Genes encoding aromatases in teleosts: Evolution and expression regulation

General and Comparative Endocrinology 205 (2014) 151–158

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Genes encoding aromatases in teleosts: Evolution and expression regulation Yang Zhang 1, Shen Zhang, Huijie Lu, Lihong Zhang ⇑, Weimin Zhang ⇑ School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, PR China

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Article history: Available online 20 May 2014 Keywords: Teleost Cyp19a1a Cyp19a1b Evolution Expression regulation

a b s t r a c t Cytochrome P450 aromatases, encoded by cyp19a1 genes, catalyzes the conversion of androgens to estrogens and plays important roles in the reproduction of vertebrates. Vertebrate cyp19a1 genes showed high synteny in chromosomal locations and conservation in sequences during evolution. However, amphioxus cyp19a1 does not show synteny to vertebrate cyp19a1. Teleost fish possess two copies of the cyp19a1 gene, which were postulated to result from a fish-specific genome duplication. The duplicated copies of fish cyp19a1 genes evolved into the brain and ovarian forms of cytochrome P450 aromatase genes, cyp19a1a and cyp19a1b, respectively, with different regulatory mechanisms of expression, through subfunctionalization under long-term selective pressure. In addition to the estradiol (E2) auto-regulatory loop, there may be other mechanisms responsible for the high expression of aromatase in the teleost brain. The study of the two cyp19a1 copies in teleost fish will shed light on the general evolution, function, and regulation of vertebrate cyp19a1. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Aromatase is an enzyme complex composed of a cytochrome P450 aromatase, the product of the cyp19a1 gene, and an NADPHdependent cytochrome P450 reductase known as a ubiquitous flavoprotein (Simpson et al., 1994) that catalyzes the biosynthesis of estrogens from androgens. Thus, the expression or suppression of cyp19a1 and aromatase activities could alter the ratio of gonadal sex steroids produced, which has been shown to control sexual differentiation and development in non-mammalian vertebrates (Kitano et al., 2000; Kwon et al., 2000; Chardard and Dournon, 1999; Rhen and Lang, 1994; Wibbels and Crews, 1994; RichardMercier et al., 1995; Elbrecht and Smith, 1992). In contrast to most other vertebrates, teleosts have two cyp19a1 genes, cyp19a1a, encoding the ovarian form of aromatase, and cyp19a1b, encoding the brain form of aromatase (Tchoudakova and Callard, 1998; Tong et al., 2001). The duplicated cyp19a1 genes are located on different chromosomes in the Nile tilapia, Oreochromis niloticus ⇑ Corresponding authors. Address: Institute of Aquatic Economic Animals, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, PR China. Fax: +86 20 84113327 (W. Zhang). E-mail addresses: [email protected] (L. Zhang), [email protected] (W. Zhang). 1 Present address: Key Laboratory of Marine Bio-resources Sustainable Utilization, Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, PR China. http://dx.doi.org/10.1016/j.ygcen.2014.05.008 0016-6480/Ó 2014 Elsevier Inc. All rights reserved.

(Harvey et al., 2003), and each isoform has its own distinct regulatory mechanisms and physiological relevance (Kishida et al., 2001; Callard et al., 2001). There are several excellent reviews on aromatases in fish (Cheshenko et al., 2008; Diotel et al., 2010; Guiguen et al., 2010; Le Page et al., 2010). The present paper is intended to briefly summarize the current advancements in the study of cyp19a1 in teleosts, including the origin, evolution, and regulation of cyp19a1 genes. 2. Two copies of cyp19a1 genes in teleosts To date, studies have shown that there is only one copy of cyp19a1 in most tetrapods, including mammals (Simpson et al., 1997) except pigs and peccaries (Corbin et al., 2007), chicken (McPhaul et al., 1988), Xenopus (Miyashita et al., 2000), and alligator (Gabriel et al., 2001). In addition, only one copy of cyp19a1 was identified in Atlantic stingray (Ijiri et al., 2000), a cartilaginous fish. In teleosts, duplicated copies of cyp19a1 were first identified in goldfish (Tchoudakova and Callard, 1998) and subsequently confirmed in many other teleosts including zebrafish (Kishida and Callard, 2001; Tong et al., 2001), rainbow trout (Valle et al., 2002), orange-spotted grouper (Zhang et al., 2004b), channel catfish (Kazeto and Trant, 2005), medaka (Kuhl et al., 2005), and tilapia (Chang et al., 2005). One cyp19a1 isoform is designated as cyp19a1a and the other as cyp19a1b. The former is mainly expressed in the ovary and encodes the ovarian aromatase P450aromA; the latter

