Molecular cloning of two isoforms of 11b-hydroxylase and their expressions in the Nile tilapia, Oreochromis niloticus Wei-Li Zhang a,1 , Lin-Yan Zhou b,1 , Balasubramanian Senthilkumaran b,c , Bao-Feng Huang a , Cheni Chery Sudhakumari b,c , Tohru Kobayashi b,d , Yoshitaka Nagahama b , De-Shou Wang a,b, * a Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Science, Southwest University, Chongqing 400715, China b Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan c Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, Andhra Pradesh, India d Tarumi Station, Research Group for Reproductive Physiology, South Ehime Fisheries Research Center, Ehime University, Matsuyama 790-8566, Japan article info Article history: Received 14 February 2009 Revised 22 May 2009 Accepted 27 May 2009 Available online 3 June 2009 Keywords: P450(11b) Alternative splicing Testis Sex steroids Nile tilapia abstract P450 11b-hydroxylase, encoded by P450(11b) gene, is a key mitochondrial enzyme to produce 11b- hydroxy testosterone, substrate for the production of 11-ketotestosterone (11-KT), which has been shown to be potent androgen in several fish species. In the present work, two alternative splicing iso- forms i.e. P450(11b)-1 and P450(11b)-2 cDNAs were cloned from the Nile tilapia, Oreochromis niloticus. They were 1614 and 1227 bp in length with open reading frames encoding proteins of 537 and 408 amino acids, respectively. In contrast to P450(11b)-1, which derived from 9 exons of the P450(11b) gene, the 7th and 8th exons were absent in P450(11b)-2. Tilapia P450(11b)-1 shares the highest homology with that of medaka, Oryzias latipes. Expressions of P450(11b)-1 and -2 were detected in the kidney and head kidney of both sexes, and in the testis but not in the ovary, with P450(11b)-2 lower than P450(11b)-1. Ontogenic expressions of both isoforms were detected in testis from 50dah onwards. P450(11b)-1 and -2 were strongly expressed in sex reversed XX testis after fadrozole and tamoxifen treatment, but completely inhibited in 17b-estradiol induced XY ovary. The existence of two alternatively spliced isoforms and the sexual dimorphic expression of P450(11b)s were further confirmed by Northern blot. Strong expres- sion signals in Leydig cells and weak signals in spermatogonia were detected by in situ hybridization and immunohistochemistry. Taken together, our data suggest a role for P450(11b) in the spermatogenesis of tilapia through the production of 11-KT in testis, in addition to cortisol production in head kidney. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction In several teleosts, 11-ketotestosterone (11-KT), capable of reg- ulating male secondary sexual characters, reproductive behavior (Mayer et al., 1990) and spermatogenesis (Miura et al., 1991; Naga- hama, 1994), has been recognized as one of the major androgens. Consistently, application of exogenous 11-KT to genetic female fry at the narrow window of sex differentiation can induce female-to-male sex reversal (Cardwell and Liley, 1991). Steroidogenic enzyme, 11b-hydroxylase (encoded by P450(11b) gene), is one of the key enzymes (involved in the production of 11b-hydroxy testosterone) in the process of 11-KT biosynthesis in the testis, as well as synthesis of the cortisol in head kidney of teleosts (Jiang et al., 1996, 1998; Kusakabe et al., 2002). Accord- ingly, the P450(11b) were detected in testes during spermatogen- esis in rainbow trout, Oncorhynchus mykiss (Liu et al., 2000), Japanese eel, Anguilla japonica (Miura et al., 1991), medaka (Yokota et al., 2005), Atlantic salmon, Salmo salar (Maugars and Schmitz, 2008) and the Nile tilapia (Ijiri et al., 2008). During the spermato- genesis, expression level of P450(11b) was comparatively low at the early spermatogenesis and sharp increased during spermiogen- esis, finally, reached its highest levels (Maugars and Schmitz, 2008). Furthermore, the expression of P450(11b) can be influenced by chemical substances which can induce sex reversal. The long- term exposure with high concentrations of 4-tert-pentylphenol (4-PP) completely inhibited expression of P450(11b) mRNA in the gonads of XY sex-reversed female medaka (Yokota et al., 2005). By in situ hybridization, P450(11b) expression was detected in ste- roid producing cells, such as Leydig cell in testis and inter-renal cells in head kidney in rainbow trout (Kusakabe et al., 2002). Most researches indicated that P450(11b) was not expressed in germ cells. However, recently in sea bass (Dicentrarchus labrax), P450(11b) was detected in spermatogonia and peaked during the 0016-6480/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2009.05.018 * Corresponding author. Address: Key Laboratory of Freshwater Fish Reproduc- tion and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Science, Southwest University, Chongqing 400715, China. Fax: +86 23 68252365. E-mail address: [email protected](D.-S. Wang). 1 Wei-Li Zhang and Lin-Yan Zhou contributed equally to this work. General and Comparative Endocrinology 165 (2010) 34–41 Contents lists available at ScienceDirect General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen
Transcript
General and Comparative Endocrinology 165 (2010) 34–41
Contents lists available at ScienceDirect
General and Comparative Endocrinology
journal homepage: www.elsevier .com/locate /ygcen
Molecular cloning of two isoforms of 11b-hydroxylase and their expressionsin the Nile tilapia, Oreochromis niloticus
a Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Science,Southwest University, Chongqing 400715, Chinab Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki 444-8585, Japanc Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, Andhra Pradesh, Indiad Tarumi Station, Research Group for Reproductive Physiology, South Ehime Fisheries Research Center, Ehime University, Matsuyama 790-8566, Japan
a r t i c l e i n f o
Article history:Received 14 February 2009Revised 22 May 2009Accepted 27 May 2009Available online 3 June 2009
0016-6480/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.ygcen.2009.05.018
* Corresponding author. Address: Key Laboratory otion and Development (Ministry of Education), Key LabChongqing, School of Life Science, Southwest UniversiFax: +86 23 68252365.
