Androgen rather than estrogen up-regulates brain-type cytochrome P450 aromatase (cyp19a1b) gene via tissue-specific promoters in the hermaphrodite teleost ricefield eel Monopterus albus

Molecular and Cellular Endocrinology 350 (2012) 125–135

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Androgen rather than estrogen up-regulates brain-type cytochrome P450 aromatase (cyp19a1b) gene via tissue-specific promoters in the hermaphrodite teleost ricefield eel Monopterus albus Yang Zhang a,1,2, Shen Zhang a,1, Wenliang Zhou a, Xing Ye c, Wei Ge d, Christopher H.K. Cheng e, Haoran Lin a,b, Weimin Zhang a,b,⇑, Lihong Zhang a,⇑ a

Department of Biology, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, PR China State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, PR China c Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510380, PR China d Department of Biology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong e School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong b

a r t i c l e

i n f o

Article history: Received 14 June 2011 Received in revised form 1 December 2011 Accepted 2 December 2011 Available online 9 December 2011 Keywords: Monopterus albus cyp19a1b Tissue-specific promoter Estrogen Androgen

a b s t r a c t CYP19A1 in the brain and pituitary of vertebrates is important for reproductive and non-reproductive processes. In teleosts, it is broadly accepted that estradiol (E2) up-regulates cyp19a1b gene via a positive autoregulatory loop. Our present study, however, showed that E2 did not up-regulate ricefield eel cyp19a1b in the hypothalamus and pituitary, whereas dihydrotestosterone (DHT) or testosterone (T) stimulated cyp19a1b expression only in the pituitary. Two tissue-specific promoters, namely promoter I and II directing the expression in the brain and pituitary respectively, were identified. Promoter I contained a non-consensus estrogen response element (ERE), and consequently did not respond to E2. Promoter II contained an androgen response element (ARE) and consequently responded to DHT. Taken together, these results demonstrated a novel steroidal regulation of cyp19a1b gene expression and an alternative usage of tissue-specific cyp19a1b promoters in the brain and pituitary of a teleost species, the ricefield eel. Ó 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction In vertebrates, estradiol, the main form of estrogens and a pleiotropic sexual steroid hormone, is crucial for reproduction by modulating the hypothalamic–pituitary–gonadal (HPG) axis (Naftolin et al., 2007). The biosynthesis of estradiol from testosterone is catalyzed by aromatase, an enzyme complex consisting of a specific cytochrome P450arom (the product of CYP19A1 gene) and a ubiquitous NADPH-dependent cytochrome P450 reductase (Simpson et al., 1994). In addition to gonads, cyp19a1 gene has also been shown to be expressed in other tissues including the brain and pituitary of vertebrates from teleosts to rat and human (Galmiche et al., 2006a; Kadioglu et al., 2008; Kamat et al., 2002; Nocillado ⇑ Corresponding authors. Address: Department of Biology, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, PR China. Tel.: +86 20 8411 0828; fax: +86 20 8411 3327 (L. Zhang); tel.: +86 20 8411 3327 (W. Zhang). E-mail addresses: [email protected] (W. Zhang), [email protected] (L. Zhang). 1 These authors contributed equally to this work. 2 Present address: The Key Laboratory of Applied Marine Biology of Guangdong Province and Chinese Academy of Sciences, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, PR China. 0303-7207/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2011.12.001

et al., 2007; Zhang et al., 2008), where the estrogen receptors (ERs) are also present (Childs et al., 2001; Kitahashi et al., 2007; Weiser et al., 2008). In the brain, aromatase is only expressed in radial glial cells of fish (Diotel et al., 2010) whereas mainly in neurons of birds and mammals under normal conditions and in reactive astrocytes after brain injury (Roselli, 2007). In the pituitary, aromatase is expressed in the gonadotropes of rats (Galmiche et al., 2006b). It has been shown that in the pituitary of mice, testosterone must be aromatized to suppress luteinizing hormone beta subunit (Lhb) expression, as its suppressive effects on Lhb could be observed only in wild-type castrates but not in estrogen receptor-a knockout castrates (Lindzey et al., 1998), suggesting that locally produced estradiol may also play important regulatory roles in the hypothalamic–pituitary–gonadal axis via paracrine and/or autocrine pathways. Thus the elucidation of the regulatory mechanisms of cyp19a1 gene expression in each part of HPG axis is of great significance. In mammals except pigs and peccaries (Corbin et al., 2007), cyp19a1 is a single-copy gene and its tissue-specific expression patterns are driven by different tissue-specific promoters (Kamat et al., 2002; Silandre et al., 2007), with promoter I.f used in the brain, and promoter II in the ovary and testis of human (Bulun

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et al., 2004), and promoters II and I.f in rat pituitary (Galmiche et al., 2006a, 2006b). In most teleosts, however, duplicated cyp19a1 genes have been identified, with cyp19a1a predominantly expressed in the ovary while cyp19a1b predominantly in the brain (Chang et al., 2005; Nocillado et al., 2007; Sawyer et al., 2006; Tchoudakova and Callard, 1998; Zhang et al., 2004, 2008). The duplicated cyp19a1 genes are located on different chromosomes (Harvey et al., 2003), and each isoform has its own distinct regulatory region (Kazeto et al., 2001; Nocillado et al., 2007; Tchoudakova et al., 2001; Tong and Chung, 2003). The transcription of cyp19a1b was shown to be initiated at different sites tissue-specifically in rainbow trout (Toffolo et al., 2007), and Intron I of grey mullet cyp19a1b had promoter activities in vitro (Nocillado et al., 2007), however, whether tissue-specific promoters drive the expression of cyp19a1b in teleosts or not remained to be an interesting question. The ricefield eel, a protogynous hermaphrodite fish inhabiting still- or slow-current waters with muddy bottoms where it burrows (Matsumoto et al., 2011), changes sex from functional female, through an intersexual stage when the gonad consisting of degenerating ovarian tissues and germinating testicular tissues, to the functional male phase during its life cycle (Chan and Phillips, 1967; Liem, 1963; Liu, 1944). Our previous study showed that cyp19a1b expression was up-regulated transiently in the hypothalamus whereas down-regulated in the pituitary of ricefield eels during sex changes (Zhang et al., 2008), suggesting possible differential regulatory mechanisms of cyp19a1b expression in the hypothalamus and pituitary of this species. In the present study, alternative tissue-specific promoters for cyp19a1b expression in the brain and pituitary of ricefield eel were identified. Previous studies in teleosts demonstrated that cyp19a1b gene was upregulated by estradiol (Callard et al., 2001; Le Page et al., 2008; Mouriec et al., 2008; Strobl-Mazzulla et al., 2008), and an estrogen response element (ERE) site specific to the promoter region of teleost cyp19a1b gene (Gardner et al., 2005; Kazeto et al., 2001; Nocillado et al., 2007; Tchoudakova et al., 2001; Tong and Chung, 2003) has been reported to confer its estrogen responsiveness (Sawyer et al., 2006). However, our present study showed that the expression of ricefield eel cyp19a1b gene failed to respond to E2 stimulation due to the alterations of the conserved ERE site in the 50 flanking sequence. Furthermore, it is shown that a functional androgen response element (ARE) in the pituitary-specific promoter confers a direct up-regulation of cyp19a1b expression by androgens in the pituitary of the ricefield eel.

