Molecular Cloning And Sequence Analysis Of Southern Catfish Androgen Receptor

Androgen receptor(AR),a ligand-dependent transcription factor,belongs to a large family of nuclear receptors.Molecular cloning and structure/function analyses have revealed that the members of the superfamily share a similar structure mainly consisting of three functional domains:a variable N-terminal domain(NTD),a highly conserved DNA binding domain(DBD) and a Ligand binding domain(LBD).AR,the mediator of androgens,plays incisive roles in the development and the maturation of the animals.In some fish species,androgen treated during the sensitive period of sex determination and differentiation can reverse the sex phenotype.However,previous experiments from our laboratory revealed that the androgen-treatment had no effects on sex differentiation of the Southern catfish,Silurus meridionalis.In order to elucidate the mechanism of the phenomenon at the molecular level,the cDNA of Southern catfish AR(scAR)was isolated by reverse transcription polymerase chain reaction(RT-PCR)followed by rapid amplification of cDNA ends (RACE).The scAR cDNA contains 3116 bp encompassing a 5'UTR of 72 bp,an open reading frame(ORF)of 2415 bp and a 3'UTR of 629 bp including the poly(A)tail.The ORF encodes a protein of 804 amino acids.Analysis on the expression pattern of scAR mRNA revealed that it was expressed in all tissues examined,including brain,pituitary,gill,heart,liver,intestine,spleen, kidney,testis/ovary and muscle.In order to elucidate the phylogenetic relationship among Southern catfish and other 7 Siluriform fishes(S.asotus,C.gariepinus,P brevicaudatus,P. truncates,P.vachelli,L.longirostris and M.macropterus.),the 3' end of each AR cDNA was cloned and phylogenetic tree based on the deduced C-terminus of these ARs was constructed.As fish experienced the fish-specific genome duplication(FSGD)during evolution,two ARs coded by two different genes were presumed to exist in fish species.We constructed a phylogenetic tree using the deduced amino acid(aa)sequence of scAR and AR aa sequences obtained from GenBank.The result revealed that ARs can be devided into three clades,the tetropod AR,fish AR-αand fish AR-β.The scAR,together with the ARs from the goldfish, zebrafish and the fathead minnow,clustered in the fish AR-βbranch.With the analysis of known fish ARs,we presumed that there might be only one functional AR existed in the Siluriform and Cypriniform fishes.The reasons are as follows:1)Up to now,only one AR had been isolated in these Cypriniform fishes and Siluriform fish(Southern catfish);2)analysis of zebrafish genome revealed that there are one AR on Chromosome 5 and one on Chromosome 14,respectivly. However,the NTD of the AR on Chromosome 14 is missing,and the similarity of the two ARs is up to 99%(NTD excluded);3)the expression pattern of scAR is similar to that of zebrafish AR which can be detected in all the tested tissues,whereas in fish species with two different ARs,ARαand ARβshowed different expression patterns in certain tissues only;4)no fragment of ARαcould be amplified from the Southern catfish using primers designed at the conserved regions of fish ARα.Multi-alignment of scAR and those from other vertebrates revealed that the scAR possessed the variable NTD and the highly conserved DBD and LBD.Interestingly,an extended sequence of 20 aa which is absent in all the other ARs detected at the C-turminus in scAR.This is caused by a 4-bp insertion before the stop codon(TGA)common in other vertebrates,which lead to the extension of the ORF to the present stop codon(TAA)of scAR.In order to make clear whether the phenomena generally exists in Siluriform fishes,fragments containing the C-terminus of ARs from 8 Siluriform species which belong to 6 genera of Siluridae,Bagridae and Clariidae were amplified and sequenced.Analysis of these sequences revealed that the extended sequence existed in all Siluriform fishes investigated.Therefore,we presumed that these extra sequences might be existed in all Siluriform fishes specifically.Moreover,comparison of the partial AR sequences from 5 bagrid catfishes(P.brevicaudatus,P.truncatus,L.longirostris,P.vachalli and M.macropterus) revealed that the 4 bp insertions and the extended sequences are exactly the same in ARs of P.brevicaudatus,P.truncatus and L.longirostris,while different from those of P.vachalli and M. macropterus,suggesting that P.brevicaudatus and Ptruncatus should be grouped in the genus Leiocassis as proposed by some other Chinese researchers.This was further confirmed by phylogenetic analysis of ARs of Siluriform species investigated in the present study.It has been reported that sex reversal was successfully obtained in several teleost,such as tilapia and rainbow trout,by androgen administration on the fry during sex differentiation via inhibiting the expression of aromatse(encoded by Cyp19a),which is highly responsible for estrogen synthesis.However,different from the situations in many other fishes, methyltestosterone(MT)was found unable to induce female-to-male sex reversal in Southern catfish.In order to shed light on the molecular mechanism of this phenomena,all female catfish fry were treated with MT from 5 to 25dah and the gonads were dissected immediately;five month-old fishes were given intraperitoneal injection with MT and the gonads were collected 7 days later.Then the expression of Cyp19a mRNA under MT treatment was examined.The result revealed that MT treatment did not show significant effect on the expression of Cyp19a,indicating that the production of estrogen can not be inhibited.This might be one of the reasons why the female-to-male sex reversal can not be obtained by MT treatment in Southern catfish.Whether this is caused by the specifically extra aa sequences of AR that Southern catfish contains,further studies are needed.