Molecular Cloning And Characterization Of Amh, Dax1and Cyp19a1a Genes And Their Response To17α-methyltestosterone In Pengze Crucian Carp

Pengze crucian carp, Carassius auratus var. Pengze endemic to pengze county, JiangxiProvince China, is a kind of triploid gynogenetic fish and reproduces by gynogenesis a specialreproductive model, which makes the offsprings possess a striking life cycle withmono-female development. The proteins AMH, CYP19A1A and DAX1encoded respectivelyby amh, dax1and cyp19a1a play important roles in gonad differentiation. Their functionshave been far less studied in teleosts. In order to study the roles of amh, dax1and cyp19a1a inthe development of fish gonad, in the present study, the full length cDNAs of amh, dax1andcyp19a1a were cloned and characterized in a triploid gynogenic fish, the pengze crucian carp.Full length cDNAs of amh, dax1and cyp19a1a of pengze crucian carp are1261bp,825bpand1922bp, respectively. The corresponding open reading frames (ORFs) of amh, dax1andcyp19a1a are1140bp,585bp and1557bp encoding379,194and518amino acids,separately. Multiple alignment and phylogenetic tree analysis indicated pengze crucian carpAMH, CYP19A1A and DAX1shared the highest amino acid identities (73.1%,98.1%and90.7%) with their counterparts of three cyprinidae species Danio rerio AMH, Carassiusauratus CYP19A1A and Danio rerio DAX1, respectively.Quantitative real-time PCR (qRT-PCR) was carried out to investigate the tissue-specificdistribution of amh, dax1and cyp19a1a in eight tissues of adult pengze crucian carp includingthe brain, eye, intestine, kidney, liver, muscle, ovary and spleen. The tissue distribution resultsindicated that amh and cyp19a1a were predominantly expressed in ovary and dax1wasdominantly expressed in liver. Meanwhile, Gene profilings of amh, dax1and cyp19a1a in thedevelopmental stages20-52days post hatching (dph) were obtained by the method ofqRT-PCR. Gene profilings in the developmental stages showed that all the three target geneshad a consistent highest expression at48dph. The period of48dph appeared to be a key timeduring the process of the gonad development of pengze crucian carp.In order to better understand the functions of amh, dax1and cyp19a1a in the process ofgonad development in pengze crucian carp, we investigated the changes of the three targetgenes expression patterns on conditions of100ng/L MT exposure during the earlydevelopmental period (20-62dph) of the fish. Results showed that100ng/L MT significantly increased the mRNA expression of amh at2-and4-week exposures and enhanced dax1andcyp19a1a at6-week exposure. The present study indicated that MT could influence the gonaddevelopment in pengze crucian carp by disturbing sex-differentiation associated geneexpression. Furthermore, the present study will be of great significance to broaden theunderstanding of molecular mechanisms of the physiological processes of the gonadalsex-differentiation and development in fish.