Study On Sex Related Genes Sox9, Ftz-F1, And P450arom In Yellow Catfish, Pelteobagrus Fulvidraco

Yellow catfish (Pelteobagrus fulvidraco) is a valuable carnivore.that has good flesh quality.There is a market potential for this fish in China, Japan, Korea and SE Asia. In naturalwater, yellow catfish grows slowly, with a small marketable size, seriously influencing itsmarket development. In term of production, the most effective way to increase the yield andefficiency of yellow catfish consists in utilizing the obvious growth difference betweenmale and female yellow catfish, and to maintain mono-sexual cultures, since males grow30％faster than females. However, studies on the molecular mechanism of yellow catfishsex determination still remain unavailable. Especially studies on the integrative regulationmodel. Here we isolated and cloned Sox9 Ftz-F1 and P450arom cDNA, which played keyfunction in vertebrate sex differentiation and gonad development, using RT-PCR and RACEfrom yellow catfish. And then we analyzed their tissue expression pattern, the expressionlevels response to the gonad development period, and their relationship using fluorescentreal time RT-PCR andβ-actin as internal control. Wish. to reveal their tissue expressionmodel and the function in gonad development through our study.Sox9 is one of the important genes related to mammal sex differentiation and determination.Two cDNAs encoding sox9 were derived from brain, testis and ovary of yellow catfishusing reverse transcriptase-polymerase chain reaction (RT-PCR) and RACE. Sequenceanalysis revealed the cDNA from testis is as same as from brain, using Blast analysis wecalled it Sox9a1. The cDNA from ovary called Sox9a2. Yellow catfish Sox9a1 cDNA was 1604 bp with 70 bp 5'UTR, 142 bp 3'UTR(excluding poly[A]), and 1 392 bp ORF, whichencodes 464 amino acids and has a Predicted mol wt of 50.62 kDa. Thereinto the stretchfrom 99 to 176 of residues is the highly conserved sox-TCF_HMG-box, from 305 to 351 isPro-and Gin-rich (P/Q) region, 352 to 464 is TA region. Moreover, the Sox9a2 cDNA was1843bp with 313bp 5'UTR, 159bp 3'UTR[excluding poly(A)], and 1371 bp ORF, whichencodes 457 amino acids and has a predicted mol wt of 50.24 kDa. Thereinto the stretchfrom 111 to 188 of residues is the highly conserved sox-TCF_HMG-box, from 321 to 358is the P/Q rich region which is shorter than in Soxal and without the characteristic Pro-andGin-rich, from 359 to 457 residues is TA region which is shorter than Sox9a1. The whole sequence identity is 64％between Sox9a1 and Sox9a2, but the ORF of Yellow catfishSox9a1 shares 71％sequence identity with Sox9a2. The amino, acids sequence identity is65％between Sox9a1 and Sox9a2. Sox9a1 shares average 73％sequence identity withother fish speciesand animals including rice field eel (M. albus),common carp (C. carpio),medaka (O.latipes),rainbow trout (O.mykiss),zebrafish (D. rerio),red fin east puffer fish(T.rubripes),and X. laevis, A.mississippiensis, chicken (G. gallus), and human (H. sapiens),with the most smallest similarity 61％with zebrafish Sox9a. And Sox9a2 shares 67％sequence identity with above mentioned animals. So the identities between Sox9a1 andother animals were higher than those of Sox9a2 and other animals.The phylogenetic tree of Sox9 consists three obvious branches, one of which includingSox9 of amphibian species, reptilian specie, birds and mammals; teleosts Sox9 weredivided into two other.branches, one including common carp Sox9b, yellow catfish Sox9a2and zebrafish Sox9b, the other branch has two smaller branches, which comprised otherfishes Sox9a or Sox9a1 and Sox9b or Sox9a2 including rainbow trout, rice field eel,medaka, pufferfish separated, yellow catfish Sox9a1 and zebrafish Sox9a belong to thesetwo smaller branches respectively. According to the unrooted tree, the farthest distance wasbetween zebrafish Sox9b and other animals Sox9, and thenext is yellow catfish Sox9a2.Yellow catfish Sox9a1 also. had farther distance with Sox9a1 of rainbow trout, medaka andrice field eel which were in the same branch. The yellow catfish Sox9a1 was detected inbrain, heart, liver, spleen and gonad of female and male fish using RT-PCR, but not inmuscle, otherwise Sox9a2 was only detected.in