P450 11b-hydroxylase, encoded by P450(11b) gene, is a key mitochondrial enzyme to produce 11b-hydroxy testosterone, substrate for the production of 11-ketotestosterone (11-KT), which has beenshown to be potent androgen in several fish species. In the present work, two alternative splicing iso-forms i.e. P450(11b)-1 and P450(11b)-2 cDNAs were cloned from the Nile tilapia, Oreochromis niloticus.They were 1614 and 1227 bp in length with open reading frames encoding proteins of 537 and 408 aminoacids, respectively. In contrast to P450(11b)-1, which derived from 9 exons of the P450(11b) gene, the 7thand 8th exons were absent in P450(11b)-2. Tilapia P450(11b)-1 shares the highest homology with that ofmedaka, Oryzias latipes. Expressions of P450(11b)-1 and -2 were detected in the kidney and head kidneyof both sexes, and in the testis but not in the ovary, with P450(11b)-2 lower than P450(11b)-1. Ontogenicexpressions of both isoforms were detected in testis from 50dah onwards. P450(11b)-1 and -2 werestrongly expressed in sex reversed XX testis after fadrozole and tamoxifen treatment, but completelyinhibited in 17b-estradiol induced XY ovary. The existence of two alternatively spliced isoforms andthe sexual dimorphic expression of P450(11b)s were further confirmed by Northern blot. Strong expres-sion signals in Leydig cells and weak signals in spermatogonia were detected by in situ hybridization andimmunohistochemistry. Taken together, our data suggest a role for P450(11b) in the spermatogenesis oftilapia through the production of 11-KT in testis, in addition to cortisol production in head kidney.
� 2009 Elsevier Inc. All rights reserved.
1. Introduction
In several teleosts, 11-ketotestosterone (11-KT), capable of reg-ulating male secondary sexual characters, reproductive behavior(Mayer et al., 1990) and spermatogenesis (Miura et al., 1991; Naga-hama, 1994), has been recognized as one of the major androgens.Consistently, application of exogenous 11-KT to genetic femalefry at the narrow window of sex differentiation can inducefemale-to-male sex reversal (Cardwell and Liley, 1991).
Steroidogenic enzyme, 11b-hydroxylase (encoded by P450(11b)gene), is one of the key enzymes (involved in the production of11b-hydroxy testosterone) in the process of 11-KT biosynthesisin the testis, as well as synthesis of the cortisol in head kidney of
ll rights reserved.
f Freshwater Fish Reproduc-oratory of Aquatic Science of
ty, Chongqing 400715, China.
).ly to this work.
teleosts (Jiang et al., 1996, 1998; Kusakabe et al., 2002). Accord-ingly, the P450(11b) were detected in testes during spermatogen-esis in rainbow trout, Oncorhynchus mykiss (Liu et al., 2000),Japanese eel, Anguilla japonica (Miura et al., 1991), medaka (Yokotaet al., 2005), Atlantic salmon, Salmo salar (Maugars and Schmitz,2008) and the Nile tilapia (Ijiri et al., 2008). During the spermato-genesis, expression level of P450(11b) was comparatively low atthe early spermatogenesis and sharp increased during spermiogen-esis, finally, reached its highest levels (Maugars and Schmitz,2008). Furthermore, the expression of P450(11b) can be influencedby chemical substances which can induce sex reversal. The long-term exposure with high concentrations of 4-tert-pentylphenol(4-PP) completely inhibited expression of P450(11b) mRNA in thegonads of XY sex-reversed female medaka (Yokota et al., 2005).By in situ hybridization, P450(11b) expression was detected in ste-roid producing cells, such as Leydig cell in testis and inter-renalcells in head kidney in rainbow trout (Kusakabe et al., 2002). Mostresearches indicated that P450(11b) was not expressed in germcells. However, recently in sea bass (Dicentrarchus labrax),P450(11b) was detected in spermatogonia and peaked during the
W.-L. Zhang et al. / General and Comparative Endocrinology 165 (2010) 34–41 35
early stages of spermatogenesis (Viñas and Piferrer, 2008). Thesereports suggested that P450(11b) was probably involved in theproduction of 11-KT and cortisol not only in the traditional steroidproducing cells but probably also in germ cells, which in turn, iscritical to spermatogenesis.