2. Materials and methods 2.1. Animals and tissue collection The ricefield eel used in the present study were obtained from a local dealer in Guangzhou, Guangdong Province, PR China. All procedures and investigations were reviewed and approved by the Center for Laboratory Animals of Sun Yat-Sen University, and were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals. Fish were sacrificed by decapitation, and tissues were dissected out, frozen immediately in liquid nitrogen and stored at 80 °C until RNA or DNA extraction. The phenotypic sex and gonadal developmental stages of ricefield eel were verified by histological sectioning of gonads and microscopic analysis as reported previously (Zhang et al., 2008). The ovaries of the female fish used in the present study were at vitellogenic stages (EV and MV with oocytes at early and mid-vitellogenic stages, gonadosomatic index (GSI) 1–2.5%), and the gonads of the intersexual fish at middle and late intersexual stages (IM and IL) (Zhang et al., 2008).

2.2. In vitro incubation of the hypothalamus and pituitary The hypothalami and pituitary glands of female (vitellogenic stage) or intersexual (mixed with some male, about 2.5%) ricefield eels (n = 120) were dissected out and washed in M199 media (Sigma, St. Louis, MO) on ice. Since female protogynous hermaphroditic ricefield eels are sex reversed to males, it is usually hard to obtain enough male fish to establish a separate male group. Thus the tissues from a few number of male fish were included in the group of intersexual fish for the in vitro incubation experiments in the present study. The hypothalamus was cut into 1 mm thick fragments and washed twice with M199. Approximately 30 mg of hypothalamic minces (pooled from 120 individuals) or five pituitary glands from each group were placed in each well of a 24-well tissue culture dish with 1 ml of M199 media (Sigma) containing 100 U/ml penicillin (Gibco, Gaithersburg, MD) and 0.1 mg/ml streptomycin (Gibco), and then incubated at 25 °C in a humidified incubator under 5% CO2. E2 and DHT stock solutions were prepared in 100% ethanol at concentrations 103-fold higher than the final, respectively. After pre-incubation for about 6 h, the hypothalamus fragments and pituitary glands were treated with E2 (1, 10, and 100 nM), DHT (1, 10, and 100 nM) or ethanol (0.1%, control group) for 24 h. Triplicates were performed for each treatment. After completion of incubation, tissues were collected and total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA) for subsequent real-time PCR analysis of cyp19a1b mRNA levels. 2.3. In vivo treatment of ricefield eels with sex steroids The experiment was conducted in the October 2010. Ricefield eels at the intersexual stages were acclimatized to laboratory conditions at temperatures 24–26 °C under a natural photoperiod and fed with Chironomus larvae. After anaesthesia with tricaine methanesulphonate (MS222; 0.5 g/L) in the water, 50 intersexual ricefield eels (mean body weight 35.2 ± 1.2 g, 10 fish for each treatment group) were implanted in the intraperitoneal cavity with a control (no steroid) or solid silastic implant containing estradiol (E2, 50 and 100 lg g1 body weight) or testosterone (T, 50 and 100 lg g1 body weight). The fish were sacrificed 24 h after implantation, and tissue samples were obtained. The hypothalamus and pituitary were dissected out, snap-frozen in liquid nitrogen, and kept at 80 °C until RNA extraction. The blood was collected, kept at 4 °C for about 4 h, and centrifuged at 10,000g for 5 min to obtain the serum. The serum samples were extracted with ethyl ether, and estradiol or testosterone concentrations were assayed using RIA kits with 125I-labelled ligands (Beijing North Institute of Biological Technology, Beijing, China) according to the manufacture’s protocols. The Radioactivity was counted on a Wallac Wizard 1470 automatic gamma counter (73% efficiency. PerkinElmer, Turku, Finland). The ligand extraction efficiencies were about 92% and 91% for estradiol and testosterone, respectively. The detection limit of the estradiol RIA kit (B05PZB, lot number 101020) was about 2 pg/ml, and the cross-reactivity of the anti-estradiol antibody was 0.016% with estriol, 0.01% with progesterone, and 0.01% with testosterone. The detection limit of testosterone RIA kit (B10PZB, lot number 101025) was about 0.02 ng/ml, and the cross-reactivity of the anti-testosterone antibody was 0.011% with dihydrotestosterone, 0.021% with estradiol, 2  1015% with estriol, 0.032% with progesterone, and 1.2  105% with androstenedione. The E2 and T-treated fish had significantly higher serum estradiol and testosterone levels as compared to the control fish, respectively (data not shown). 2.4. Real-time PCR analysis of cyp19a1b mRNA levels Real-time PCR assay for quantification of cyp19a1b mRNA levels in the present study was performed as previously reported (Zhang