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Fig. 1. Schematic diagram showing the relative positions of introns and exons of teleost cyp19a1 genes, as modified from our previous report (Zhang et al., 2008). The exons are boxed and indicated by Roman numerals at the bottom, and the introns are depicted by lines. The translated exons are shaded. The genomic structure was determined by comparing the sequences of the corresponding cDNA and genomic DNA, and the following sequences were downloaded from Entrez (NCBI): Nile tilapia cyp19a1a gene (AF472620) and cDNA(U72071); Nile tilapia cyp19a1b gene (AF472621) and cDNA(AF295761); zebrafish cyp19a1a gene (NM131154) and cDNA(NC007129); zebrafish cyp19a1b gene (NM131642) and cDNA (NC007136); ricefield eel cyp19a1a gene (EU841366) and cDNA (EU252487); ricefield eel cyp19a1b gene (EU840259) and cDNA (EU252488); orange-spotted grouper cyp19a1a gene (Li, 2006) and cDNA (AY510711); orange-spotted grouper cyp19a1b gene (Li, 2006) and cDNA (AY510712).

is mainly expressed in the brain and encodes the brain aromatase P450aromB. In Japanese eel and European eel, teleosts belonging to an ancient group of elopomorphs (Ijiri et al., 2003; Jeng et al., 2005), only one copy of cyp19a1 has been identified (Tzchori et al., 2004), most likely due to the loss of the other copy of cyp19a1 during the evolution of this lineage (Cheshenko et al., 2008). Although it was once postulated that only one copy of cyp19a1 is present in ricefield eel (Yu et al., 2008; Guiguen et al., 2010), we recently identified duplicated copies of cyp19a1 in ricefield eel: cyp19a1a, predominantly expressed in the ovary, and cyp19a1b, predominantly expressed in the brain (Zhang et al., 2008). The sizes of cyp19a1b and cyp19a1a genes vary highly among teleosts, mostly due to the length of introns; however, the gene structures are highly conserved, with 8 introns and 9 exons in cyp19a1a and 9 introns and 10 exons in cyp19a1b (Fig. 1). 3. Syntenic analysis of cyp19a1 loci in vertebrates The evolutionary relationship of cyp19a1 genes was examined with syntenic analysis in gnathostome vertebrates, with the exception of the platypus due to the incompleteness of its sequences. As information on cyp19a1 in agnathostome vertebrates is still lacking, it was not included in this analysis. Among the representative vertebrates examined, the cyp19a1 loci showed a highly conserved synteny, with the same neighboring genes and arrangement and without the insertion of other genes in most vertebrates (Fig. 2). The conserved syntenic region around the cyp19a1 loci could be categorized into two conserved gene blocks: AP4E1/Tnfaip813/ CYP19/GLDN/DMXL2 and SCG3/LysMD2/TMOD2/TMOD3/LED1/ MAPK6/GNB5. In teleosts, duplicated copies of cyp19a1 are present on different chromosomes and are associated with the conserved gene blocks. The syntenic region of cyp19a1a in teleosts contains gene blocks Tnfaip813/cyp19a1a/GLDN/DMXL2 and CG3/LysMD2/ TMOD2/TMOD3/LED1/MAPK6, whereas the syntenic region of