In order to elucidate the function of P450(11b) in the sex differ-entiation and spermatogenesis, P450(11b) cDNA was cloned fromthe testes of the Nile tilapia in the present study. Interestingly,two alternatively spliced isoforms of P450(11b) were isolated. Tis-sue distribution, ontogenic and cell specific expression patternswere also investigated by RT-PCR, in situ hybridization (ISH) andImmunohistochemistry (IHC). The influences of aromatase inhibi-tor fadrozole (F), the estrogen receptor antagonist tamoxifen(TAM), and the estrogen 17b-estradiol (E2) (Kobayashi et al.,2003) on the expression levels of two P450(11b)s in the Nile tilapiawere also studied. These data may allow new insight and perspec-tives into the function of P450(11b) in teleosts.
2. Materials and methods
2.1. Animals
The Nile tilapias (Oreochromis niloticus) were maintained in re-circulating freshwater tanks at 26 �C. All genetic females (XX) andmales (XY) were obtained by artificial fertilization of eggs fromnormal females (XX) with sperm from either sex reversed males(XX) or super males (YY), respectively (Wang et al., 2008).
2.2. Drug treatment
All-female (XX) and all-male (XY) tilapia fry (300 fry/aquarium)were reared in aerated 30 � 30 � 50 cm aquaria at 26 �C. Fish feedwere sprayed with 95% ethanol containing F, 100 lg/g; TAM,25 lg/g; E2, 25 lg/g. Control fishes were fed with 95% ethanolsprayed feed. Drug treatment was applied to the fry from 5 to30 day, the critical period of the Nile tilapia sex differentiation(Kobayashi et al., 2003). The E2 was purchased from Sigma(USA), and the F and TAM were from Novartis Company (AG, Swit-zerland) and Egis (Budapest, Hungary) respectively.
2.3. Cloning of P450(11b) cDNAs
Total RNA from the Nile tilapia testis was prepared using RNA-iso (Takara, Japan). The first strand cDNA was synthesized with2 lg total RNA from testis using oligo-dT18 primer and M-Mulvreverse transcriptase (Promega, USA) according to the manufac-turer’s instructions. A 644 bp cDNA fragment of P450(11b) wasamplified from the testis by RT-PCR with degenerate primers
(Table 1) designed using the conserved region of known cDNA se-quences from medaka, zebrafish (Danio rerio) and rainbow trout.PCR were performed as follows: 94 �C (2 min), followed by 30 cy-cles of 94 �C (30 s), 60 �C (45 s), and 72 �C (1 min) and final exten-sion at 72 �C (10 min). PCR was performed on a PTC-100 thermalcycler (Bio-Rad). All the PCR products were electrophoresed on1.2% agarose gel with ethidium bromide to visualize the bands fol-lowed by purification using QIAquick Gel Extraction Kit (Qiagen).The fragments were then cloned into pGEM-T vector (Promega)and sequenced using an ABI PRISM 377 DNA genetic analyzer.
Gene specific primers (Table 1) were used to obtain the 50- and 30-cDNA ends of P450(11b) using the SMART RACE Kit (Clontech)according to the manufacturer’s instructions. After sequencing, apair of gene specific primers (ORF primers, which was located inthe 1st and 9th exons, Table 1) was designed to amplify the entirecoding regions of P450(11b) from the testis. Surprisingly, two bandswith the size of�1.2 and�1.6 kb were observed after gel electropho-resis of the PCR products. Both bands were excised, purified sub-cloned and then sequenced. The �1.6 kb product is the normalP450(11b), designated as P450(11b)-1), while the �1.2 kb productis the alternatively spliced isoform of P450(11b), named asP450(11b)-2.
2.4. Phylogenetic analysis
Multiple alignment of deduced amino acid sequences were per-formed based on 15 complete and 3 partial sequences of P450(11b)using the software Lasergene and ClustalX. ClustalX was also em-ployed to construct the tree using the neighbor-joining method.TREEVIEW was used to display the phylogenetic tree. The valuesrepresent bootstrap scores out of 1000 trials, indicating the credi-bility of each branch. All the P450(11b) amino acid sequences, ex-cept the tilapia (FJ713103) were obtained from GenBank: human,X54741; baboon, Q29527; sheep, L34337; cattle, BAA00347;mouse, P15539; rat, NP_036669; pig, AAD01633; Tetraodon,CAF97877; fugu (lcl|SINFRUP00000066166); zebrafish, BC155806;medaka, EF025509; rainbow trout, AF179894; sea bass,AF449173; salmon, DQ352841; bullfrog, D10984; Xenopus,AF449175 and eel (Jiang et al., 1996; not deposited in GenBank).Zebrafish P450 aromatase (NM_131154) was used as outgroup.