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et al., 2008) except gapdh was used as an internal control. The sequences of oligonucleotides used in the present study are listed in Supplementary Table S1. The primers used for real-time PCR analysis were CYP19A1BQF1 and CYP19A1BQR1 for cyp19a1b (EU252488), GAPDHQF1 and GAPDHQR1 for gapdh (FJ873738). The real-time PCR was performed on the Rotor-Gene 3000 Detection System (Corbett Research, Sydney, Australia) in a volume of 20 lL containing 0.2 lM of each primer, 10 lL of 2 SYBR Green Master Mix (Toyobo, Osaka, Japan), l lL of cDNA template which was transcribed from 1 lg total RNA using ThermoScript™ RT-PCR System (Invitrogen). The PCR cycling conditions were: 95 °C for 1 min; 40 cycles of 95 °C for 15 s, 55 °C for 15 s, 72 °C for 30 s; 85 °C for 20 s for signal collection in each cycles. Data were analyzed by the Rotor-gene version 6-0-22 software. The quantification of the mRNA level was performed using a standard curve with 10-fold serial dilution of plasmids containing corresponding DNA fragments from 101 to 108 copies. The correlation coefficients and PCR efficiencies were not less than 0.997% and 91%, respectively. To minimize variation due to differences in RNA loading, each sample was normalized to the expression level of the house keeping gene gapdh, and the relative expression levels of cyp19a1b were calculated as the copy number ratios to gapdh. The results were presented as fold induction relative to controls. 2.5. Cloning and analysis of 50 flanking region of cyp19a1b Genomic DNA was extracted from the liver of female ricefield eel by phenol/chloroform method and a GenomeWalker library was constructed using a Universal GenomeWalker™ Kit (Clontech, Mountain View, CA) according to the manufacturer’s protocol. The 50 flanking region of ricefield eel cyp19a1b was isolated from the GenomeWalker library by a PCR-based genomic walker technique, using adaptor primer AP1 and gene-specific primer CYP19A1BW1R for the first PCR, and adaptor primer AP2 and gene-specific primer CYP19A1BW2R for the nested PCR. The PCR cycling conditions were: 94 °C for 3 min; 40 (the first) or 36 (the nested) cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 3 min; and final 30 min extension. The targeted PCR products were gel-purified using Gel Extraction System (Omega Bio-Tek, Inc., Norcross, GA), subcloned into pGEM-T Easy Vector (Promega, Madison, WI) and sequenced. Promoter sequences were analyzed with the web-based software Transcription Element Search System (TESS) (http://www.cbil.upenn.edu/tess) to search for putative transcription binding sites. The sequence homology was analyzed using the Megalign of the DNAstar software package. The alignment of ERE-containing regions of cyp19a1b promoter in teleosts was performed with web-based software BIPAD (Bi and Rogan, 2006). 2.6. RLM-50 RACE analysis of cyp19a1b transcripts in the brain and pituitary To determine 50 ends of ricefield eel cyp19a1b transcripts in the brain and pituitary, RNA ligase-mediated rapid amplification of cDNA 50 ends (RLM-50 RACE) was performed using the GeneRacer™ kit (Invitrogen), which can specifically amplify cDNAs only from full-length 50 -capped mRNA. Briefly, five micrograms of total RNA isolated from the brain or pituitary of female fish was processed according to the manufacturer’s protocol to obtain the RACE-ready cDNA. The RACE-ready cDNA was PCR-amplified using primers GR5P and CYP19A1BR1 for the first PCR, and GR5NP and CYP19A1BR2 for the nested PCR. The PCR products were gel-purified, ligated into TA cloning vector pGEM-T Easy (Promega) and sequenced.

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2.7. RT-PCR analysis of cyp19a1b transcripts of different 50 untranslated regions (UTRs) Total RNA (2 lg) isolated from different tissues of female ricefield eel was first treated with DNase I, and then reversetranscribed as previously (Zhang et al., 2008). The first-strand reaction (0.5 ll) was amplified for cyp19a1b gene with either CYP19A1BF1 (located in Exon I) or CYP19A1BF2 (located in Exon II) as upstream primer and CYP19A1BR2 as downstream primer, and for bactin gene (AY647143) with primer pair b-actinF2 and b-actinR2 using the Biometra TGRADIENT thermal cycler. PCR was performed in 15 lL final volume containing 1.5 ll 10 reaction buffer, 1.5 mM MgCl2, 0.2 mM dNTP, 0.2 lM of each primer, and 1.0 U Platinum Taq DNA Polymerase (Invitrogen). Water was used as a negative control in the RT-PCR reaction. The PCR reaction mixture was heated at 94 °C for 2 min, followed by 34 cycles of amplification for the cyp19a1b, and 28 cycles for bactin. The PCR products for cyp19a1b produced with the two sets of primers were analyzed on the same agarose gel (1.5%) with two rows of loading wells and stained with ethidium bromide (0.5 lg/ml) to compare the abundance of the amplification products. The bactin gene was used as a control for integrity of the RNA samples and the efficiency of reverse transcription. The gel image was captured on the Gel-Doc 2000 and analyzed with the software Quantity One (Bio-Rad). The RT-PCR products from the pituitary using different primers were presented qualitatively as the difference between the two sets of primers was phenomenal. To further confirm the authenticity of PCR amplification, the PCR products for cyp19a1b on the agarose gel after electrophoresis were transferred onto a nylon membrane by the capillary method. The membrane was hybridized overnight at 48 °C with probes of cyp19a1b (nt 18-745), which was randomly labeled with [a-32P]dCTP using the Random Primer DNA Labeling kit (TaKaRa, DaLian, China). After washing twice with low stringency wash solution (equal to 2 SSC, 0.1% SDS) at 48 °C for 10 min and high stringency wash solution (equal to 0.1 SSC, 0.1% SDS) twice at 48 °C for 15 min, the membrane was exposed to a phosphor storage screen and visualized with a Typhoon 8600 Variable Mode Imager (Molecular Dynamics, CA, USA). 2.8. Reporter plasmid construction The putative promoter region, the 50 flanking region of transcript in the brain (designated as promoter I) or Intron I (putative promoter for transcript in the pituitary, and designated as promoter II) of the ricefield eel cyp19a1b, was inserted upstream of the Firefly luciferase gene of the pGL3-basic vector (Promega) to generate reporter plasmid, and Renilla luciferase pRL-Tk vector (Promega) was used as an internal control. For promoter I, the fragment 2022/+103 (+1 corresponding to the transcription initiation site in the brain) was amplified by PCR with primers CYP19A1BPF1 and CYP19A1BPR1, in which restriction enzyme sites Kpn I and Xho I were introduced, respectively. The PCR fragment of promoter I or pGL3-basic vector were digested with Kpn I and Xho I, and ligated to generate the reporter plasmid pGL3-PI. The promoter II reporter plasmid pGL3-PII containing a fragment of 1519/+124 (+1 corresponding to the transcription initiation site in the pituitary) was constructed as above with primers CYP19A1BPF2 and CYP19A1BPR2. The identity of insert was verified by sequencing. To assess the functionalities of ERE-like motif in the promoter I and ARE motif in the promoter II to hormonal stimulation, ERE-like and ARE motifs were mutated by fusion PCR with a pair of complementary primers containing the desired nucleotides (Supplementary Table S1). Three types of mutations were generated for ERE-like motif (AGATCAgtcTGACCA): (1) 5 A ? G with primers CYP19A1BmF1 and CYP19A1BmR1; (2) +7 A ? C with primers CYP19A1BmF2 and CYP19A1BmR2; (3) 5 A ? G plus +7 A ? C by