cyp19a1b contains gene block cyp19a1b/AP4E1/GNB5. Compared to tetrapods, the genes in the syntenic regions of cyp19a1a and cyp19a1b in teleosts appear to be complementary, and the sum of genes in both syntenic regions of cyp19a1a and cyp19a1b in teleosts (except the stickleback) is equal to the genes in the two conserved gene blocks around the cyp19a1 loci of tetrapods. The syntenic analysis of Cyp19a1 loci also revealed some particular chromosomal rearrangements in mammals. In the horse genome, the two conserved gene blocks in the syntenic region around the Cyp19a1 locus are inversed. Tandem duplication of Cyp19a1 occurred in the cow genome, another mammalian species, in addition to pigs and peccaries (Corbin et al., 2007), which contains multiple copies of Cyp19a1. In the mouse genome, the syntenic region around the Cyp19a1 locus only contains the conserved gene block Tnfaip813/CYP19/GLDN/DMXL2 but not the other one, implying that the Tnfaip813/CYP19/GLDN/DMXL2 gene block has undergone transposition (Chiang et al., 2001). Similarly in the genome of stickleback, the syntenic region around the cyp19a1a locus only contains the same gene block, Tnfaip813/CYP19/GLDN/DMXL2, as in mouse but not the other conserved gene block as in other teleosts. This finding suggests that the gene block containing cyp19a1a has also undergone transposition and the underlying mechanisms for the transposition of cyp19a1 may be conserved in stickleback and mouse. 4. The origin and evolution of cyp19a1 in teleosts The well-conserved synteny of both cyp19a1a and cyp19a1b in teleosts with cyp19a1 in tetrapods supports the idea that the teleost-duplicated cyp19a1 genes arose from a whole-genome duplication event (Chiang et al., 2001). Furthermore, cyp19a1 in gnathostome vertebrates appears to have evolved from the same ancestral cyp19 gene, around which there are presumably two conserved gene blocks, AP4E1/Tnfaip813/CYP19/GLDN/DMXL2 and

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Fig. 2. Physical maps of the genomic environment around cyp19a1 in vertebrates by synteny analysis. The cyp19a1 genes are indicated by blue bars and other genes around cyp19a1 by orange bars. The conserved DNA blocks are denoted by using different colored lines. The green- and yellow line-boxed DNA blocks includ almost the same gene content or gene order from teleosts to mammals, and the purpleline-boxed DNA blocks are present specifically in teleosts. All the genomic data and software used were downloaded from http://genome.ucsc.edu/cgi-bin/hgBlat.

SCG3/LysMD2/TMOD2/ TMOD3/LED1/MAPK6/GNB5 (Fig. 3). These two gene blocks are highly conserved in tetrapods, whereas in teleosts, fish-specific genome duplication (Meyer and Van de Peer, 2005) resulted in the duplication of cyp19a1 and the neighboring gene blocks. Following duplication, one copy retained most of these genes while losing AP4E1 and GNB5, whereas the other copy lost most of these genes while retaining AP4E1 and GNB5 in the two conserved blocks. It is likely that through subfunctionalization (Force et al., 1999; Postlethwait et al., 2004) under selective pressure, the former copy of cyp19a1 evolved as cyp19a1a encoding the aromatase mainly in the ovary and the latter as cyp19a1b encoding the aromatase mainly in the brain (Fig. 3).

Where did the ancestral cyp19a1 gene originate? Cyp19a1 has not been identified in the sequenced genomes of urochordates, echinoderms, or protostomes (Markov et al., 2009; Reitzel and Tarrant, 2010). The earliest presence of the cyp19a1 gene was identified in cephalochordate amphioxus (Castro et al., 2005; Mizuta and Kubokawa, 2007; Callard et al., 2011), suggesting a possible invertebrate origin of cyp19a1. However, although the amphioxus genome shares a high degree of synteny with those of vertebrates (Putnam et al., 2008), the cyp19a1 locus in the amphioxus genome does not show any synteny to either teleosts or tetrapods and was tandemly duplicated in the same chromosome (Fig. 2). This suggests that teleost cyp19a1 genes could not have directly originated

