Muhammad Shafi,WANG Yanan,ZHOU Xiaosu,MA Liman,Faiz Muhammad,QI Jie,and ZHANG Quanqi

College of Marine Life Sciences, Key Laboratory of Marine Genetics and Breeding of Ministry of Education of China,Ocean University of China,Qingdao 266003,P.R.China

1 Introduction

FTZ-F1 (fushi tarazu factor-1) is a conserved member of nuclear receptor superfamily,which regulates the expression of homeobox segmentation genefushi tarazu(Uedaet al.,1990; Lavorgnaet al.,1991).Previous studies have demonstrated its important role in steroidogenesis and sex differentiation.FTZ-F1 hosting family belongs to a group of orphan nuclear receptors that can be divided into two subgroups based on their sequence homology and function,LRH/FTF (Liver receptor hormone/a-fetoprotein transcription factor,subgroup (1) plays a critical role in regulating development,cholesterol transport,bile acid homeostasis and steroidogenesis and expresses in pancreas,liver,intestine and ovary (Galarneauet al.,1996; Nittaet al.,1999; Luet al.,2001; Fayardet al.,2004),while SF-1/Ad4BP (steroidogenic factor-1/adrenal 4 binding protein,subgroup (2) regulates the development of hypothalamic pituitary adrenal and gonadal axis and serves as an essential factor in sex differentiation (Parker and Schimmer,1997; Hammer and Ingraham,1999).Sex steroid-hormones are vital steroids that are found in steroidogenic organs.SF-1 plays a key role in development and differentiation of steridogenic tissues by controlling the expression of all steroidogenic enzymes and cholesterol transporters functioning in steroidogenesis (Mangelsdorf and Evans,1995; Nordqvist,1995).

Several transcriptional factors identified in orphan nuclear receptor superfamily play pivotal roles in gonadal development (Mangelsdorf and Evans,1995; Nordqvist,1995).Since the isolation ofFTZ-F1for the first time inDrosophila(Uedaet al.,1990),the conservedFTZ-F1has been isolated in vertebrates and invertebrates including mammals (Lalaet al.,1992),tetrapods (Ellinger-Ziegelbaueret al.,1994),aves (Kudo and Sutou,1997),crustaceans (Chan and Chan,1999),nematode (Gissendanner and Sluder,2000) and some teleosts (Liuet al.,1997; Itoet al.,1998; von Hofstenet al.,2002; Zhanget al.,2004; Denget al.,2008).

Unlike mammals,the sex determination and differentiation are greatly diverse among fish species (Devlin and Nagahama,2002; Zhanget al.,2007).The sex determination researches in fish will provide important insight into the plasticity of sex determination in vertebrates; the mechanism underlining such variability among fish species is unknown.The black rockfishS.schlegeliiis a commercially important species in marine culture.In Ko-rea,the production ofS.schlegeliiranked the second,only lower than that ofParalichthys olivaceus(Lee,2002).Although artificial hatching and large scale culture have met success in this ovoviviparous species,little is known about its sex determination and differentiation.The aim of this study is to isolateFTZ-F1cDNA of black rockfish and analyze its expression in order to understand the mechanisms underlining its sex differentiation.

2 Materials and Methods

2.1 RNA Extraction and Multi-Tissue RT-PCR

Black rockfish were collected from a rockfish hatchery near Yantai city,China.Whole larvae 5,10,20 and 33 d post spawn (dps) were collected.Juveniles (105 d old) and mature individuals (3 years old) were sacrificed with their gonad,liver,spleen,kidney,brain,heart,muscle,gill,intestine tissues dissected.The gonad tissue of juveniles was not referred to the ovary or testis; they were morphologically indefinable at this stage.All collected samples were frozen immediately in liquid nitrogen and stored at ?80℃ until use.The total RNA was extracted from either whole body of fingerlings or tissues with Trizol reagent (Invitrogen,USA) and treated with RNasefree DNase (Tiangen,China) according to manufacture’s instructions.