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sequential mutation using the above primers. The functional ARE (AGAACAatcTGTTCA) were mutated to nonfunctional forms (GT ? CC) (Spady et al., 2004) with primers CYP19A1BmF3 and CYP19A1BmR3. Finally, the PCR fragments were inserted into pGL3-basic vector and verified by DNA sequencing. As controls, the reporter plasmids for the putative promoter of the orange-spotted grouper cyp19a1b (FJ914569), with either its original ERE or ricefield eel ERE-like motif, were constructed similarly. The orange-spotted grouper ERE was mutated to ricefield eel ERE-like motif with primer set EcEREm-F and EcEREm-R (Supplementary Table S1) using QuikChange Site-Directed Mutagenesis Kit (Stratagene). 2.9. Cell culture U251-MG (human astrocyte) and LbT2 (mouse gonadotrope, generously provided by Dr. Pamela Mellon, University of California-San Diego, La Jolla, CA) cells were grown in high-glucose DMEM (Gibco) containing 10% fetal calf serum (Gibco) and 1 mg/ ml penicillin–streptomycin (Gibco) at 37 °C in a humidified incubator under 5% CO2. 2.10. Transfection and dual-luciferase reporter assay Plasmids for transfection were prepared from overnight bacteria culture using PureLink™ HI-Pure Plasmid DNA Purification Kit (Invitrogen) according to the manufacturer’s protocol. Twenty-four hours prior to transfection, U251-MG or LbT2 cells were plated to 24-well plates (105 cells/well) in fresh phenol red-free high-glucose DMEM (Gibco) containing 10% charcoal/dextran fetal calf serum (Gibco), and transiently co-transfected with pGL3 reporter vectors (380 ng/well) and an internal control vector pRL-TK (20 ng/well) using Lipofectamine 2000 (Invitrogen, 1 lL/well) according to the manufacturer’s instruction. The transfection medium was replaced with fresh medium 4 h later, and luciferase activities were measured 48 h after transfection. To assess the functionalities of ERElike and ARE motifs, the cells were also co-transfected with goldfish ESR1 (Jiao and Cheng, 2010) or ricefield eel AR (FJ873736) expression vector (50 ng unless otherwise specified), and E2 or DHT was added 24 h after transfection. Each transfection reaction was carried out in triplicates, and the experiments were repeated at least three times. The Firefly and Renilla luciferase activities were measured for each sample using Dual-Luciferase Reporter Assay System (Promega). The Firefly luciferase data were corrected for transfection efficiency with Renilla luciferase activity. The experimental results are expressed as fold changes relative to pGL3-basic vector unless otherwise specified. 2.11. Statistical analysis All data are presented as means ± SEM. The significance of observed differences between groups was determined by one-way ANOVA followed by the Dunnett test (for comparing treatment groups with the control group) or Tukey multiple comparison test (for comparing all pairs of groups) as specified in figure legends using the SPSS software package. Where only two groups were compared in a data set, Student’s t-test was used instead. Significance was set at P < 0.05.

of intersexual (Fig. 1) and female (Fig. 2) ricefield eel. E2 did not significantly increase cyp19a1b mRNA levels in either the pituitary (Figs. 1A and 2A) or the hypothalamus (Figs. 1B and 2B) of ricefield eel at either the intersexual or female stage. On the other hand, DHT stimulated cyp19a1b expression in a dose-dependent manner only in the pituitary (Figs. 1C and 2C) but not hypothalamus (Figs. 1D and 2D) of intersexual or female ricefield eel. In agreement, the in vivo experiments with implantation of E2 or T showed that only the expression of cyp19a1b in the pituitary was significantly increased by T treatment (Fig. 3B). 3.2. Ricefield eel cyp19a1b gene is driven by different promoters in the brain and pituitary In order to clarify why androgens stimulated the expression of ricefield eel cyp19a1b in the pituitary but not the hypothalamus, we examined the 50 ends of cyp19a1b transcripts in the pituitary and brain (including hypothalamus). In the brain of ricefield eel, three cyp19a1b transcripts of different 50 UTRs, 50 UTR-type 1, 50 UTR-type 2, and 50 UTR-type 3, were identified (Fig. 4), which had a common Exon I and 50 splice donor site (50 SS) within intron I, but different 30 splice acceptor sites (30 SS). The transcript of 50 UTR-type 1 was identical to ricefield eel cyp19a1b cDNA (EU252488), which was used to define the boundaries of intron I. The transcript of 50 UTR-type 2 was spliced to a 30 SS within intron I, which led to the inclusion of a 75-bp fragment of intron I. The transcript of 50 UTR-type 3 was spliced to a 30 SS within exon II, which led to the deletion of majority of exon II including the initial AUG. Therefore, a putative AUG for the transcript of 50 UTR-type 3 can be only identified in exon IV, which may encode for a nonfunctional aromatase. In the pituitary of ricefield eel, however, a novel cyp19a1b transcript, 50 UTR-type 4, was identified (Fig. 4). 50 UTR-type 4 transcript was transcribed from transcription start site 2 (Tss2) within intron I (Fig. 4), which leads to the exclusion of exon I but all other exons. Like cyp19a1b transcripts of 50 UTR-type 1 and 2 in the brain, cyp19a1b transcript of 50 UTR-type 4 in the pituitary may encode a functional aromatase. The cyp19a1b transcripts in the brain and pituitary of ricefield eel were also examined by RT-PCR coupled with Southern blotting (Fig. 5). PCR with the primer set spanning exon I and exon VI (Fig. 5A), which presumably amplifies transcripts of 50 UTR-type 1, 2, and 3 in the brain, revealed that the expression of cyp19a1b gene was mostly restricted to the brain, especially the olfactory bulb, telencephalon, hypothalamus and optic tectum-thalamus. However, PCR with the primer set spanning exon II and exon VI (Fig. 5B), which presumably amplifies transcripts of 50 UTR-type 4 in the pituitary as well as transcripts of 50 UTR-type 1 and 2 in the brain, revealed high expression of cyp19a1b in the pituitary as well as the brain. Furthermore, primer extension assay detected one single extended product in the pituitary, which was consistent with transcript of 50 UTR-type 4 (Supplementary Fig. S1). Thus, it could be speculated that the 50 flanking region of cyp19a1b (designated as promoter I) directed the expression in the brain, whereas intron I (designated as promoter II) directed the expression of cyp19a1b in the pituitary of ricefield eel. 3.3. The ERE site is not conserved in the 50 flanking region (promoter I) of ricefield eel cyp19a1b gene

3. Results 3.1. DHT or T instead of E2 up-regulated ricefield eel cyp19a1b gene only in the pituitary The effects of E2 or DHT on cyp19a1b expression were firstly examined in vitro with incubated hypothalami and pituitary glands

A 2022-bp cyp19a1b 50 flanking sequence (Supplementary Fig. S2, FJ873735) was isolated from GenomeWalker libraries by PCR. Surprisingly, no consensus TATA box at an appropriate distance from the 50 end of cyp19a1b cDNA was identified in this region. However, two overlapped downstream core promoter elements (DPE)-like sequences (Smale and Kadonaga, 2003) were

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Fig. 1. Effects of E2 and DHT on cyp19a1b mRNA levels in the in vitro incubated pituitary (A and C) and hypothalamus (B and D) of intersexual ricefield eel. The pituitary glands or hypothalami were taken out and incubated for 24 h in the presence or absence (control, containing 0.1% ethanol) of E2 or DHT (concentration at 1, 10 and 100 nM). The mRNA level of cyp19a1b was determined by real-time PCR. Data are expressed as fold induction relative to control. Each bar represents mean ± SEM of triplicates. ⁄P < 0.05 relative to the control (one-way ANOVA followed by the Dunnett test).

