Zebrafish (Danio rerio) androgen receptor: Sequence homology and up-regulation by the fungicide vinclozolin

Comparative Biochemistry and Physiology, Part C 151 (2010) 161–166

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Zebrafish (Danio rerio) androgen receptor: Sequence homology and up-regulation by the fungicide vinclozolin Amanda N. Smolinsky, Jennifer M. Doughman, Liên-Thành C. Kratzke, Christopher S. Lassiter ⁎ Roanoke College, Department of Biology, Salem, VA 24153, USA

a r t i c l e

i n f o

Article history: Received 28 July 2009 Received in revised form 1 October 2009 Accepted 2 October 2009 Available online 7 October 2009 Keywords: Testosterone Androgen receptor Zebrafish Gene homology Embryo Vinclozolin

a b s t r a c t Steroid hormones regulate gene expression in organisms by binding to receptor proteins. These hormones include the androgens, which signal through androgen receptors (ARs). Endocrine disrupters (EDCs) are chemicals in the environment that adversely affect organisms by binding to nuclear receptors, including ARs. Vinclozolin, a fungicide used on fruit and vegetable crops, is a known anti-androgen, a type of EDC that blocks signals from testosterone and its derivatives. In order to better understand the effects of EDCs, further research on androgen receptors and other hormone signaling pathways is necessary. In this study, we demonstrate the evolutionary conservation between the genomic structure of the human and zebrafish ar genes and find that ar mRNA expression increases in zebrafish embryos exposed to vinclozolin, which may be evolutionarily conserved as well. At 48 and 72 h post-fertilization, vinclozolin-treated embryos express ar mRNA 8-fold higher than the control level. These findings suggest that zebrafish embryos attempt to compensate for the presence of an anti-androgen by increasing the number of androgen receptors available. © 2009 Elsevier Inc. All rights reserved.

1. Introduction Androgens consist of a subgroup of steroid hormones that regulate genes by binding to androgen receptors (ARs) (Mangelsdorf et al., 1995). Androgenic hormones influence animal behavior and play important roles in sex differentiation and maturation in vertebrates, including fish (Borg, 1994). In humans, androgens help control many male secondary sex characteristics, specifically by inhibiting adipose tissue deposition (Singh et al., 2006), promoting enlarged skeletal muscle cells (Sinha-Hikim et al., 2004), and influencing brain patterns (Giammanco et al., 2005). Androgens also serve as the precursors of all estrogens, a group of steroid hormones that affects various tissues and organ systems in both embryonic and adult vertebrates (Lassiter et al., 2002). Thus far, full-length forms of ar genes have been cloned in many fish species including rainbow trout, Onchoryncus mykiss (Takeo and Yamashita, 1999), Japanese eel, Anguilla japonica (Ikeuchi et al., 2001), and western mosquitofish, Gambusia affinis (Ogino et al., 2004). In some fish, two receptors have been cloned and termed ar-alpha and ar-beta. Contemporary with our work, an androgen receptor for zebrafish (Danio rerio) was recently described (Jorgensen et al., 2007; Hossain et al., 2008). The Cypriniform fish, including zebrafish, have lost ar-beta and retain only ar-alpha (Douard et al., 2008). In zebrafish, the androgen receptor is expressed in the developing brain during embryogenesis (Gorelick et al., 2008). Adult expression includes regions of the brain ⁎ Corresponding author. Department of Biology, Roanoke College, 221 College Lane, Salem, VA 24153, USA. Tel.: +1 540 375 2460; fax: +1 540 375 2447. E-mail address: [email protected] (C.S. Lassiter). 1532-0456/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2009.10.001