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Fig. 3. Evolutionary model for the origin of cyp19a1 genes in teleosts. The putative ancient paralog is composed of two conserved DNA blocks (refer to Fig. 2), which are indicated by green and yellow boxes. After the fish-specific genome duplication, two paralogs appeared, and their genes underwent mutual loss, except for the cyp19a1 gene. Finally, the reserved cyp19a1 genes underwent subfunctionalization and formed the ovary- and brain-specific aromatases in teleosts.

from amphioxus and are most likely derived from an ancestor evolving in parallel with amphioxus. It is also possible that substantial rearrangements in the chromosomal regions around the cyp19a1 locus during evolution have occurred at the base of the vertebrate ancestral group closely related to amphioxus. 5. Transcriptional regulation of cyp19a1 genes in teleosts In most mammals, the cytochrome P450 aromatase is encoded by a single Cyp19a1 gene, and tissue-specific expression is achieved by tissue-specific promoter usage and alternative splicing (Simpson et al., 1997; Bulun et al., 2003). In contrast, most teleosts have duplicated cyp19a1 genes, and each isoform has its own distinct regulatory region and mechanism (Kishida et al., 2001; Callard et al., 2001). Promoters for cyp19a1a and/or cyp19a1b have been isolated and functionally characterized in some fish species, including catfish (Kazeto and Trant, 2005), Humpback grouper, Broad-barred goby, and Barramundi (Gardner et al., 2005), gilthead seabream (Wong et al., 2006), goldfish (Tchoudakova et al., 2001), grey mullet (Nocillado et al., 2007), Japanese medaka (Kuhl et al., 2005; Nakamoto et al., 2007; Tanaka et al., 1995), Nile tilapia (Yoshiura et al., 2003; Chang et al., 2005), rainbow trout (Kanda et al., 2006; Toffolo et al., 2007), rare minnow (Wang et al., 2010b), red-spotted grouper (Huang et al., 2009), sea bass (Galay-Burgos et al., 2006), gobiid fish (Kobayashi et al., 2005), zebrafish (Kazeto et al., 2001; Tong and Chung, 2003), and orangespotted grouper (Zhang et al., 2012). The recent progress in knowledge of transcriptional regulation of teleost cyp19a1a and cyp19a1b is summarized in the following sections. 5.1. Promoters for cyp19a1 genes in teleosts Sequence analysis of the promoters for cyp19a1a and cyp19a1b in some teleosts has revealed that the proximal promoter regions are relatively conserved, particularly the positions of the Foxo1/4 binding site and the two Ftz-f1 binding sites in the cyp19a1a promoter, and the positions of the ERE motif, STAT1 binding site, and RFX binding site in the cyp19a1b promoter (Zhang et al., 2012). The functionalities of several of these transcription factor binding sites have been characterized in a few teleost species.

5.2. Up-regulation of cyp19a1a in the gonad The expression of cyp19a1a and the biosynthesis of estrogens play important roles in the ovarian differentiation and development in teleosts. It has been shown that the expression of cyp19a1a in the ovary is activated by many factors, including gonadotropins and related signaling pathways, Ftz-f1, and Foxl2. 5.2.1. Gonadotropins and related signaling pathways In mammals, gonadotropins regulate Cyp19a1 expression and steroidogenesis in the gonads by binding to their cognate receptors in somatic cells and activating the cAMP/PKA/CREB pathway (Richards, 1994). Similarly, gonadal steroidogenesis in teleosts is also under the control of gonadotropins. GtHs increase cAMP concentrations in the ovarian fragments from eels (Salmon et al., 1985) and rainbow trout (Idler et al., 1975, 1984). cAMP/PKA signals stimulate estradiol (E2) production in tilapia (Bogomolnaya and Yaron, 1984) and goldfish (Van Der Kraak, 1992). Through in vitro studies, hCG, PMSG, and cAMP-inducing agents were found to promote aromatase activity in goldfish (Kagawa et al., 1984) and medaka (Nagahama et al., 1991). In an in vivo study, hCG increased cyp19a1a transcript and enzyme activity in the ovary of catfish during the preparatory and prespawning phases (Rasheeda et al., 2010). In addition, Fsh was shown to be the main inducer of E2 synthesis in salmon (Cheshenko et al., 2008). It has been established that cAMP/PKA signaling pathway can phosphorylate cAMP response element (CRE)-binding protein (CREB), which binds CREs and activates the transcription of target genes. CREs were found to be conserved in the promoters of cyp19a1a genes of many teleosts (Kanda et al., 2006; Kazeto et al., 2001; Tchoudakova et al., 2001; Wong et al., 2006; Zhang et al., 2013). CRE mutation abolished the activation of the cyp19a1a promoter by cAMP in Japanese flounder (Yamaguchi et al., 2007) and ricefield eel (Zhang et al., 2013). Thus, as in mammals, gonadotropins in teleosts activate cyp19a1a transcription through the cAMP/PKA pathway. 5.2.2. Ftz-f1 Among the cis-elements in the cyp19a1 promoter directing its expression in the ovary, the SF-1/Ad4BP binding site, to which Ftz-f1 (NR5A) homologues bind, is conserved across vertebrates