Fig. 2. Effects of estradiol (E2) and dihydrotestosterone (DHT) on cyp19a1b expression in the in vitro incubated pituitary (A and C) and hypothalamus (B and D) of female ricefield eel. The pituitary glands were taken out and incubated for 24 h in the presence or absence (control, containing 0.1% ethanol) of E2 or DHT (concentration at 1, 10 and 100 nM). The mRNA level of cyp19a1b was determined by real-time PCR. Data are expressed as fold induction relative to control. Each bar represents mean ± SEM of triplicates. ⁄P < 0.05 relative to the control (one-way ANOVA followed by the Dunnett test).

predicted around position +20. Some of the putative transcription factor binding sites identified with TESS analysis (Supplementary Fig. S2) include one ERE, one half-ERE (TGACC), one cAMP responsive elements (CRE), one NF-kappa B binding site, two signal transducers and activators of transcription (STATs), and one serum response factor (SRF). It is very interesting to note that the ERE sequence (50 -AGATCAgtcTGACCA-30 , designated as ERE-like) in the 50 flanking region

of ricefield eel cyp19a1b is non-consensus as containing altered nucleotides at positions 5(G ? A) and +7(Y ? A) when compared to the optimal ERE sequence (Driscoll et al., 1998) (Fig. 6A). Furthermore, ricefield eels obtained from different geographical locations of China contained the same ERE-like sequences (Supplementary Fig. S3), suggesting that the alterations of an ERE to an ERE-like was not an artifact. In contrast, the highly conserved ERE motifs (50 -RGGTCAnnnTGACCY-30 ), particularly the underlined

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at position 23 (+1 relative to the 50 end of the transcript in the pituitary) resembling the consensus TATA box. Furthermore, the sequence around position +1 was similar to the initiator (Inr), and two DPEs (Smale and Kadonaga, 2003) were predicted at position +27 and +37, respectively. TESS analysis revealed many putative transcription factor binding sites in the first intron of ricefield eel cyp19a1b gene which, of particular interest, included one ARE, five POU transcriptional factor binding sites, one CRE, one Egr-1 binging site, two NF-Y binding sites, and two consensus binding motifs for SF-1/Ad4BP. 3.5. E2 failed to up-regulate ricefield eel cyp19a1b promoter I and II in vitro

Fig. 3. The in vivo effects of E2 and testosterone (T) on cyp19a1b expression in the hypothalamus and pituitary. The ricefield eels at the intersexual stages were treated with E2 or T (50 and 100 lg g1 body weight) by peritoneal implantation for 24 h. The mRNA levels of cyp19a1b in the hypothalamus (A) and pituitary (B) were quantified with real-time PCR, and data are expressed as fold induction relative to the control. Each bar represents mean ± SEM (n = 5–7). ⁄P < 0.05 relative to the control group (one-way ANOVA followed by the Dunnett test).

perfect 13-bp core consensus sequences, were observed in the 50 flanking regions of cyp19a1b in other teleosts (Fig. 6B). 3.4. An ARE identified in Intron I (promoter II) of cyp19a1b gene Sequence analysis of Intron I of ricefield eel cyp19a1b gene (Supplementary Fig. S4, EU840259) identified a sequence TAAAAA

E2 (10 nM) had no direct effects on the activities of both promoter I and II in either U251-MG or LbT2 cells (Fig. 7A and B). Even at a higher concentration (100 nM), E2 could not stimulate the promoter I activity (Supplementary Fig. S5A). However, mutation of non-consensus ERE-like sequence of promoter I back to optimal ERE restored the E2 responsiveness of promoter I in cultured U251-MG cells (Fig. 7C). At the concentration of 10 nM, E2 resulted in 4-fold increase in the activity of mutated promoter I when ERElike motif on the wild-type promoter I was mutated at position +7 from A to C, and about 11 folds when mutated at position 5 from A to G. As expected, dual mutations at both position 5 and +7 showed much higher effects (about 25 folds) than single mutation at either position (Fig. 7C), and E2 up-regulated the promoter activities dose-dependently (Supplementary Fig. S5A). In addition, the mutation of the ERE in the orange-spotted grouper cyp19a1b promoter to ERE-like substantially attenuated its responsiveness to E2 (10 nM), from about 40-fold to only 3-fold induction (Fig. 7C). 3.6. DHT up-regulated promoter II through the ARE motif with a higher potency in a gonadotrope cell line LbT2 DHT (10 nM) did not stimulate the activity of promoter I in either U251-MG cells or LbT2 cells, however, it up-regulated the activity of promoter II both in U251-MG cells and in LbT2 cells, with higher potencies in LbT2 cells (Fig. 8A and B). The stimulation by DHT of promoter II in LbT2 cells was dose-dependent, with a

Fig. 4. Different 50 untranslated regions (50 UTRs) of cyp19a1b transcripts obtained from the brain and pituitary, and a map of ricefield eel cyp19a1b gene. 50 UTR-type 1, 2, and 4 were revealed by RLM 50 RACE, and 50 UTR-type 3 revealed by RT-PCR analysis. 50 UTR-type-1–3 are possibly generated with transcription start site 1 (Tss1) in the exon I coupled with different 30 splice acceptor sites. 50 UTR-type 4 is possibly generated with transcription start site 2 (Tss2) in the intron I. The translation start codons (AUG) are located in exon II for 50 UTR-type 1, 2, and 4, whereas a potential AUG (pAUG) for 50 UTR-type 3 located in exon IV. Positions and orientations of primers used for 50 RACE are shown above the gene map as black arrows. The table indicates the numbers of clones isolated from the brain and pituitary. Gene structure was determined by alignment of the genomic sequence with cDNA sequence of cyp19a1b. Translated and untranslated exons are indicated as filled and empty boxes, respectively, and indicated by Roman numerals inside. The translation stop codon TGA in exon X is also indicated.