and testis (de Waal et al., 2008; Gorelick et al., 2008). Complete ar cDNAs have also been cloned in several amphibians, including xenopus, Xenopus laevis (Fischer et al., 1995), as well as in many mammals. Although androgen receptors have been cloned and studied in a number of species, relatively little is known about how the androgen receptor gene is affected by external compounds during development. This information is of critical importance given the increasing amount of endocrine disrupting compounds (EDCs) found in the environment (Courant et al., 2007) and determining when embryos may be responsive to EDCs. EDCs are natural or synthetic chemicals that interfere with normal hormone signaling pathways by stimulating or inhibiting interactions, and are known to cause physiological, reproductive, and behavioral defects. EDCs in the environment originate from a variety of sources, including pesticides (McKinlay et al., 2008). Vinclozolin is an EDC that limits the body's ability to respond to changes in the hormone levels of androgens such as testosterone and has been confirmed as an anti-androgen in fish (Katsiadaki et al., 2006; Martinovic et al., 2008). In both adult fish (Martinovic et al., 2008) and mammals (Loutchanwoot et al., 2008), vinclozolin has been shown to upregulate the androgen receptor. Vinclozolin is used primarily as a fungicide on golf course grasses and crops both grown in and imported into the United States. Human health may be impacted by the presence of vinclozolin. Workers can be exposed to vinclozolin through the respiratory tract or skin contact, while the general population can ingest residual amounts of the chemical remaining on foods (Euling and Kimmel, 2001). Vinclozolin's action as an anti-androgen involves the ability of its metabolites, M1 and M2, to competitively inhibit androgens by fitting into the hormone binding domain on the AR protein (Kelce and

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Wilson, 1997). The AR normally functions by binding an androgen, undergoing a conformational change, binding the promoter region of a gene at Androgen Response Elements (ARE), and expediting the transcription of that gene. However, when vinclozolin or one of its metabolites is present, the androgen can be outcompeted for binding sites on the AR. In this case, vinclozolin binds the AR but inhibits the conformational change that allows binding to the ARE (Euling and Kimmel, 2001; Euling et al., 2002; Kavlock and Cummings, 2005). Androgen-dependent genes are not transcribed as efficiently when vinclozolin is present. In this study, the tropical zebrafish was chosen as a model organism to study the sequence homology of the androgen receptor (ar) and embryonic exposure to vinclozolin. We used a competitive RT-PCR assay to measure the relative quantities of ar mRNA with an internal standard. We hypothesized that zebrafish embryos, similar to other vertebrates, may try to compensate for interference in androgen signaling by up-regulating transcription of the receptor gene.

2. Materials and methods 2.1. Fish husbandry and vinclozolin treatment Adult live zebrafish were obtained from Carolina Biological Supply Company (Burlington, NC, USA) and raised on a diet of TetraMin tropical flakes and Omega One Freeze Dried Brine Shrimp. Tanks were kept on a 14 h ay/10 h night cycle. Embryos were collected, rinsed, and placed in Petri dishes containing 10 mL of 0.3× Danieau buffer (1× Danieau is 58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca (NO3)2, 5 mM HEPES, pH 7.6). Experimental groups composed of 100 embryos were exposed 10 μg/L vinclozolin dissolved in ethanol; controls received 0.1% ethanol as a vehicle control. The treatment concentration was lower than the 700 μg/L used on fathead minnows to cause complete reproductive failure (Villeneuve et al., 2007; Martinovic et al., 2008). Our aim was to investigate for more subtle molecular effects at a lower concentration. Solutions were replaced every 24 h. Embryos were incubated at 28.5 °C. Embryonic RNA was extracted at 0, 24, 48, and 72 h post-fertilization (hpf) using Qiagen's RNEasy Mini Kit. Reverse transcription using an oligo dT primer was performed using Thermo's Verso cDNA Kit.

2.2. ar sequence comparisons We analyzed the probable DNA- and ligand-binding domain locations by aligning the zebrafish ar sequence with other vertebrate ar sequences using ClustalW (http://www.ebi.ac.uk/clustalw/). Sequence similarities of various androgen receptor binding domains were established using the percentages of positives found on BLAST (http://www.ncbi.nlm.nih.gov/blast/). Intron and exon lengths of the zebrafish and human ar genes were determined by searching genomes (Zebrafish v7 and Human v36) on Ensembl (http://www. ensembl.org/index.html). The phylogenetic tree was constructed using amino acid sequences from GenBank. The amino acid sequences were aligned using the neighbor joining test on ClustalW, and TreeView 1.6.6 was used to draw the phylogenetic tree.