2.5. Analysis of P450(11b) expression by RT-PCR and semiquantitativeRT-PCR
Total RNAs (2.0 lg) were isolated from various tissues (brain,pituitary, gill, heart, spleen, liver, intestine, ovary, kidney, muscle,testis and head kidney) of adult (10-month-old) and gonads at dif-ferent developmental stages (5–90 days after hatching, dah) by
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RANiso (Takara, Japan) according to the manufacture’s instructionsand treated with DNase I (RNase free) (Invitrogen, Carlsbad, CA,USA). Reverse transcription was carried out using SuperScript IIby Oligo-dT18 at 42 �C for 1 h. The reverse transcribed cDNAs werediluted 10 times before use. A pair of gene specific primers (Table1), which were located in the 6th and 9th exons of the P450(11b),were used to analyze the expressions of both mRNA isoforms. Thesetwo primers allow us to amplify 723 and 431 bp cDNA fragment ofP450(11b)-1 and -2, respectively. PCR was performed as follows:94 �C (2 min), followed by 30 cycles of 94 �C (30 s), 60 �C (45 s),and 72 �C (1 min) and final extension at 72 �C (10 min). A 342 bpb-actin fragment was amplified (27 cycles) to test the quality ofthe cDNA with a pair of b-actin primers (Table 1). b-Actin waswidely used as an internal control for tilapia gene expression stud-ies, including sex reversed fish (Kobayashi et al., 2008). b-Actin geneexpression was found to be unaffected by the drugs used for treat-ment in the present study. Positive and negative controls were setup with P450(11b)-1 and -2 plasmid DNA and water, respectively.
For drug treatment study, gonads of five individuals were col-lected from each drug treated (and control) group when thesefishes were adult. Semi-quantitative RT-PCR (Jiao et al., 2006)was performed to measure the mRNA levels after drug treatmentusing P450(11b)-1 and -2 specific primers (Table 1). A series ofPCRs with different cycle numbers (from 22 to 36, with an intervalof 2) were performed to determine the linear phase of the amplifi-cation. Based on these pilot experiments, 27 cycles for b-actin and32 cycles for the target genes were chosen and applied to the sub-sequent semi-quantitative RT-PCR analyses. Band intensitiesresulting from the PCR amplification were analyzed using the im-age analysis software Quantity One (Bio-Rad). P450(11b)-1 and -2mRNA levels were expressed relative to that of b-actin in eachsample. Data analyses were performed using one-way ANOVAand the least significant difference on the GraphPad Prism 4 soft-ware (GraphPad Software, San Diego, CA, USA).
2.6. Northern blot analysis
Northern blot was performed as described previously (Wanget al., 2002; Jiang et al., 2003). Total RNAs were extracted fromovary, testis and brain of adult fish. Poly (A) + RNAs were purifiedusing Oligotex-dT30 (Takara, Japan). mRNA from each tissue(5 lg) were electrophoresed on a 1.5% formaldehyde agarose geland transferred onto a nylon membrane (Hybond-N+, Amersham,UK). Hybridization was carried out overnight at 42 �C with 32P-la-beled P450(11b) fragments as probe, followed by washing twice at60 �C with a series SSC-SDS solution. Signals were analyzed using aBAS 2000 Imager (Fuji Co., Ltd., Japan), and quantified using theFuji-MacBAS software (V1.0). The membrane was stripped and fur-ther hybridized with 32P-labeled tilapia b-actin probe to serve as apositive control for loading variations.
2.7. In situ hybridization (ISH)
Gonads of adult fish were dissected and fixed in 4% paraformal-dehyde in 0.1 M phosphate buffer (pH 7.4, 4% PFA) at 4 �C over-night. After fixation, the gonads were embedded in paraffin andcross-sections were cut at 5 lm. Probes of sense and antisensedigoxigenin (DIG)-labeled RNA strands were transcribed in vitrofrom linearized pGEM-T easy-P450(11b)-1 ORF cDNA using a RNAlabeling kit (Roche Diagnostics GmbH, Mannheim, Germany). ISHwas performed as described previously (Kobayashi et al., 2000).
