the ovary, which is approximately 40 times the testis. The expression of amhrII in adult age 3 fish are significantly higher than juveniles(23, 30, 40, 60 and 80da), and the expression in ovary was significantly higher than testis(P <0.01). The amhrII expression level in female juveniles is low, and its express quantity rise rapidly in 60 da, and the expression decreased sharply in 80 da. The above datas show that amhrII in T. rubripes male sex differentiation, gonadal development and germ cell formation process may play a decisive role.In this study, we used the 3Y fishs' testis and ovary as a material for the transcriptome sequencing analysis. Through raw data filtering filtering, in the testis and ovary respectively 38,464,975 and 36,092,955 reads comparison to the T. rubripes genome database. In testis and ovary, a total of 21468 genes were assigned with one or more KEGG annotated, they mapped to 39 KEGG pathways of these total numbers of annotated genes. Total metabolic pathways were divided into six categories. Each path contains differentially expressed genes(DE gene), in which the two most number of genes for Signal transduction, Infectious diseases. In addition, filtered through Varscan all possible SNP locus on chromosome, in the testis and ovary samples found 23879 and 23879 cSNPs, respectively. Through the difference between samples cSNP statistics obtained in the testis and ovary, we found 9553 cSNP in testis and ovary exist, testis-specific cSNP 14326, and ovary unique cSNP 46122.By HTSeq statistical comparison to the value of the gene, and then analysis differentially expressed genes by DESeq, finally in accordance with the expression of multiple differences and express significant difference p-value screened differentially expressed genes were identified 680 differentially expressed genes, of which 124 gene expression in the ovary, 556 genes highly expressed in the testis. We selected agrp1, sstr2, foxn4(male high expression) and gdf3, gdf15(female expression) 5 differentially expressed genes, and the accuracy of the sequencing results of the transcriptional group was verified by the qRT-PCR method with three age fish as the template. The results of RNA sequencing and qRT-PCR are basically identical, suggests that the expression of the transcriptome sequences analysis more reliable.Study its expression in different developmental stages of T. rubripes.(1) The results shows that in the critical period of T. rubripes ovarian differentiation(about 35 days) agrp1 while highly expressed in the testis and ovary, testis is about 48 times the amount of the ovaries. From this we can speculate that agrp1 may be involved in ovarian differentiation process of T. rubripes.(2) Sstr2 mainly expressed in juvenile gonads(23da to 40da), and the expression of other periods are low. And sstr2 expressed in testis were higher than ovary, and all express the amount the highest in the 40 days of age. It can be speculated, sstr2 gene plays an important role in T. rubripes ovarian differentiation process.(3) Foxn4 expressed only in adults, and the expression of male in foxn4 was higher than female. Foxn4 genes involved rubripes testis and ovary development process.(4) Gdf3 expressed in the whole developmental stages of females, and the expression was low before 80 da, 2Y and 3Y were higher. The highest expression occured in 3Y ovary, and males only appear in their adult fish. It can be seen, gdf3 participate in the formation of gonads development and egg cells forming process, and may also play a role in ovarian upkeep.(5) Gdf15 always express at different developmental stages of T. rubripes, the expression of 40 da is the highest, and testis is about 48 times the amount of the ovaries, the expression of other periods are less. This indicates gdf15 may be involved in ovarian differentiation. |
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