Studies On Function Of Vasa、Follistatin And Anti-Mullerian Hormone In Reproductive Cycle Of Cynoglossus Semilaevis Günther

Half-smooth tongue sole (Cynoglossus semilaevis Günther) is a Chinese inshorespecies with great potential for aquaculture.This paper mainly study on the functionof Vasa,follistatin and anti-mullerian hormone in reproductive cycle of Half-smoothtongue sole through molecular biology and mRNA in suit hybridization technology,aiming to clarify their function in gametogenesis of fishes. The main results andconclusions are as follows:1. Cloning of Vasa and expression analysis during the reproductivecycle in Cynoglossus semilaevis GüntherFull sequence of Vasa gene was cloned from Cynoglossus semilaevis. The lengthof the cDNA is2602bp and this gene encodes a protein of722amino acids. Analternative isoform of Vasa was found. The deduced amino acid sequence contains8conserved motifs of DEAD-box family protein,5RGG repeats and10RG repeats inN-terminal region. Tissues expression analysis showed that csVasa mRNA expressedstrongly in gonad and little in heart.Expression patterns of csVasa in reproductive cycle studied by methods ofRT-PCR and mRNA in suit hybridization. The transcript level of Vasa gene in theprimary yolk stage and tertiary yolk stage is higher than peri-nucleolus stage andatretic follicles stage in ovary. The transcript level of spermatogenesis stage isobviously higher than that of spermatocyte stage and spermiation stage. Theseresults suggest that Vasa gene may play an important role in spermatogenesis andoogenesis during reproductive. Vasa mRNA in suit hybridization in primary yolkstage ovary showed that Vasa mRNA were dispersed uniformly throughout the cytoplasm, and strong at cortical region in stage Ⅲ oocytes.2. Cloning of follistatin and expression analysis during thereproductive cycle in Cynoglossus semilaevis GüntherFull sequence of follistatin gene was cloned from Cynoglossus semilaevis withRACE technology. The length of follistatin cDNA is1455bp, and this gene encodesa protein of321amino acids, which belongs to TGF-βfamily. Tissues expressionanalysis showed that follistatin mRNA expressed in all13tissues detected, stronglyin gonad and less strongly in gill and brain.This result shows follistatin has a widefunction.Expression patterns of follistatin in adult Cynoglossus semilaevis reproductivecycle studied by methods of RT-PCR. The transcript level of follistatin gene in theprimary yolk stage is highest, in tertiary yolk stage and peri-nucleolus stage ishigh.We concluded that follistatin suppresses Follicle-StimulatingHormone(FSH),which stimulates the development of oocyte. No evident relationshipwas found with LH. In testes, follistatin geng is expressed in all stage. Expressionlevel is lower in spermatogenesis stage, which may result from the regulationfunction of follistatin to aromatase.3. Cloning of anti-mullerian hormone and expression analysis duringthe reproductive cycle in Cynoglossus semilaevis GüntherPart of anti-mullerian hormone gene sequence was cloned from Cynoglossussemilaevis with RT-PCR technology. Its length is955bp, encodes318amino acidsand belonged to TGF-β family. Tissues expression analysis showed that AMHmRNA expressed only in gonad, and in testis is richer than that in ovary.Expression patterns of AMH in adult Cynoglossus semilaevis reproductive cyclestudied by methods of RT-PCR. The transcript level of AMH gene in tertiary yolkstage and peri-nucleolus stage is high, and highest in atretic follicles stage. Itsuggests that AMH can suppress Follicle-Stimulating Hormone(FSH) in fish just likein mammal. Besides, AMH may play a role in suppresses the expression of LHR in Female ovary during reproductive cycle. In testes, the transcript level of AMHreaches to summit in spermatocyte stage, decreases in reproductive season. This iscoincide with mammal AMH is suppressed by testosterone in adolescence. Itsuggests that AMH in fish is also suppressed by testosterone. Besides, AMHpossesses a physiological function of restraint spermatogenesis...