2.8. Immunohistochemistry (IHC)
The gonads and head kidney of adult fish were dissected andfixed in Bouin’s solution at room temperature overnight with
gentle shaking. Specimens were embedded in paraffin and sec-tioned at 5 lm thickness. Paraffin sections were deparaffinizedand hydrated, and IHC were carried out according to theinstructions of Histofine SAB-PO (multi) kit (Nichirei, Tokyo, Ja-pan). The antiserum was raised against frog P450(11b) (Jianget al., 1998). Freshly diluted primary antibody by 100 timeswas used for this study. After overnight incubation with pri-mary antibody at 4 �C, the slides were washed in 1�PBS for10 min, then, the slides were incubated with anti-rabbit IgGat room temperature for 1 h. After washing, Diaminobenzidinetetrachloride was applied for the color reaction, the slides werethen counterstained in hematoxylin, dehydrated, mounted andanalyzed on Zeiss confocal microscope. As a negative control,the primary antibody was substituted with normal rabbit serumand there was no significant immunostaining of controlsections.
3. Results
3.1. Molecular cloning of tilapia P450(11b)s
The P450(11b) genomic DNA and two isoforms of P450(11b)were cloned from the Nile tilapia. The genomic DNA (7236 bp)(FJ713105) comprises of 9 exons (Fig. 1). The full-lengthP450(11b)-1 cDNA (1859 bp) (FJ713103) encompassing a 50-untranslated region (UTR) of 60 bp, a 30-UTR of 185 bp and anORF of 1614 bp encode a protein of 537 aa. Whereas the alterna-tively spliced isoform P450(11b)-2 (FJ713104), amplified by thesame pair of P450(11b)-1 ORF primers, is 387 bp shorter than theORF of P450(11b)-1 because of the absence of the 7th and 8th exonswhich resulted in an early termination of P450(11b)-2 ORF (encod-ing 408 aa) at the beginning of the 9th exon due to the readingframe shift. Based on the 30- and 50-RACE results, the 30-UTR and50-UTR sequences of both P450(11b)s were found to be identical(data not shown).
3.2. Sequence and phylogenetic analysis
Multiple alignment of the amino acids sequences of the tila-pia P450(11b)-1 and those from other vertebrates revealed thatP450(11b) was conserved through evolution (Fig. 2), especiallyat the characteristic regions of the P450 enzymes, i.e. (A) ste-roid binding site, (B) oxygen-binding site, (C) Ozols’, (D) aro-matic, and (E) heme-binding site. Regions A, B and E werehighly conservative, 92–100% homology with those of other fishP450(11b), �84% with amphibian and �58% with mammalianP450(11b). In contrast, region D shared the lowest homologywith those of other vertebrates, 58–83% homology with fish,�58% and �41% with its amphibian and mammalian counter-parts, respectively, while, region C shared the moderately con-served homology with those of other vertebrates, 79–89%homology with fish, 73% and 57% with its amphibian and mam-malian counterparts, respectively (Fig. 2). Based on an align-ment of 19 P450(11b) sequences, a phylogenetic tree wasconstructed using the zebrafish p450 aromatase as the out-group. Tilapia P450(11b) showed the highest similarity (80.5%in nucleotide sequence and 84.2% in aa sequence) to that ofmedaka. The high homology of this protein between tilapiaand medaka, as shown in the phylogenetic tree, the 18 specieswere clustered into three groups, representing fish, amphibiansand mammals, respectively. The phylogenetic tree clearly re-flects the evolutionary relationship of these selected animals,and basically agrees with the known taxonomic relationshipsamong these species. For example, fish P450(11b) has a relativeshort phylogenetic distances with amphibians when compared
Fig. 2. Alignment of the amino acid sequences of P450(11b) from tilapia and other vertebrates. P450 enzymes characteristic regions are boxed: (A) Steroid binding; (B)oxygen-binding; (C) Ozol’; (D) aromatic and (E) heme-binding regions. Refer to Section 2 for GenBank accession numbers. BOXSHADE (http://www.ch.embnet.org/software/BOX_form.html) was used to make this figure.
Fig. 1. Gene structure of the Nile tilapia P450(11b) (GenBank Accession No. FJ713105) and the two isoforms of P450(11b)s. Only the coding sequences of the gene weredepicted. The white box indicated the ORF of P450(11b), and the black box of P450(11b)-2 indicated the un-translated region of the 9th exon which belong to the ORF ofP450(11b)-1; I, intron; E, exon; The numbers indicated the nucleotide numbers.
W.-L. Zhang et al. / General and Comparative Endocrinology 165 (2010) 34–41 37
with mammals. Two sub-clusters, i.e. the relatively primitive eel(anguillids), salmon (salmonids) and zebrafish (cyprinids), andthe relatively advanced fugu (tetraodontids), tilapia (cichlids)fishes, including tilapia can be clearly distinguished from thetree (Fig. 3).