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(Zhang et al., 2013). In teleosts, members of the NR5A subfamily, including mdFtz-f1 (NR5A4) in medaka (Watanabe et al., 1999), tiAd4BP/Sf-1 (NR5A4) in tilapia (Yoshiura et al., 2003; Wang et al., 2007), and rtFtz-f1 (NR5A2) in rainbow trout (Kanda et al., 2006), were shown to bind the Ftz-f1 sites in the cyp19a1a gene and activate cyp19a1a transcription in vitro. The expression of tiAd4BP/Sf-1 and cyp19a1a was co-localized to the granulosa cells of the vitellogenic ovary in tilapia (Wang et al., 2007). These results suggest that NR5A homologs are potentially involved in the transcriptional regulation of cyp19a1a in the ovarian follicles of teleosts. The NR5A subfamily in vertebrates comprises four members, NR5A1-NR5A4 (Fayard et al., 2004). A previously isolated Ftz-f1 homolog (NR5A4) from orange-spotted grouper (Zhang et al., 2004a) could not activate cyp19a1a transcription in vitro (Zhang et al., 2012), suggesting that the activation of the cyp19a1a promoter by Ftz-f1 homologs in teleosts may be of structural preference. NR5A2 is an Ftz-f1 homolog conserved across vertebrates (Fayard et al., 2004), was suggested to activate Cyp19a1 expression in the mammalian ovary (Hinshelwood et al., 2003; Fayard et al., 2004). The possibility that NR5A2 regulates the ovarian expression of cyp19a1a in teleosts warrants further study. In mammals, Sf-1 and cAMP act synergistically to up-regulate the expression of Cyp19a1 (Carlone and Richards, 1997a,b). However, it remains to be clarified whether such synergism in the activation of the cyp19a1a promoter by cAMP and Ftz-f1 homologs is also true in teleosts. 5.2.3. Forkhead transcription factors Foxl2 (forkhead transcription factor gene 2), one of the forkhead transcription factors, can recognize and bind to the 7-nucleotide core DNA sequence 50 -(G/A)(T/C)(C/A)AA(C/T) A-30 (Pisarska et al., 2004) and was shown to be important for ovarian differentiation and development in vertebrates via the inhibition of the male sexual differentiation pathway (Uhlenhaut et al., 2009). The binding site for Foxl2 has been identified in both PII of mammalian Cyp19a1 and the promoters of cyp19a1a in teleosts (Pannetier et al., 2006; Wang et al., 2007; Yamaguchi et al., 2007). Foxl2 was shown to activate the transcription of cyp19a1a from Japanese flounder (Yamaguchi et al., 2007) and tilapia (Wang et al., 2007) in vitro in several cell lines. Foxl2 could activate the transcription of cyp19a1a synergistically with Sf-1 (Wang et al., 2007). Foxl2 deficiency in XX tilapia resulted in varying degrees of oocyte degeneration, significantly decreased aromatase gene expression, and even caused complete sex reversal in some fish (Li et al., 2013). These results suggest that Foxl2 plays important roles in ovarian differentiation and development by up-regulating cyp19a1a expression in teleosts. In addition, binding sites for Foxo 1/4, the other forkhead transcription factors, were found to be conserved in the proximal promoter of cyp19a1a in some teleosts, particularly in Perciformes (Zhang et al., 2012). In rat primary granulosa cell cultures, Foxo1 was shown to repress the expression of aromatase (Park et al., 2005). In contrast, deletion of the region containing Foxo1/4 binding sites in the cyp19a1a promoter of orange-spotted grouper, a Perciformes teleost fish, reduced promoter activity by approximately threefold, suggesting the possible involvement of Foxo1/4 binding sites in the regulation of the cyp19a1a promoter in vitro (Zhang et al., 2012). Nonetheless, it remains to be elucidated whether Foxo transcription factors are in fact involved in the regulation of fish cyp19a1a genes.

