| In the process of artificial breeding,zig-zag eels(Mastacembelus armatus)usually occur the imbalance between sexes and abnormal gonadal development,restricting the efficiency of artificial propagation.It has been confirmed that both of aromatase(CYP19)and gonadotropins receptor were involved in steroid hormone biosynthesis pathways and played a crucial role in sex differentiation and gonadal development.They have been identified in different teleosts,but little is known about their mechanisms and regulatory relationships involved in gonadal development in zig-zag eels.In this study,we obtained immature(stage II)and mature(stage IV)gonad samples of the zig-zag eels by histological analysis,and the gene transcripts of testes and ovaries at different stages during the maturation process were analyzed using high-throughput sequencing.We prepared the brain type aromatase(Cyp19b)monoclonal antibody of the zig-zag eels for the first time and analyzed the changes of gonadal type aromatase(Cyp19a)and Cyp19 b during gonadal maturation at protein levels.The core c DNA sequences of cyp19 a,cyp19b,and luteinzing hormone / chorionic gonadotropin receptor(lhcgr1 and lhcgr2),and the 3’ untranslated region(3’UTR)sequences of lhcgr1 and lhcgr2 were cloned from the gonad and brain,respectively,and the mi RNAs regulating lhcgr1 and lhcgr2 were predicted and identified.This study is helpful to explore the unique physiological processes of gonadal development in zig-zag eels,providing a vital theoretical basis for revealing the reproductive mechanism.The results were as follows:(1)Gonadal transcriptome showed that 68 Gene ontology(GO)terms related to gonadal development were enriched,including cellular response to gonadotropin stimulus(GO:0071371),response to glucocorticoid(GO: 0051384),and developmental process involved in reproduction(GO: 0003006),etc.There were six KEGG signaling pathways directly related to gonadal development,including steroid hormone biosynthesis,cholesterol metabolism,relaxin signaling pathway,cytochrome P450 metabolism,and ovarian steroidogenesis.Ultimately,we screened 26 candidate genes related to gonadal development,including cytochrome P450 superfamily members(cyp17a1,cyp11a1,and cyp19a1)and hydroxysteroid dehydrogenases(hsd17b3,hsd11b2,and hsd17b1).(2)There were two isoforms of aromatase in the genome of zig-zag eels,namely Cyp19 a and Cyp19 b.The expression of Cyp19 a in the ovaries was higher than that of the testes at the protein level,while Cyp19 b was only expressed in the brain.There was no significant difference of cyp19 a expression between stage Ⅱ ovaries and testes at the m RNA level,but its in stage Ⅳ ovaries was significantly higher than that in stage Ⅱ ovaries(P<0.05),which was about 40 times higher.(3)There were two isoforms of luteinzing hormone / chorionic gonadotropin receptor in the genome of zig-zag eels,namely Lhcgr1 and Lhcgr2.lhcgr1 was expressed to varying degrees in the most tissues,whereas lhcgr2 was only highly expressed in the mature gonads.q PCR and transcriptome results showed that the expression of lhcgr1 and lhcgr2 increased significantly as the ovaries developed from stage Ⅱ to stage Ⅳ(P<0.05).The expression of lhcgr1 in stage Ⅳ ovaries was significantly higher than that in stage Ⅳ testes(P<0.05),while the expression of lhcgr2 in stage Ⅱ ovaries was significantly lower than that in stage Ⅱ testes(P<0.05).(4)The 3’ UTR of lhcgr1 and lhcgr2 of zig-zag eels were cloned and predicted that lhcgr1 and lhcgr2 were regulated by 33 and 28 mi RNAs,respectively.Both of mi R-30-y and mi R-737-x targeted lhcgr1 and lhcgr2,but mi R-9-z targeted lhcgr2.As the gonads developed from stage Ⅱ to stage Ⅳ,the expression of mi R-30-y was increased in the ovaries and testes.But the expression of mi R-737-x was increased only during the testes maturation,with no significant change in the ovaries.The expression of mi R-9-z was decreased during the ovaries maturation,while increased in the testes. |
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