3.3. Ontogenic expression of two isoforms of P450(11b) in testis by RT-PCR
P450(11b) mRNAs were detectable only from 50dah in male tes-tes and increased thereafter, with highest expression in testis of
Fig. 3. Phylogenetic tree of vertebrate P450(11b)s, using zebrafish P450 aromatase as the outgroup. The values represent bootstrap scores out of 1000 trials, indicating thecredibility of each branch. Partial sequences were indicated by*. Refer to Section 2 for GenBank accession numbers.
Fig. 4. Ontogenic analysis of P450(11b) expression in different developmental stages of tilapia by RT-PCR. The numbers indicates the day after hatching numbers; A, adult; M,markers. Lower panel, b-actin as the internal control.
Fig. 5. Expression pattern of P450(11b)s in various tissues of adult fish. B, brain; P, pituitary; G, gill; H, heart; S, spleen; L, liver; I, intestine; O, ovary; K, kidney; M, muscle; T,testis; HK, head kidney; 1, 2 and 3, markers; PC, positive control; NC, negative control. Lower panel, b-actin as the internal control.
Fig. 6. Northern blot analysis on the expression of P450(11b) in tilapia testis (T) andovary (O) and brains of male and female (Bm and Bf). The sizes of transcripts areindicated on the left of the blot. The lower panel shows the same membrane afterstripping and hybridization with the tilapia b-actin probe.
38 W.-L. Zhang et al. / General and Comparative Endocrinology 165 (2010) 34–41
adult fish, while no expression was detected in any developmentalstages of the ovary (Fig. 4).
3.4. Tissue distribution analysis of two isoforms of P450(11b) by RT-PCR
In various tissues examined, both P450(11b)-1 and P450(11b)-2were found to be expressed in the kidney and head kidney of bothXX and XY fish, and in the testis of the XY fish. However, theexpression level of P450(11b)-1 was several times stronger thanthat of P450(11b)-2 (Fig. 5) .
3.5. Northern blot
Consistent with the RT-PCR results, Northern blot analysis dem-onstrated sexually dimorphic expression pattern of P450(11b) intilapia gonads as well, with two bands of 1.8 kb (very strong) and1.5 kb (weak), respectively, detected in the testis, while no bandin the ovary. In contrast, no signals were detected in the brain ofboth sexes (Fig. 6).
3.6. Effect of drug treatment on gene expression
In the present study, E2 (XY), F and TAM (XX) treatmentresulted in complete sex reversal in tilapia, respectively. On
the one hand, in the ovary of adult XX control fish, noexpression of P450(11b)-1 and -2 mRNA were detected,whereas in the gonad (testis) of the sex reversed XX fish
Fig. 7. Relative expression levels of tilapia P450(11b)s in gonad of control and sex reversed adult tilapia by semi-quantitative RT-PCR. (A) Female-to-male sex reversal inducedby fadrozole (F) and tamoxifen (TAM) and (B) male-to-female sex reversal induced by 17b-estradiol (E2). XX control in Fig. 7A and XY E2 in Fig. 7B were the background.Results were expressed as mean values ± S.E.M. from five individual fishes. (**P < 0.01 significantly different as compared with the respective control by one-way ANOVA.) Thedosages of the drugs used were described in Section 2.
Fig. 8. Expression of tilapia P450(11b) by in situ hybridization (A and B), and by immunohistochemistry (C–F). A–E, testis; F, head kidney; E, negative control with out the firstantibody; Asterisk, Leydig cells; arrow, spermatogonia; arrow head, inter-renal cells. Scale bar, A, C, E and F, 100 lm; B and D 50 lm.
W.-L. Zhang et al. / General and Comparative Endocrinology 165 (2010) 34–41 39
(groups treated with F and TAM), strong expression, similarto that of the control XY gonad, were detected by semi-quantitative RT-PCR (Fig. 7A). On the other hand, in the tes-tis of adult XY control fish, strong expression of P450(11b)-1and -2 was detected. However, the expression was com-pletely inhibited in gonad (ovary) of E2 treated XY fish aftermale-to-female sex reversal (Fig. 7B).
3.7. In situ hybridization
To ascertain the P450(11b) positive cell population, ISH was per-formed using ovaries and testes from tilapia. Specific signals wereobserved in the Leydig cells (strong) and spermatogonia (weak) ofthe testis (Fig. 8A and B), while no signals was detected in the testiswith probes of sense as the negative control (data not shown),which are consistent with the results from RT-PCR and Northernblot.
3.8. Immunohistochemistry
By IHC, strong expression signals of P450(11b) were de-tected in Leydig cells, weak signals were detected in sperma-togonia of the testis (Fig. 8C and D) and inter-renal cells ofhead kidney (Fig. 8F), and no expression signal was detectedwithout the primary antibody in the testis of the negativecontrol (Fig. 8E).