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physiologically relevant E2 levels. The down-regulation of cyp19a1a was found to be essential for male sexual differentiation in many teleosts (Guiguen et al., 2010). Thus, the identification of physiological factors responsible for down-regulating cyp19a1a expression is of great interest. 5.3.1. Dmrt1 Dmrt1, double sex and mab-3 related transcription factor 1, is an important transcription factor for testicular development and is conserved across vertebrates. In tilapia, Dmrt1 was shown to bind the DNA sequence ‘‘TACATATGTA’’ in the cyp19a1a promoter and inhibit the basal as well as Ftz-f1- and Foxl2-activated cyp19a1a transcription (Wang et al., 2010a), Moreover, the overexpression of Dmrt1 in XX tilapia decreased cyp19a1a expression and resulted in the delay of ovarian formation, ovarian degeneration, and even sex reversal in some fish (Wang et al., 2010a), suggesting that Dmrt1 is a suppressive regulator of cyp19a1a during sexual differentiation in tilapia. In red-spotted grouper, however, expression of dmrt1 was found to be localized in germ cells, including spermatogonia, and primary and secondary spermatocytes, but not Sertoli cells (Xia et al., 2007). This result raises the question of how Dmrt1 in germ cells represses the expression of cyp19a1a, which is expressed mainly in somatic cells in teleosts. 5.3.2. Dax1 Dax1, an orphan nuclear receptor in the NR0b1 subfamily, was shown to inhibit the activation of Cyp19a1 transcription by SF-1 in mammals (Wang et al., 2001). In medaka, a teleost fish, Dax1 could also inhibit Ftz-f1-activated cyp19a1a transcription (Nakamoto et al., 2007), suggesting that the suppression of cyp19a1 expression by Dax1 may be conserved in vertebrates. Dax1 is only expressed in postvitellogenic follicles (Nakamoto et al., 2007), suggesting its possible roles in down-regulating cyp19a1a expression after vitellogenesis. In protandrous black porgy fish, the expression of cyp19a1a is correlated positively with NR5A1 but negatively with NR0b1, suggesting the involvement of NR0b1 in the down-regulation of cyp19a1a during sexual differentiation (Wu et al., 2008). 5.3.3. Epigenetic modifications Recent studies suggest that epigenetic mechanisms may regulate Cyp19a1 expression in mammals including human (Knower et al., 2010), sheep (Fürbass et al., 2008), buffalo (Monga et al., 2011), and rat (Lee et al., 2013). In the European sea bass, a species with sex determination controlled by both genetic and temperature effects, Navarro-Martín et al. (2011) showed for the first time that increased temperature during a critical period in early development was able to increase promoter DNA methylation and prevent gonadal expression of cyp19a1a. The authors suggested that cyp19a1a promoter methylation is most likely part of the longsought-after mechanism connecting temperature and environmental sex determination in vertebrates. In ricefield eel, a protogynous sex-changing teleost, DNA methylation and histone de-acetylation and methylation may abrogate the stimulation of cyp19a1a by gonadotropins in a male-specific fashion and are associated with female to male sex change (Zhang et al., 2013). These results suggested that the epigenetic control of cyp19a1a gene expression could play an important role in the gonadal differentiation of teleosts and possibly other lower vertebrates as well. 5.4. Regulation of cyp19a1b

5.3. Down-regulation of cyp19a1a in the gonad As a powerful regulator of many physiological processes, the synthesis of E2 must be adequately controlled to ensure

Cyp19a1b is highly expressed in the brain and pituitary of teleosts, but the regulatory mechanism is less understood compared to cyp19a1a in the ovary.