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Fig. 5. Tissue-specific expression of different 50 UTRs of cyp19a1b in female ricefield eel. (A) RT-PCR spanning exon I and exon VI of cyp19a1b. (B) RT-PCR spanning exon II and exon VI of cyp19a1b. (C) Tissue expression of reference gene bactin. The PCR products of cyp19a1b were stained by ethidium bromide (the top panels of A and B), and further analyzed by Southern blotting (the middle panels of A and B). The positions and orientations of primers used for cyp19a1b analysis are shown above the gene map as black arrows (the bottom panels of A and B). Ob, olfactory bulb; Te, telencephalon; Hy, hypothalamus; Ot, optic tectum-thalamus; Ce, cerebellum; Mo, medulla oblongata; RT, RT minus; NC, negative control (water as PCR template).

Fig. 6. Sequence analysis of ERE-like in the 50 flanking region of ricefield eel cyp19a1b. (A) Comparison with the optimal ERE showing the alterations at positions 5 and +7. R represents a nucleotide A or G, and Y represents a nucleotide T or C. (B) Alignment with ERE sites of other teleost cyp19a1b promoters shows that the nucleotides at 5 and +7 positions are not conserved in the ERE-like sequence of ricefield eel cyp19a1b. The proximal region from 310 to 276 of 50 flanking region of ricefield eel cyp19a1b was aligned by web-based software BIPAD (Bi and Rogan, 2006) to the proximal regions containing ERE of cyp19a1b promoters in other teleosts, including orange-spotted grouper (FJ914569), tilapia (AF472621), humpback grouper (AY686694), barramundi perch (AY686693), goby (AY686695), medaka (AY705086), grey mullet (AY859424), goldfish (AF324893), zebrafish (AF406756), and catfish (AY780360). The conserved nucleotides of ERE sequences are shadowed in gray.

significant induction at a concentration as low as 0.1 nM, reaching a plateau at the concentration of 1 nM and a maximal stimulation of 5.2-fold at the concentration of 10 nM (Supplementary Fig. S5B).

The stimulatory effects of DHT on cyp19a1b promoter II through the putative ARE motif were verified by site-directed mutagenesis using LbT2 cells (Fig. 8C). A 2-bp mutation at positions +3 and +4

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Fig. 7. Effects of E2 on the activities of promoter I (PI) and promoter II (PII) in U251-MG (A) or LbT2 (B) cell lines. Cells were transiently co-transfected with the cyp19a1b promoter I or II reporter plasmid together with goldfish ESR1 expression construct (gfESR1), and treated with E2 (10 nM) or 0.1% DMSO vehicle (as control) for 24 h before luciferase activities were measured. Data are expressed as fold change relative to promoterless pGL3-basic vector. Each bar represents mean ± SEM of triplicates. No significant difference was observed among each group (P > 0.05, one-way ANOVA followed by the Tukey multiple comparison test). (C) Site-directed mutagenesis analysis of ERE-like motif on PI of ricefield eel cyp19a1b and ERE motif on the orange-spotted grouper cyp19a1b promoter in U251-MG cells. The sequences of ERE or ERE-like motifs are shown above the promoter-depicting lines respectively, with site-mutated bases boxed. U251-MG cells were co-transfected with promoter reporter plasmids containing ERE, ERE-like, or site-mutated ERE-like motifs together with goldfish ESR1 expression construct, and treated as above. EBAPIwt and EBAPImutERE: reporter constructs containing promoter I of ricefield eel cyp19a1b with wild-type ERE-like motif and mutated ERE-like motifs, respectively; EcCyp19a1bwt and EcCyp19a1bmutERE: reporter constructs containing promoter of the orange-spotted grouper cyp19a1b with ERE motif and ERE-like motif, respectively. Data are expressed as fold changes relative to the control (E2 at 0 nM). Each bar represents mean ± SEM of triplicates. ⁄P < 0.05 for differences between wild-type and mutants of ricefield eel cyp19a1b promoter I in response to E2 at 10 nM (one-way ANOVA followed by the Dunnett test). #P < 0.05 for the difference between the wild-type and mutant of the orange-spotted grouper cyp19a1b promoter in response to E2 at 10 nM (Student’s t-test).

from GT to CC of the putative ARE motif completely abolished DHTinduced reporter gene expression. In addition, flutamide, an antiandrogen, significantly reduced DHT-induced promoter II activity (Fig. 8D). 4. Discussion In the brain of vertebrates, emerging evidence suggests the important roles of CYP19A1 in regulating reproductive as well as non-reproductive processes (Garcia-Segura, 2008; Roselli, 2007). In the pituitary, CYP19A1 was shown to be involved in the regulation of Lhb by testosterone in mice (Lindzey et al., 1998), and in teleostean species such as catfish (Kazeto and Trant, 2005), suggesting that the involvement of aromatase in the regulation of pituitary lhb may be common in vertebrates. However, the molecular mechanisms underlying the regulation of cyp19a1 gene in the brain, and particularly in the pituitary, are still poorly understood. In vertebrates, the expression of cyp19a1 gene is tissue-specific, which ensures the physiologically relevant production of estrogens. In some species like pigs and teleosts, gene duplication provided a mechanism for tissue-specific regulation of cyp19a1 gene (Kamat et al., 2002). On the other hand, a single copy of CYP19A1 gene was identified in human and rat, and the tissue-specific expression of CYP19A1 gene was controlled by tissue-specific promoters (Kamat et al., 2002; Silandre et al., 2007). In teleost, different transcripts of cyp19a1b with different 50 ends were identified in tilapia (Chang et al., 2005), and tissue-specific transcription start

sites of cyp19a1b were observed in rainbow trout (Toffolo et al., 2007). These results suggested that alternative promoters may be utilized in tissue-specific expression of cyp19a1b gene in fish (Nocillado et al., 2007), and the possible existence of specific zebrafish cyp19a1b promoter in pituitary cells was also raised recently (Vosges et al., 2010). However, our present study demonstrated the presence and functionalities of the tissue-specific promoters of cyp19a1b gene in ricefield eel, a teleost fish. Our results showed that promoter I, located in the 50 flanking region, directed the expression of cyp19a1b in the brain whereas promoter II, located in the first intron, directed the expression in the pituitary of ricefield eel. To our knowledge, this is the first report showing tissue-specific promoter regulation of cyp19a1 gene in the pituitary of nonmammalian vertebrates. In contrast to the case in mammals where promoters directing cyp19a1 expression in the pituitary may also be used in the brain or ovary (Galmiche et al., 2006a, 2006b), the high specificity of ricefield eel cyp19a1b promoters in the brain and pituitary may offer a potential for the tissue-specific inhibition of cyp19a1b expression to gain insight into its regulation and specific roles. The regulation by sex steroids of cyp19a1 expression in the brain of vertebrates varies depending upon species, brain regions, and methods utilized (Kamat et al., 2002). In rats, the effects of E2 on brain aromatase activities and gene expression are still inconsistent, with either up-regulation (Zhao et al., 2008) or no effect (Roselli et al., 1997) reported, and the presence of ERE upstream of Cyp19a1 gene remains to be identified (Zhao et al., 2007). In teleosts, however, it is well known that the expression of cyp19a1b gene was