4. Discussion
In the present study, the P450(11b) gene, consisting of 9 exons,was sequenced from the Nile tilapia. Two alternatively spliced iso-forms of P450(11b) cDNAs were cloned from tilapia testis.P450(11b)-1 cDNA contains all the 9 exons common to all verte-brates, whereas P450(11b)-2 contains only 7 exons, with the 7thand 8th exons spliced off. Two bands were detected in testis, headkidney and kidney when using the primers located on the 6th and9th exons, whereas only one band could be detected using theprimers located on other exons (exons 1–6). The existence of twoalternative spliced forms of P450(11b) transcripts was further con-firmed by Northern blot using testis mRNA, where two bands (1.8and 1.5 kb) could be detected. In sea bass, two mRNA bands (2 and3.9 kb) in testis and head kidney were also detected by Northernblot, and were hypothesized to be two mRNAs having differentlength of 3’-UTR due to alternative polyadenilation signals (Socorroet al., 2007). While in rainbow trout, two P450(11b)s encoded bytwo genes, which were supposed to be resulted from the recentfish specific genome duplication, have been reported (Liu et al.,2000). Different from these reports, the two isoforms of tilapiaP450(11b) cDNAs were proved to be resulted from alternativesplicing in the ORF by using different exons as our data indicated.It is well known that P450 enzymes have five characteristic regions(Gotoh et al., 1983; Morohashi et al., 1987; Poulos et al., 1987;Nonaka et al., 1995), i.e. (A) steroid-binding; (B) oxygen-binding;
40 W.-L. Zhang et al. / General and Comparative Endocrinology 165 (2010) 34–41
(C) Ozol’; (D) aromatic and (E) heme-binding regions. TheP450(11b)-1 possesses all these five regions as other P450 en-zymes, however, the P450(11b)-2 protein only possesses the com-plete steroid binding and oxygen-binding region, but lost thearomatic, heme-binding and part of Ozol’ regions for the absenceof the 7th and 8th exons. This indicates that though P450(11b)-2is able to recognize and bind the substrate (such as testosterone),but lacks hydroxylase function due to the absence of the redox cen-ters which is located in the heme-binding region. As a result, differ-ent from P450(11b)-1, P450(11b)-2 is likely to act as a bindingprotein to regulate the substrate concentration in steroid produc-ing cells. The cell type in which P450(11b)-2 expresses should bedetermined by ISH or IHC. However, no suitable probe can be de-signed because of exact sequence homology among both isoforms,except for missing 7th and 8th exons. As for mammals, the case ofalternatively spliced forms of P450(11b) has rarely been reported,except for ovine species (Anwar et al., 1998).
The expressions of tilapia P450(11b)-1 and -2 displayed a sexu-ally dimorphic pattern in gonad, only in the testis but not in ovaryof both the developing and adult fish. In fact it has been reported inseveral teleosts, such as the Japanese eel (Jiang et al., 1998), rain-bow trout (Liu et al., 2000; Kusakabe et al., 2002) and medaka(Yokota et al., 2005), that P450(11b) was not detected in the ovaryin the mRNA level, even though positive P450(11b) staining by IHChas been reported in the ovary of the protogynous honeycombgrouper (Alam et al., 2005). Furthermore, the temporal and spatialexpression of the two P450(11b)s were basically the same, excepttheir difference in expression level. Functional feminization ormasculinization can be obtained in most fish species using estro-gen (Baron et al., 2008), estrogen receptor antagonist and aroma-tase inhibitor treatment due to the high sensitivity of gonadalphenotype of fish to sex steroid. In medaka, morphologically sex-reversal was observed in XY and testicular expression ofP450(11b) was inhibited by estrogenic chemicals treatment (4-tert-pentylphenol) (Yokota et al., 2005). To determine if the expres-sions of P450(11b)s were affected by estrogen, estrogen receptorantagonist and aromatase inhibitor treatment, the expressions ofP450(11b)s were investigated by drug treatment (E2, F and TAM).In the present study, E2 (XY), F and TAM (XX) treatment resultedin complete sex reversal in tilapia, respectively. F, TAM and E2were very effective drugs for inducing female-to-male and male-to-female sex reversal, respectively, in the Nile tilapia. F andTAM treatment caused a down-regulation of all steroidogenic en-zymes, including aromatase, and the transcription factor Foxl2(‘‘pro-ovary” gene), up-regulation of Dmrt1(‘‘pro-testis” gene);while E2 treatment resulted in the up-regulation of aromataseand Foxl2, down-regulation of Dmrt1 in the Nile tilapia (Kobayashiet al., 2003; Bhandari et al., 2006; Wang et al., 2007; Ijiri et al.,2008). Simultaneously, strong expression signals of P450(11b)-1and -2 were detected in F and TAM treated gonad (XX), whereascompletely inhibited in E2 treated gonad (XY), displaying a tes-tis-dependent expression pattern. As it was not a direct down-stream gene of estrogens, its up- and down-regulations wereprobably dependent on transcription factors, such as Ad4BP/SF-1,which was reported to regulate the expression of P450(11b) inmammals (Morohashi and Omura, 1996), and it probably also reg-ulates P450(11b) in fish. Nevertheless, the molecular mechanismsof steroid action during feminization or masculinization in fish stillvaguely understood. Ontogenic expression showed that P450(11b)-1 and -2 were detected in testis from 50dah onwards, rather closelycorrelates with the initiation of spermatogenesis (70dah onwards)(Kobayashi et al., 2000). Furthermore, strong expression signal ofP450(11b)-1 was detected in Leydig cells of the testis, co-localizedwith other steroidogenic enzymes (P450c17, 11b-HSD and SCC)(Kusakabe et al., 2003; Kobayashi et al., 2005; Zhou et al., 2007)responsible for the biosynthesis of 11-KT as reported in several
teleosts (Kusakabe et al., 2002; Wang et al., 2002; Yazawa et al.,2008). Taken together, our data suggested that the P450(11b)-1of tilapia is the key enzyme for the biosynthesis of 11-KT in testis,which in turn, has key roles in initiating spermatogenesis asproved in the Japanese eel (Miura et al. 1991). Therefore,P450(11b) is essential for the spermatogenesis and maintenanceof testis function.