2.2 Isolation and Sequencing of FTZ-F1 cDNA

One microgram total RNA fromS.schlegeliitestis was reversely transcribed using RTase M-MLV (RNase H?)(TaKaRa,Shiga,Japan) reverse transcriptase and OligodT15 primer (Tiangen,China) into the first-strand cDNA which was then used as templates for amplifying the middle fragment ofFTZ-F1cDNA with degenerate primers FTZ-F and FTZ-R (Table1) designed from the conserved regions of FTZ-F1 genes ofOryzias latipes,Acanthopagrus schlegelii,Oncorhynchus mykissandCynoglossus semilaevis(accession Nos.BAA32394,AAS75792,AAW83490 and ABQ41307).The amplification was carried out by denaturing at 94℃ for 5 min,followed by 30 cycles of denaturing at 94℃for 30 s,annealing art 57℃ for 30 s and extending at 72℃ for 1min and an extra extension at 72℃ for 7 min.The product was separated in 1.2% agarose gel,recovered with PCR purification kit (Biomiga),ligated to pMD18-T vector (TaKaRa,Dalian,P.R.China)and transferred into competentE.coliDHα.The positive recombinants survived screening with M13 forward and reverse primers were sequenced.The sequences were analyzed with Blast program (http://blast.ncbi.nlm.nih.gov).

After isolation of a partial fragment ofssFTZ-F1cDNA,its 5’ and 3’ ends were obtained by rapid amplification of cDNA ends (RACE).The 3’ end was amplified by denaturing at 94℃ for 5 min followed by 30 cycles of denaturing at 94 ℃for 30 s,annealing at 60℃ for 30 s and extending at 72℃for 45 s and an extra extension at 72℃ for 7 min.The primers were FTZ-3’R and adopter primer(UPM).A second semi-nested PCR was carried out with the conditions above and FTZ-3’RN and adaptor primer(NUP).The 5’ end was amplified with FTZ-5’R and adapter primer (UPM) by denaturing at 94℃ for 5 min,followed by 30 cycles of denaturing at 94℃ for 30 s,annealing at 58℃ for 30 s and extending at 72℃ for 30 s and a final extension at 72℃ for 7 min.The amplified ends were separated and recovered with Biomiga Gel Extraction Kit,ligated into PMD18-T vector (TaKaRa)and propagated inE.coliDHα.

Table 1 The primer sequence

2.3 Phylogenetic Analysis

Multiple alignment of FTZ-F1 was conducted with known sequences using clustal X (1.81).The sequences aligned included those of other teleosts fish,amphibian,human,chicken and fruit fly.The phylogenetic tree was constructed with neighbor-joining method (Saitou and Nei,1987) and MEGA 4 software (Tamuraet al.,2007)with 1000 bootstrap replicates.

2.4 Quantitative Real-Time PCR

A pair of gene-specific primers FTZ-RTF and FTZRTR was designed to amplify a 128-bp ofssFTZ-F1for quantitative analysis (Table 1).A pair of forward and reverse primers designed to amplify a 128 bp fragment of 18S rRNA (18S-FP and 18S-RP) was used as the internal normalizer.A standard curve was constructed by using various copy numbers of a plasmid containingssFTZ-F1cDNA.The qRT-PCR amplifications were carried out in total volumes of 20 μL containing 10 μL 2 × SYBR premix Ex Taq (TaKaRa,Dalian,China),1 μL cDNA,0.8 μL each of forward and reverse primers,0.4 μL Rox reference Dye II (50 ×) and 7 μL PCR graded water.Real-time PCR was carried out with an ABI7500 and programmed for an initial step of 95℃ for 2 min followed by 40 thermal cycles of 95℃ for 15 s and 60℃ for 1 min.Fluorescent detection was used after each extension step.A dissociation step was included after thermo cycling,through which only a single product was amplified.The ratio betweenFTZ-F1and 18S rRNA was used for comparison.Statistical analysis was performed by one-way analysis of variance (ANOVA)with SPSS13.0 software.A probability value ofP< 0.05 was considered to indicate statistical significance.