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Fig. 8. Effects of DHT on the activities of promoter I (PI) and promoter II (PII) in U251-MG (A) or LbT2 (B) cell lines. Cells were transiently co-transfected with the cyp19a1b promoter I or II reporter plasmid together with ricefield eel AR expression construct (eAR), and treated with DHT (10 nM) or ethanol vehicle (as control) for 24 h before luciferase activities were measured. Data are expressed as fold change relative to promoterless pGL3-basic vector. Each bar represents mean ± SEM of triplicates. ⁄P < 0.05 relative to DHT at 0 nM (one-way ANOVA followed by the Tukey multiple comparison test). (C) The reduction of ARE-dependent DHT response of PII activity in LbT2 cells by site-directed mutagenesis. The sequences of ARE motifs are shown above the promoter-depicting lines respectively, with site-mutated bases boxed. LbT2 cells were cotransfected with promoter II reporter plasmids containing wild-type or site-mutated ARE motifs together with ricefield eel AR expression construct, and treated as above. EBAPIIwt and EBAPIImutARE: reporter constructs containing promoter II of ricefield eel cyp19a1b with ARE motif and mutated ARE motif, respectively. Data are expressed as fold induction relative to the control (DHT at 0 nM). Each bar represents mean ± SEM of triplicates. #P < 0.05 for difference between the wild-type and mutant in response to DHT at 10 nM (Student’s t-test). (D) The inhibition by the anti-androgen flutamide of DHT-stimulated PII activities. Cells were transiently co-transfected with promoter II reporter plasmids together with ricefield eel AR expression construct (25 ng). Cells were treated with 0.1% ethanol or DHT (1 nM) plus flutamide (100, 1000, or 10,000 nM) for 24 h before luciferase activities were measured. Flutamide was added to the culture medium 2 h earlier than DHT. Data are expressed as fold changes relative to the control (DHT at 0 nM). Each bar represents mean ± SEM of triplicates. (a) P < 0.05 compared with DHT at 0 nM, and (b) P < 0.05 compared with flutamide at 0 and 100 nM (One-way ANOVA followed by Tukey multiple comparison test).

up-regulated by estradiol (Le Page et al., 2008; Mouriec et al., 2008), and this induction was shown to be regulated through conserved ERE on the promoter of cyp19a1b gene (Menuet et al., 2005). Surprisingly, the expression of ricefield eel cyp19a1b gene was not up-regulated by E2 in both the hypothalamus and pituitary in vitro as well as in vivo. As E2 significantly up-regulated the salmon gonadotropin-releasing hormone (sgnrh) mRNA levels in the hypothalamus of ricefield eel in vitro (Supplementary Fig. S6A), the hypothalamus fragments survived the incubation procedure and maintained the sensitivity to E2 stimulus in the present study. In addition, esr1 and esr2a mRNA was detected by RT-PCR in the incubated hypothalamus and pituitary (Supplementary Fig. S6B), suggesting that a lack of responsiveness of ricefield eel cyp19a1b gene toward E2 might not be due to the absence of estrogen receptors. Admittedly, evidence that estrogen receptors colocalize with cyp19a1b in the same cell of the hypothalamus or pituitary will further

support this notion, which will be the focus of our future study. Interestingly, however, a non-consensus ERE-like motif was identified on the promoter I of ricefield eel cyp19a1b gene when compared to those conserved EREs in other teleosts. Particularly, there is a mutation from A to G at 5 position in the 13-bp core consensus sequences of ERE of ricefield eel cyp19a1b promoter I, which is in striking contrast to the perfectly conserved core consensus sequences in other teleosts examined. It has been shown that the 13-bp core consensus ERE is the minimum length for binding ER and mediating the induction of estrogen-responsive genes (Klein-Hitpass et al., 1986). Only when appropriate immediate flanking nucleotides are present, could ERE with changes in core consensus sequence bind ER (Driscoll et al., 1998). In vitro transfection assay showed that ricefield eel cyp19a1b promoter I did not respond to E2 stimulation either in U251-MG or LbT2 cells. However, back mutations of that ERE-like motif to optimal ERE restored its responsiveness to E2. A parallel

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control experiment revealed that a replacement of the conserved ERE motif on the promoter of orange-spotted grouper, a teleots in Perciformes, with the ERE-like sequence on cyp19a1b promoter I of the ricefield eel, substantially attenuated its responsiveness to E2. These results suggest that the non-consensus ERE-like sequence might be the major reason for the unresponsiveness of ricefield eel cyp19a1b promoter I to E2 stimulation. These lines of evidence suggest that unlike other teleosts, the expression of cyp19a1b in the brain and pituitary of ricefield eel (at least at female and intersexual stages) was not stimulated by estrogens, and the high mRNA level of cyp19a1b gene observed in the brain of ricefield eel in our previous study (Zhang et al., 2008) could not be explained by the positive autoregulatory loop of E2 as hypothesized for other teleostean species (Mouriec et al., 2008), which warrants further study. In rats, androgens have been shown to up-regulate the expression of Cyp19a1 gene in the brain, although the presence of ARE upstream of Cyp19a1 gene remains to be identified (Roselli, 2007; Zhao et al., 2007). In teleosts such as zebrafish and catfish, androgens were also shown to up-regulate the expression of cyp19a1b gene, and this induction appeared to be in fact due to aromatization of androgens to estrogens and involve estrogen receptors rather than androgen receptors (Kazeto and Trant, 2005; Lassiter and Linney, 2007; Mouriec et al., 2009). However, our present study showed that in ricefield eel, androgens (DHT or T) stimulated the expression of cyp19a1b gene only in the pituitary but not the hypothalamus in vitro as well as in vivo. As in the pituitary, androgen receptor (ar) mRNA was also detected in the hypothalamus of ricefield eels (Supplementary Fig. S6B). This evidence suggested that the un-responsiveness of cyp19a1b to DHT in the hypothalamus might not be due to the absence of androgen receptors. Admittedly, colocaliztion of ar and cyp19a1b in the same cells will also further support this point of view, which will be studied in the future. Interestingly, the pituitary-specific promoter II of ricefield eel cyp19a1b contained an ARE motif, and the stimulatory effects of DHT on promoter II was observed with higher potencies in LbT2 cells (a mouse gonadotrope cell line) than in U251-MG cells. This differential activation of promoter activity by DHT in different cell lines was supported by the similarities of the transcription factor binding sites found on the cyp19a1b promoter II and the promoters of mammalian follicle-stimulating hormone beta subunit (fshb) and lhb genes (Ciccone et al., 2008; Zhu et al., 2007) (Supplementary Fig. S7). These results further strengthened the pituitary-specific characteristics of ricefield eel cyp19a1b promoter II. DHT has been reported to be actively metabolized into 5-alpha-androstane-3-beta-17-betadiol and act through estrogen receptors (Handa et al., 2008; Mouriec et al., 2009; Pak et al., 2005), which may complicate the explanation of androgenic effects of DHT. In our present study, however, the effects of DHT or testosterone on ricefield eel cyp19a1b expression were examined in parallel with estradiol both in vitro and in vivo, and a lack of response of ricefield eel cyp19a1b to estradiol treatment suggested that the stimulation of ricefield eel cyp19a1b expression by androgens was not likely to be mediated through estrogen receptors. Furthermore, an anti-androgen (flutamide) significantly decreased the stimulatory effects of DHT, and mutation of ARE completely abolished its effects on promoter II activities in vitro. These lines of evidence suggested that the stimulation of promoter II by DHT was mediated directly through the ARE motif, which is in striking contrast to the understanding that androgens must be aromatized to estrogens to up-regulate the expression of cyp19a1b in other teleosts (Kazeto and Trant, 2005; Mouriec et al., 2009). As to the expression of ricefield eel cyp19a1b gene in the pituitary, further experiments on characterizing cellular localization and the factors responsible for the cell-differential activation of promoter II by DHT are highly warranted. Collectively, the data provided in the present study indicated that two tissue-specific promoters, namely promoter I in the 50