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5.4.1. Regulation of cyp19a1b by steroidal hormones Studies in several teleosts have demonstrated that the cyp19a1b gene is up-regulated by estradiol (Callard et al., 2001; Le Page et al., 2008; Mouriec et al., 2008). An estrogen response element (ERE) specific to the promoter region of the 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). Cyp19a1b is also highly expressed in the brain of ricefield eel (Zhang et al., 2008). The expression of cyp19a1b in the brain and pituitary of ricefield eel was found to be driven by tissuespecific promoters, and E2 did not stimulate cyp19a1b expression in the brain or brain-specific cyp19a1b promoter activity in vitro (Zhang et al., 2012). These findings suggest that in addition to the positive auto-regulation by E2 (Diotel et al., 2010), there may be other mechanisms responsible for the high levels of cyp19a1b in the brain of some teleosts. In teleosts such as zebrafish and catfish, androgens were also shown to up-regulate the expression of the cyp19a1b gene, which appeared to be mediated by estrogen receptors via conversion to estrogens (Kazeto and Trant, 2005; Mouriec et al., 2009; Lassiter and Linney, 2007). However, the pituitary-specific promoter of ricefield eel cyp19a1b contained an ARE motif that mediated the direct stimulation by androgens of cyp19a1b expression in the pituitary of ricefield eel (Zhang et al., 2012). Thus, there are two mechanisms mediating the steroidal up-regulation of cyp19a1b in the pituitary of teleosts: a positive androgen-feedforward regulation identified in ricefield eel (Zhang et al., 2012), and a positive estrogen-feedback regulation in other teleosts (Kazeto and Trant, 2005; Mouriec et al., 2009). 5.4.2. Regulation of cyp19a1b by NR5As and Foxl2 In medaka, lrh-1 and cyp19a1b mRNA were co-localized in the hypothalamus, and Lrh-1 increased the expression of a cyp19a1b reporter gene in several mammalian cell lines including HEK293, TM3, TM4, and 3T3-L1, possibly by binding to the putative site TGGCCTTGA in the cyp19a1b promoter (Ohmuro-Matsuyama et al., 2007). In catfish, cyp19a1b, ftz-f1, and foxl2 exhibit synchronous expression patterns, and Ftz-f1 and Foxl2 could bind to the cyp19a1b promoter to enhance transcription of the cyp19a1b gene (Sridevi et al., 2012). However, whether such a mechanism is conserved in other teleosts remains to be elucidated. 6. Summary and future perspectives Duplicated copies of cyp19a1 in teleosts are considered to arise from fish-specific genome duplication, followed by the subfunctionalization of the two cyp19a1 genes and mutual loss of neighboring genes associated with each cyp19a1 locus. However, the origin of the ancient paralog of teleost cyp19a1 remains elusive. The Cyp19a1 locus in amphioxus did not show any synteny to teleosts and other vertebrates. A Blast analysis did not revealed a cyp19a1 gene or orthologs within the sequenced genome of lamprey, or invertebrates including urochordates, echinoderms, and protostomes (Callard et al., 2011). Markov et al. (2009) proposed that cytochrome P450 aromatase most likely originated from xenobiotic-detoxifying CYPs. Regardless, which CYP is the ancestor of cyp19a1 and in which taxon the ancient paralog of cyp19a1 originated remain to be investigated. The expression of cyp19a1a may be up-regulated by Foxl2 and Sf-1 during ovarian differentiation and down-regulated by Dmrt1 and epigenetic modifications during testicular differentiation in teleosts. However, the identification of other forkhead transcription factors and NR5A members involved in the regulation of cyp19a1 in teleosts is of interest. As estrogen signaling may also be necessary for testicular differentiation and development in