3 Results

3.1 Isolation of ssFTZ-F1 cDNA

The middle fragment,554 bp in length,was amplified,which showed a high homology with other knownFTZF1.From this fragment,gene specific primers were designed for isolating 5’ and 3’ ends.The full cDNA,1786 bp in length,has been deposited in GenBank with accession number JQ071502.The cDNA included a 1449 bp ORF,a 232 bp 5’ UTR and a 105 bp 3’ UTR (Fig.1).The predicted ssFTZ-F1,which was 482 amino acid residues in length,was 54453.99 Daltons and highly similar with those of fish but less similar with those of invertebrates.ssFTZ-F1 was 94.3%,93.4%,82.5%,80.8%,71.7%,70.1%,69.8% and 68.8% similar with that of seabream,medaka,rainbow trout,half smooth tongue sole,channel catfish,zebrafish and African catfish,respectively. In ssFTZ-F1,the conserved boxes showed more than 90%similarity with those of other vertebrate.ssFTZ-F1 had conserved AF-2 (activation function-2) motif (LLIEML,LBD amino acid 469–475) and showed more than 96%similarity with other FTZ-F1.The region I,II and III were 92%–100%,90%–100% and 60%–90% similar with their homologues,respectively.ssFTZ-F1 had P box ESCKG which was found at the base of zinc finger,and characterized in all FTZ-F1 box receptors.The D box was found in the second zinc fingers (Fig.2).

3.2 Phylogenetic Analysis

Phylogenetic tree of ssFTZ-F1 was grouped together withM.albusFTZ-F1 andA.schlegeliiFTZ-F1 first,and then with that of cichlid fish and other acanthopterygian fish.This is generally in accordance with the taxonomic relationships (Fig.3).

3.3 Expression of ssFTZ-F1

On dps 5,the abundance ofssFTZ-F1transcript was comparatively low,but it increased significantly on dps 10 (P< 0.05) and decreased after dps 20 (Fig.4A).In juveniles (after dps 105),the abundance fluctuated among different tissues.In gonad,spleen,brain and kidney,it was high with the highest abundance detected in gonad;while in gill,intestine,heart,liver and muscle tissues,the abundance was similarly lower with the lowest found in intestine and heart (Fig.4B).In mature adult (3 years old),the transcript ofssFTZ-F1transcript was detected in testis,ovary,spleen and brain.Trace amount ofssFTZ-F1transcript was detected in liver (Fig.4C).Male tissues showed higher expression than female counterparts (P<0.05).The highest expression was found in male testis (Fig.4C).

Fig.1 Nucleotide and deduced amino acid sequences of Sebastes schlegelii FTZ-F1 (JQ071502).The start codon is underlined while stop codon is showed by asterisk.

Fig.2 Multiple alignment of ssFTZ-F1 (JQ071502),Cynoglossus semilaevis hsFTZ-F1 (ABQ41307), Gallus gallus cFTF/LRH-1 (BAA22838),Homo sapiens hSF-1 (AAB53105),Danio danio zFF1A (AAK54449),Mus musculus mSF-1(AAB28338),Rana rugosa rrSF-1 (BAA36789),Oncorhynchus mykiss rtFTZ-F1 (AAW83490).The boxes show the respective regions and domains.

Fig.3 Phylogenetic tree based on the alignment of ssFTZ-F1 and other FTZ-F1.Bootstrap values (%) was calculated with 1000 permitations and the scale bar refers to a phylogenetic distance of 0.1 amino acid substitutions per site.The protein sequences were downloaded from NCBI.The accession numbers are mentioned in materials and methods.

Fig.4 Relative abundance ssFTZ-F1 transcript at different stages of fry (A),various tissues of juvenile (105 d old)(B) and mature individuals (3 years old) (C).The relative expression variance is showed as ratio (the amounts of ssFTZ-F1 mRNA normalized to 18S rRNA).Data are shown as mean ± SEM (n = 3).Values with different superscripts indicate statistically difference (P < 0.05).