flanking region and promoter II in the first intron, directed the expression of ricefield eel cyp19a1b in the brain and pituitary, respectively. Furthermore, androgens rather than estrogens upregulated the expression of cyp19a1b gene in the pituitary of ricefield eel. The differential regulation of ricefield eel cyp19a1b gene between the pituitary and hypothalamus by androgen via tissuespecific promoters may be a causal mechanism that underlies protogyny in this species, which is worth further study. Acknowledgements We thank Yifan Deng, Huiyi Yang, and Zhixin Liu for their assistance in preparation of tissue samples, and Yumei Yang and Chengxiang Wu for their assistance in the analysis of esr and ar expression. This work was supported by the Natural Science Foundation of China (30970359, 31072197), and National Key Technology Research and Development Program (2008AA09Z406). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mce.2011.12.001. References Bi, C., Rogan, P.K., 2006. BIPAD: a web server for modeling bipartite sequence elements. BMC Bioinfo. 7, 76. Bulun, S.E., Takayama, K., Suzuki, T., Sasano, H., Yilmaz, B., Sebastian, S., 2004. Organization of the human aromatase p450 (CYP19) gene. Semin. Reprod. Med. 22, 5–9. Callard, G.V., Tchoudakova, A.V., Kishida, M., Wood, E., 2001. Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish. J. Steroid Biochem. Mol. Biol. 79, 305–314. Chan, S.T.H., Phillips, J.G., 1967. The structure of the gonad during natural sex reversal in Monopterus albus (Pisces: Teleostei). J. Zool. Lond. 151, 129–141. Chang, X., Kobayashi, T., Senthilkumaran, B., Kobayashi-Kajura, H., Sudhakumari, C.C., Nagahama, Y., 2005. Two types of aromatase with different encoding genes, tissue distribution and developmental expression in Nile tilapia (Oreochromis niloticus). Gen. Comp. Endocrinol. 141, 101–115. Childs, G.V., Unabia, G., Komak, S., 2001. Differential expression of estradiol receptors a and b by gonadotropes during the estrous cycle. J. Histochem. Cytochem. 49, 665–666. Ciccone, N.A., Lacza, C.T., Hou, M.Y., Gregory, S.J., Kam, K.Y., Xu, S., Kaiser, U.B., 2008. A composite element that binds basic helix loop helix and basic leucine zipper transcription factors is important for gonadotropin-releasing hormone regulation of the follicle-stimulating hormone beta gene. Mol. Endocrinol. 22, 1908–1923. Corbin, C.J., Hughes, A.L., Heffelfinger, J.R., Berger, T., Waltzek, T.B., Roser, J.F., Santos, T.C., Miglino, M.A., Oliveira, M.F., Braga, F.C., Meirelles, F.V., Conley, A.J., 2007. Evolution of suiform aromatases: ancestral duplication with conservation of tissue-specific expression in the collared peccary (Pecari tayassu). J. Mol. Evol. 65, 403–412. Diotel, N., Le Page, Y., Mouriec, K., Tong, S.K., Pellegrini, E., Vaillant, C., Anglade, I., Brion, F., Pakdel, F., Chung, B.C., Kah, O., 2010. Aromatase in the brain of teleost fish: expression, regulation and putative functions. Front. Neuroendocrinol. 31, 172–192. Driscoll, M.D., Sathya, G., Muyan, M., Klinge, C.M., Hilf, R., Bambara, R.A., 1998. Sequence requirements for estrogen receptor binding to estrogen response elements. J. Biol. Chem. 273, 29321–29330. Galmiche, G., Corvaisier, S., Kottler, M.L., 2006a. Aromatase gene expression and regulation in the female rat pituitary. Ann. N. Y. Acad. Sci. 1070, 286–292. Galmiche, G., Richard, N., Corvaisier, S., Kottler, M.L., 2006b. The expression of aromatase in gonadotropes is regulated by estradiol and gonadotropinreleasing hormone in a manner that differs from the regulation of luteinizing hormone. Endocrinology 147, 4234–4244. Garcia-Segura, L.M., 2008. Aromatase in the brain: not just for reproduction anymore. J. Neuroendocrinol. 20, 705–712. Gardner, L., Anderson, T., Place, A.R., Dixon, B., Elizur, A., 2005. Sex change strategy and the aromatase genes. J. Steroid Biochem. Mol. Biol. 94, 395–404. Handa, R.J., Pak, T.R., Kudwa, A.E., Lund, T.D., Hinds, L., 2008. An alternate pathway for androgen regulation of brain function: activation of estrogen receptor beta by the metabolite of dihydrotestosterone, 5alpha-androstane-3beta, 17betadiol. Horm. Behav. 53, 741–752. Harvey, S.C., Kwon, J.Y., Penman, D.J., 2003. Physical mapping of the brain and ovarian aromatase genes in the Nile Tilapia, Oreochromis niloticus, by fluorescence in situ hybridization. Anim. Genet. 34, 62–64. Jiao, B., Cheng, C.H., 2010. Disrupting actions of bisphenol A and malachite green on growth hormone receptor gene expression and signal transduction in seabream. Fish Physiol. Biochem. 36, 251–261.

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