some teleosts (Guiguen et al., 2010), it is worth studying the site of estrogen synthesis and how it is regulated in male teleosts. The regulation of cyp19a1b is less understood compared with that of cyp19a1a in teleosts. The positive auto-regulatory loop of E2 was proposed to explain the high levels of aromatase in the brain of teleosts (Diotel et al., 2010); however, the co-localization of ER and cyp19a1a in radial glial cells in the brain remains to be demonstrated (Mouriec et al., 2009). Moreover, cyp19a1b is also highly expressed in the brain in ricefield eels, though without the positive E2-autoregulation (Zhang et al., 2012), suggesting that there may be other mechanisms maintaining high levels of cyp19a1b in the brain of teleosts. The regulation of cyp19a1b expression by STAT1 and RFX signaling, for which binding sites are conserved in the proximal promoters of cyp19a1a genes in some teleosts (Zhang et al., 2012), is worth further exploration. Acknowledgments This work was supported by the Special Fund for Agro-scientific Research in the Public Interest (201403008), the National Key Technology Research and Development Program (2012AA10A4 07), the Natural Science Foundation of China (30471346, 3077 1651, 31072197, 31172088, 31372513), the Research Fund for the Doctoral Program of Higher Education of China, and Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science. References Bogomolnaya, A., Yaron, Z., 1984. Stimulation in vitro of estradiol-17beta secretion by the ovary of a cichlid fish, Sarotherodon aureus. Gen. Comp. Endocrinol. 53, 187–196. Bulun, S.E., Sebastian, S., Takayama, K., Suzuki, T., Sasano, H., Shozu, M., 2003. The human CYP19 (aromatase P450) gene: update on physiologic roles and genomic organization of promoters. J. Steroid Biochem. Mol. Biol. 86, 219–224. 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. Callard, G.V., Tarrant, A.M., Novillo, A., Yacci, P., Ciaccia, L., Vajda, S., Chuang, G.Y., Kozakov, D., Greytak, S.R., Sawyer, S., Hoover, C., Cotter, K.A., 2011. Evolutionary origins of the estrogen signaling system: insights from amphioxus. J. Steroid Biochem. Mol. Biol. 127, 176–188. Carlone, D.L., Richards, J.S., 1997a. Evidence that functional interactions of CREB and SF-1 mediate hormone regulated expression of the aromatase gene in granulosa cells and constitutive expression in R2C cells. J. Steroid Biochem. Mol. Biol. 61, 223–231. Carlone, D.L., Richards, J.S., 1997b. Functional interactions, phosphorylation, and levels of 30 ,50 -cyclic adenosine monophosphate-regulatory element binding protein and steroidogenic factor-1 mediate hormone-regulated and constitutive expression of aromatase in gonadal cells. Mol. Endocrinol. 11, 292–304. Castro, L.F., Santos, M.M., Reis-Henriques, M.A., 2005. The genomic environment around the Aromatase gene: evolutionary insights. MC Evol. Biol. 5, 43. 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. Chardard, D., Dournon, C., 1999. Sex reversal by aromatase inhibitor treatment in the newt Pleurodeles waltl. J. Exp. Zool. 283, 43–50. Cheshenko, K., Pakdel, F., Segner, H., Kah, O., Eggen, R.I., 2008. Interference of endocrine disrupting chemicals with aromatase CYP19 expression or activity, and consequences for reproduction of teleost fish. Gen. Comp. Endocrinol. 155, 31–62, Review. Chiang, E.F., Yan, Y.L., Guiguen, Y., Postlehwait, J., Chung, B.C., 2001. Two Cyp19 (P450 Aromatase) genes on duplicated zebrafish chromosomes are expressed in ovary and brain. Mol. Biol. Evol. 18, 542–550. 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. Elbrecht, A., Smith, R.G., 1992. Aromatase enzyme activity and sex determination in chickens. Science 255, 467–470.

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