4 Discussion

The black rock fish is a prime aquaculture species and it has important market value in China,Korea and Japan(Lee,2002; Whanget al.,2011).FTZ-F1 plays an important role in gonad differentiation (Ingrahamet al.,1994; Luoet al.,1994; Mangelsdorf and Evans,1995;Nordqvist,1995; Ikedaet al.,1994.,Zhanget al.,2004;Hanleyet al.,1999).FTZ-F1have been cloned from gonad of several other fish species such asC.semilaevis,O.mykiss,T.okinawaeandA.schlegeli(Itoet al.,1998;Nakajimaet al.,2000; Liuet al.,2004; Denget al.,2008).In this study,we cloned theFTZ-F1gene from the ovoviviparous black rockfishS.schlegeliiand designated it asssFTZ-F1.

Black rockfish FTZ-F1 showed high similarity with those of fishes and less similarity with those of invertebrates (Itoet al.,1998; Nakajimaet al.,2000; Denget al.,2008).Our ssFTZ-F1 also showed the typical structure of nuclear receptors.The three main regions,region I,II and III as well as FTZ-F1 box and AF-2 hexamer,were noted as in other examined organisms.It has been reported that these regions have characteristics of nuclear receptor superfamily and are highly conserved (Wanget al.,1989;Hondaet al.,1993).The region I with a function of DBD(DNA binding domain) has the zinc finger motifs and can bind to hormone response elements (Fig.2).These motifs of zinc finger are related to the steroid hormone receptor superfamily containing two functional domains,P and D box (Fig.2).The region I can also bind certain sequences of target genes that can regulate the nuclear receptors.

The P box is a distinguishing agent between the sequences of hormone response elements that locates at the base of zinc finger.The box (ESCKG) is found among all FTZ-F1 box-containing receptors.The D box is the second zinc finger that characterizes the spacing of those elements.The two boxes in the putative LBD (Ligand binding domain),known as region II and III,are considered as nuclear hormone receptors (Wanget al.,1989;Hondaet al.,1993). The ssFTZ-F1 consists of conserved activation function-2 (AF-2) motif (LLIEML) positioning in LBD amino acid 469–475 (Fig.2).The transcriptional activation depends on ligand binding as described previously (Toraet al.,1989).The FTZ-F1 box ofS.shlegeliishows more than 90% similarity with those of other vertebrates such as orange spotted grouper (Uedaet al.,1992); while the AF-2 shows more than 75% similarity with those of other vertebrates except of a few species.The regions I,II and III of black rock fish also share very high similarity with their vertebrate counterpart regions.The conservative functional domains mean that the function of ssFTZ-F1 may also conservative.

The expression of ssFTZ-F1in adult black rockfish was high in the gonad,spleen,kidney and brain tissues of both males and females,while expression in gill,intestine,muscle,heart and liver tissues was not detactable.ssFTZ-F1mRNA abundance in gonads (testis and ovary)was higher than in other tissues,suggesting ssFTZ-F1was a gonad development factor.The expression level in whole body of larvae is in accordance with FTZ-1 in half-smooth tongue sole (Denget al.,2008).In juveniles of black rock fish,the expression pattern of ssFTZ-1was markedly different from that of adult fish.It was detectable in all tissues assayed although it varied among tissues.The tissue distribution of FTZ-1 in juvenile fish has not been reported yet.It was investigated that FTZ-F1 was involved in tissue differentiation in zebra fish.The expression ofFTZ-F1was found in pituitary,mandibular arch,pronephric duct,liver,rostral diencephalons,hindbrain and pancreas during development (Liuet al.,1997;Chaiet al.,2000; Von Hofstenet al.,2001).The wide tissue distribution ofFTZ-F1in larvae and juveniles may relate with its unknown function during early development of fish.

Acknowledgements

This work was supported by the National High-Tech Research and Development Program (2012AA10A402)and the National Natural Science Foundation of China(31172385).The first author thanks China Government Scholarship Council for award of Ph.D research scholarship.

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