Recent investigations demonstrated that estrogen plays a keyrole in determining the direction of gonadal sex differentiation,while androgen has no direct effects, in non-mammalian verte-brates (Vizziano et al., 2007; Ijiri et al., 2008). Previous studiesfrom our group revealed that the main steroidogenic enzymes,such as P450c17, SCC and 3b-HSD were detected only in the XX go-nads, but not in the XY gonads, before and during the morpholog-ical sex differentiation (Nagahama, 2000). P450(11b) is keyenzyme for the synthesis of 11-KT, the main androgen detectedin the Nile tilapia. In the present study, the initiation ofP450(11b) expression in gonads starts from around 50dah, longafter the initiation of morphological sex differentiation (around25dah) in Nile tilapia. Taken together, we conclude that androgensincluding 11-KT were not involved in the sex differentiation in tila-pia. However, treatment of tilapia fry with exogenous androgen,such as MT, can induce sex reversal. This can be explained as bothARa and ARb have started expression before the initiation of mor-phological sex differentiation in tilapia (Sudhakumari et al., 2005).In the present study, P450(11b) were also found to be expressed inthe spermatogonia detected by ISH and IHC. Similar findings havebeen reported in sea bass too, in which relatively high level ofP450(11b) expression were detected in spermatogonia comparedwith spermatocytes and spermatids using laser capture microdis-section and quantitative-PCR, and therefore, the authors suggestedthat some functions that were characteristic of a particular celltype in mammals can be accomplished in more than one cell typein lower vertebrates (Viñas and Piferrer, 2008). These data suggestthat in fish, or at least in the sea bass and tilapia, P450(11b) is prob-ably not only predominantly expressed in the Leydig cells, but alsoexpressed in spermatogonia. Whereas, the exact role of P450(11b)in spermatogonia and spermatocyte needs further investigationand clarification.
Besides, P450(11b)s were also detected in the head kidney andkidney of both sexes by RT-PCR in our study. Expression ofP450(11b) in the head kidney has been reported in many teleosts(Liu et al., 2000; Socorro et al., 2007), and it is well known thatP450(11b) is responsible for the cortisol biosynthesis in interrenaltissue (Jiang et al., 1998). Further studies are needed to elucidatethe relationship between cortisol, androgen production and sper-matogenesis, also the possible interference between cortisol andandrogen production along the reproductive cycle. In the presentstudy, P450(11b) was also found to be expressed in the kidney intilapia. The same thing has only been reported in the air-breathingcatfish (Clarias gariepinus) and sea bass (Rasheeda et al., 2005;Socorro et al., 2007). Further studies are needed to understand inwhich cell type it is expressed and what functions it exerts in thekidney.
Acknowledgments
This work was supported by grants from the National HighTechnology Research and Development Program (863 program)of China (No. 2007AA10Z165), the National Natural Science Foun-dation of China (Nos. 30770272, 30871905 and 30831160508), theNational Basic Research Program of China (2009CB941200), theProgram for Changjiang Scholars and Innovative Research Teamin University (IRT0859), the Natural Science Foundation Projectof CQ CSTC (No. CSTC, 2008BB1006), the Science and TechnologyInnovation Fund for the Graduates of Southwest University (No.
W.-L. Zhang et al. / General and Comparative Endocrinology 165 (2010) 34–41 41
b2007006) and in part by a Grant-in-aid for Scientific Researchfrom the Solution-Oriented Research for Science and TechnologyResearch Project of Japan Science and Technology Corporation.
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