ovary. This indicated that there were tissuesspecific expression pattern of Sox9a1 and Sox9a2 and there were differences in theirregulation mechanism.The fushi tarazu factor-1 (FTZ-F1) is a member of the nuclear receptor Superfamily andwas originally found as a regulator of the Drosophila homeobox segmentation gene FTZ.These genes were nominated as a sub-superfamily by nuclear receptor committee in 1999,and this sub-family have be classified two sub-group, one is NR5A1 including SF-1/Ad4BP(steroidogenic factor-1/adrenal 4 binding protein), another is NR5A2 including LRH/FTF(liver receptor hormone/a-fetoprotein transcription factor), which regulates a-fetoproteinexpression and involved in cholesterol metabolism. The mammal SF-1/Ad4BP genes areimportant regulators of steroid biosynthesis by controlling transcription ofmany P450enzymes including P450scc,3β-HSD and P450arom. They are expressed in steroidogenic tissues and are involved in the embryonic development of adrenals and gonads. In adultmammals, LRH-1 expression is confined principally to tissues of endodermal origin, suchas the liver, pancreas and intestine-justifying its functional classification as anenterohepatic NR. Recently, in several species LRH-1 was highly expressed in ovary. Thissuggests LRH-1 and SF-1 are involved in the ovarian steroidogenesis. Here we isolatedyellow catfish Ad4BP/SF-1 cDNA, which is 1 986 bp with 79 bp 5'UTR, 461 bp3'UTR(excluding poly[A]), 1 446 bp ORF, which encodes 482 amino acids and has apredicted mol wt of 53.9 kDa. The conserved DBD domain was in residues from 22 to 88,and Ftz-F1 box was in 89 to 116 residues. Yellow catfish LRH-1 cDNA is 1945 bp with207 bp 5'UTR, 238 bp 3'UTR[excluding poly (A)], 1500 bp ORF, which encodes 500amino acids and has a predicted tool wt of 57.19 kDa. DBD domain was in residues from39 to 105, and Ftz-F1 box was in 106 to 132. At the same time of isolation LRH-1, we alsogot an inactive LRH-1'in which the zinc-finger domain and AF-2 motif is absent. TheLRH-1'is 1663 bp with 283 bp 5'UTR, 330 bp 3'UTR, 1050 bp ORF, which encodes 350amino acids and had a predicted tool wt of 39.7 kDa. Yellow catfish Ad4BP/SF-1shares48％sequence identity with P450aromB, the sequence similarities are 60％in ORE Theamino acid sequence identity is 59％between LRH-1 and Ad4BP/SF-1. The LRH-1 shares79％-85％sequence identity with LRH-1 of other animals, but only 59％-66％identity withthe SF-1. however, yellow catfish Ad4BP/SF-1 shares 57％-96％sequence identity withSF-1 of other animals, and only 57％with human SF-1 which is lower than 60％betweenyellow catfish LRH-1 and human SF-1. RT-PCR analysis indicated yellow catfish LRH-1was detected in almost all tissues except muscle and heart. Unfortunately yellow catfishAd4BP/SF-1 was not detected using RT-PCR in all tissues used in our experiment. Maybethe expression of Ad4BP/SF-1 in adult fish was hardly detected or the primers used inexperiment were not proper.Aromatase cytochrome p450 (P450arom; the product of the CYP19 gene) is therate-limiting enzyme which catalyzes the conversion of androgens to estrogens. Here wereport the cloning of two types of cDNA encoding P450arom derived.from yellow catfish(Pelteobagrus fulvidraco) brain, and ovary, using RT-PCR, and RACE. The brain-derivedcDNA Was 2260 bp with 205 bp 5'UTR, 555 bp 3'UTR[exclUding poly(A)] and 1500 bpORF, which encodes 500 amino acids and has a predicted mol wt of 56 kDa. Moreover, theovarian cDNA was 1914 bp with 13 bp 5'UTR, 362 bp 3'UTR[excluding poly(A)] and1539bp ORF, which encodes 513 amino acids and has a predicted mol wt of 58.7 kDa. Yellow catfish P450aromA shares 49％sequence identity with P450aromB, the sequencesimilarities are 62％and 3％in ORF and 3'UTR respectively. The amino acid sequenceidentity is 60％between P450aromA and P450aromB. The P450aromA shares 61％-90％sequence identity with ovarian aromatases of other fish species, but only 60％identity withthe homologous P450aromB, 51％and 52％with chicken ovarian and human beingplacenta aromatases. And P450aromB shares 59％-85％sequence identity withbrain-derived aromatases of other fish species, about 50％with human being placenta andchicken ovarian aromatases. But the percentage of identity/similarity was higher in