Anti-mullerian Hormone Gene Of The Half-smooth Tongue-sole (Cynoglossus Semilaevis): Cloning And Its MRNA Expression

In fish breeding, gonad-related gene function and transgene studies are the mostimportant research methods in aquaculture. The half-smooth tongue-sole (Cynoglossussemilaevis), which is a specific economic fish in China, has long been one of the mostimportant farmed fish, it is also found male and female growth differences in one of fish.There is a natural sex reversal of the dioecious species in the half-smooth tongue-sole,sex determination type is ZW type, which is an ideal model system for sex research.Anti-Mullerian hormone (anti-Müllerian hormone, AMH), also known as Müller’stubes inhibitor (Müllerian inhibiting substance, MIS), is based on the peptides exist inthe form of cytokines belonging to the transforming growth factor β(TGF-β)superfamily, it widely distributed in vertebrates and invertebrates. AMH gene in malegonadal differentiation period inhibited the formation of Muller’s tube, to prevent thedevelopment of female reproductive organs, leaving Wu husband’s tube develops intothe male genital organs, is an important factor in sex differentiation.In1986the gene was cloned from Cate, etc. for the first time since, has been ofgreat concern. Anti-Müllerian hormone contains all of the structural components whichare representative to the superfamily, including a AMH-N region, TGF-β domain,and anactive C-terminal region including9conserved cysteine residues.In this paper, the half-smoothe touge sole was taken as research object, we clonedAMH ORF cDNA,DNA and its promoter. The open reading frame (ORF) of thehalf-smoothe touge sole AMH gene was1563bp long encoding520amino acids (notincluding the stop codon, with the signs of structure in the TGF-β superfamily.Amplified genomic sequence of the size of2855bp, with an open reading frame cDNAsequence comparison, found AMH gene contains7exons and6introns, with thetilapia and zebrafish AMH structure similar. The promoter area of AMH gene contains aTATA box, and there exists SRY, GATA-1, Sox5binding sites, which was related to sex.Real-time quantitative PCR (real-time PCR) analysis showed that the half-smoothtongue-sole AMH mRNA was preferentially expressed in testis, however it was notclearly in ovarys. Highest expression was detected at70dph. AMH mRNA wasexpressed higher in reversal gonad than in normal gonad. There is a close relationshipbetween AMH mRNA and the development of gonad. It is maybe the importanttranscription factor in promoting the development of testis.By in situ hybridization, expression of AMH mRNA was mainly observed in gonads.At the adult stage, AMH mRNA was expressed in granulose cells surrounding theoocytes, and expressed in Sertoli cells surrounding spermatogenic cysts. Whereas, inthe ovary of Japanese eel and medaka AMH mRNA was not detected, but was detectedin zerbfish,which shows that in different species AMH may play a different role.