| Sex determination of most fish could be affected by the gender gene and the environment. Eventually the fish would differentiate into the phenotypic sex that is inconsistent with the genotypic sex. The results of most experiments have showed that the proportion of estrogen and androgen was the direct cause to phenotypic sex in gender formation stage of fish. Aromatase inhibitors (Aromatase inhibitors, AI) could cause masculinization of red grouper, also downregulate of Cypl9a gene and masculinization of salmon, rainbow trout, tilapia and zebrafish in larvae. But it showed inconsistent results in the same testing of medaka. Thus, the mechism of sex forming was complex in fish. The unisexual cultivation of fish is an important way to improve efficiency in the aquaculture industry. Not only the mechanism of masculinization induced by aromatase inhibitors was foundation of gender formation in fish, but also had significance for practical production. It would be need more experimental data to be demonstrated.Takifugu obscurus was a rare freshwater fish of the middle and lower reaches of the Yangtze River in China. But its toxic in ovaries, liver and blood have influenced on the Commercial value and the development of farming. Male of Takifugu obscurus in the artificial breeding conditions was less toxic, and androgenesis breeding has good prospects. In this study, Takifugu obscurus larvae were taken as the experimental objects, and Cypl9a, Dmrtl, Foxl2, Cypllb gene cDNA sequence were cloned respectively in gonad of Takifugu obscurus. Takifugu obscurus larvae (7dah) were exposed to aromatic inhibitors letrozole (O,25,125,625,3125μg/L), exposed for49days. Detection of Cypl9a and Dmrtl genes expression and gonadal histological changes in experimental fish larvae. Provide the basic data to explore the feasibility of androgenesis induced in Takifugu obscurus.1Cloning and analysis of Dmrtl, Cyp19a, Foxl2, Cypllb genes cDNA partial sequence in gonad of Takifugu obscurus In this study, according to the existing Genbank nucleotide sequence in fish, Degenerate primers of Cyp19a,Dmrtl,Foxl2,Cyp11b and β-actin gene were designed in the conserved region, respectively. And the cDNA sequence of gonadal Cypl9a, Dmrtl, Foxl2, Cyp11b genes were cloned in Takifugu obscurus, respectively.The cDNA partial sequence of ovarian Cyp19A,Foxl2were cloned in Takifugu obscurus, respectively, the Cyp19a cDNA partial sequence was836bp, open reading frame encoding277amino acid. Containing the conserved I-helix, aromatase specific conserved region and heme-binding domains. Result indicated the phylogenetic relationship between Takifugu obscurus and Takifugu rubripes was very similar, comparing their partial sequence, homology was100%. Yet had the genetic relationship with human, mouse, frog, chicken, the Foxl2cDNA partial sequence was501bp, Open reading frame encoding166amino acids, including105amino acids encoded the FH domain (FH-domain). Result indicated the phylogenetic relationship between Takifugu obscurus and mammalian was very similar, comparing their FH domain, homology was96%.In addition,cDNA partial sequence of Dmrtl,Cypllb were cloned from the Takifugu obscurus testis, respectively, the Dmrtl cDNA partial sequence was605bp, Open reading frame encoding200amino acids, containing66amino acids encoded conservative DM box (DM domain). Result indicated the phylogenetic relationship between Takifugu obscurus and Takifugu rubripes was very similar, comparing their partial sequence, homology was99%.Yet had the genetic relationship with human, mouse, chicken, the Cyp11b cDNA partial sequence was538bp, Open reading frame encoding156amino acids, Result indicated the phylogenetic relationship between Takifugu obscurus and other fish was very similar, comparing their Corresponding partial sequences.2The effects of LE on the gonadal differentiation in Takifugu obscurus larvaeHistologically, In42days after hatching, the control and the low LE concentration of25μg/L groups, gonadal differentiation has been completed in Takifugu obscurus, the testes and ovaries were about50%, respectively. With LE concentration increasing, the percentage of the females decreased, At LE concentration of125μg/L group, the percentage of the females dropped to30%, about20%of the samples showed intersex from female to male reversal; At LE concentration of625μg/L group showed no typical ovarian tissue. The percentage of the female was reduced to0%, the percentage of intersex were37%, and the remaining were testis; At the highest LE concentrations of3125μg/L group showed no typical ovarian structure,20%of the samples showed intersex,80%of the samples were testes. In56days after hatching,100%of the samples displayed male in the highest LE concentrations group of3125μg/L.3The effects of LE on expression of Cyp19a and Dmrtl in Takifugu obscurus larvaeRT-PCR results have showed, Obvious expression of the Cyp19a gene in female, the apparent expression of the Dmrt1gene in male. At the LE concentration of125μg/L group, the sample number of single expression of the Cyp19a reduced to about20%, about30%of samples were co-expression of Cyp19a and Dmrt1genes. With LE concentration increasing, the sample number of single expression of Cyp19a decreased.when the LE concentration of625μg/L and125μg/L groups, expression rate of Cyp19a have dropped to0. The highest LE concentration of125μg/L group, the sample number of single expression of Dmrt1from70％(42dah) up to100%(56dah).4The effects of LE on the expression level of Cyp19a and Dmrt1in Takifugu obscurus larvaeTake β-actin as a reference, At the end of the experiment (56dah), the calculation of Cyp19a and Dmrt1relative expression levels during all experimental groups in Takifugu obscurus gonadal. The results showed that:LE could downregulate gonadal Cypl9a gene expression in Takifugu obscurus, and there was a dose-dependent inhibition of the relationship between the LE concentration,At the LE concentration of125μg/L and625μg/L groups, Cyp19a gene expression was significantly lower than female of control group; At the LE concentration of3125μg/L group, Cypl9a gene expression similar to the male sample of the control group during all samples; Dmrtl gene expression upregulated with the rise of LE concentration increasing; At above concentration of625μg/L LE groups, Dmrtl expression of the male samples have no significant difference with the control group during all samples.This experiment showed that the aromatase inhibitor LE could lead to masculinization reversed in Takifugu obscurus larvae, the reversal percentage have a connect with concentration of letrozole. The reversal process resulted in the endogenous hormone balance change. |
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