theregions of high homology, including theⅠ-helix, an aromatase-specific conserved regionⅡ,and the heme-binding regionⅢ, vs. human P450arom, which were 67％～96％, 78％～86％and 78％～100％respectively. Phylogenetic tree of the P450arom shows two main branches,one of them clusters all teleosts (bonny fishes) leaving apart the elasmobranches(cartilaginous fishes) and the other branch includes the tetrapods and the elasmobranches.The teleosts group bifurcates into two clear branches, one of them containing the brainvariants of aromatase and the other one the ovarian variants. It is acceptable thatduplication of an ancestral gene early in the teleostean lineage results in the fishP450aromB and A paralogs though there is no directive evidence. The yellow catfishP450arom are the closest with channel catfish. This result is consistent with theresult ofcontradiction classification. The fluorescent real-time quantity RT-PCR was developed toanalyze tissue-specific expression of P450arom B and P450arom A in adult yellow catfish,and measure mRNA relative expression leveIs withβ-actin as internal standard. B-isoformwas preferentially expressed in brain of both males and females but also present at muchlower levels in ovary, testis, however, A-isoform expression was restricted to ovary, andthere was no expression either A-or B-isoform in liver. The high to low order of P450aromB relatively, expression level in tissues was female fish hypothermia, male fishhypothalamus, female fish pituitary, male fish pituitary, male fish fore-brain, female fishfore-brain, ovary, testis. However, there was no significant difference of the totalexpression level in brain between male fish and female fish. The comparison of P450aromA and P450arom B expression in ovary indicated the P450arom A was 18.7 times ofP450arom B.We detected the expression levels of Sox9a1,Sox9a2,LRH-1,P450aromB and P450aromAin brain, liver, kidney and gonad of yellow catfish which was at different developed phases of gonad with the approaches of fluorescent quantitative RT-PCR. Comparison the Sox9a1levels between female and male yellow catfish with the gonad development of phaseⅣandⅤshowed that Sox9a1 was high in the liver, brain and testis, but low in kidney andovaries. There is no significant difference between male and female yellow catfish brain.The Sox9a1 expression level of testis at phaseⅡwas higher than phaseⅣandⅤ. Thisindicated that Sox9a1 played important role in the early development of testis. Expressionlevel of Sox9a2 had two peaks in ovary with phaseⅢandⅤ, and low in phaseⅣandⅥ.The expression levels of LRH-1 in different organs revealed that LRH-1 expressed highquantity in ovary, liver and brain, but low in kidney. The levels were significantly higher inthe ovary with phaseⅢandⅤthan in ovary with other development phases. Thisexpression pattern was consistent with Sox9a2. The expression level of P450aromB in maleyellow catfish brain was higher than in female fish, however, the difference was no distinct.During the different developing phases of ovary, expression of this gene ascended in thelate phaseⅡand peak appeared in phaseⅣ, then the level decreased in phaseⅤand phaseⅥ. The trend is that expression in phaseⅢshould be higher than that in late phaseⅡ,however, the result didn't accord to this trend, which needed to be identify in future. Thisexpression pattern of P450aromB is the same with the result of goldfish.and rainbow trout.The expression levels of P450aromA in ovary with lateⅡandⅢphase were higher thanthe ovary withⅣ,Ⅴ, andⅥphases. There was no significant difference between earlyⅡphase ovary and ovary with other development phases. The expression levels ofP450aromA in ovary with lateⅡandⅢphase were 6 and 5 times, of earlyⅡphase ovaryrespectively, however, the expression level was below 10％inⅣ,Ⅴ, andⅥphases.It was observed from the expression pattern of Sox9a2,LRH-1 and P450aromA, Sox9a2and LRH-1 had the similar expression pattern; it indicated that regulatory relationship waspresent between them. The similar results were found in mammals. There are bindingsequences of Sox9 protein in the region of Ftz-F1 promoter. However, there is no distinctcorrelation in the expression of aromatic enzyme, Sox9 and LRH-1.