2.3. Competitive RT-PCR Competitive RT-PCR was performed on whole embryo RNA using an internal deletion control method previously described (Lassiter et al., 2002; Lassiter and Linney, 2007). Primers used for ar were ARRT1 (5′CTG ACC ACT GAA AGT AGT GAG GAG-3′) and ARRT2 (5′-CTG TAC CTT CTG AAC TCA GGT TGG-3′). A deletion construct (Celi et al., 1993) was created using primers ARRTDEL (5′-CTG ACC ACT GAA AGT AGT GAG TGA GCG AAC GAG AGG CAG G-3′) and ARRT2. PCR was done for 30cycles using GoTaq (Promega). Briefly, a deletion construct was made that consisted of a fragment of the ar gene with a 40 bp deletion. This deletion construct amplified during PCR with the same primers as the ar cDNA, but resolved on an agarose gel as a different size. A known quantity of deletion construct was placed in a PCR tube with an unknown quantity of the embryonic cDNA and a PCR reaction ran with primers ARRT1 and ARRT2. The deletion construct and embryonic sample competed for reagents. When the two samples contained equal amounts of embryo ar cDNA and deletion construct DNA, the two bands were of equal intensity (Fig. 1). All samples were processed in biological (three separate batches of embryos) and technical triplicates (three separate PCR-based measurements for each batch of embryos). The ratio of embryo cDNA:deletion construct was used to generate a relative copy number of the message. This method has been previously described (Lassiter et al., 2002). Statistical differences were analyzed by Student's t test.

Fig. 1. Example of competitive PCR using ar cDNA and an internal deletion construct standard (IS). Lanes 1–4 (0 hpf), 5–8 (24 hpf), 9–12 (48 hpf), and 13–16 (72 hpf) illustrate co-amplification of a fixed volume of ar cDNA with increasing quantities of internal standard. When band intensity is equal, the concentration of unknown ar cDNA equals that of the known deletion construct. During the competitive PCR experiment, each sample was done in biological and technological triplicate (cDNA from three clutches was each measured by the technique three times).

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3. Results 3.1. ar sequence homology The predicted zebrafish AR amino acid sequence based on GenBank EU708622 was aligned with the AR amino acid sequences of eight different fish species. Sequence similarities of the DNAbinding domain and ligand-binding domains were analyzed (Fig. 2). The highest sequence similarity of the DNA-binding domain was shared with the fathead minnow (Pimephales promelas), goldfish (Carassius auratus), and killifish (Kryptolebias marmoratus), and the highest sequence similarity of the ligand-binding domain was shared with the fathead minnow. The lowest sequence similarities of both the DNA-binding domains and ligand-binding domains were shared with the eel (A. japonica). A phylogenetic tree of vertebrate androgen receptors highlighted the relative evolutionary distance of ARs (Fig. 3). The predicted zebrafish AR amino acid sequence was compared to the AR proteins of eight different fish, two amphibians, and two avian species. Also included in the phylogenetic tree were the AR proteins of six different primates, including humans, as well as the AR proteins of six other mammals. 3.2. ar genomic structure Intron and exon lengths of the human and zebrafish ar genomic structures were determined using Ensembl (Fig. 4). Only exons containing coding sequence were examined. Both the human and zebrafish ar genomic structures consist of eight coding exons, with the 5′ untranslated region (5′UTR) present in part of the first exon and the 3′ untranslated region (3′UTR) present in part of the last exon. The lengths of the internal coding exons (exons 2–7) of the zebrafish and human ar genes are very similar. Exons 2, 3, 5, and 6 are identical in size, while exons 4 and 7 have only slight differences in length (Table 1). The intron lengths are highly variable between zebrafish

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and human, although the splice sites have been conserved (Table 2). The intron lengths range from less than 300 bp to nearly 50 kb. The zebrafish ar is found on chromosome 5. Using Ensembl Zv7, the gene is located from basepairs 24,749,584 to 24,867,333. The genes adjacent to the ar on zebrafish chromosome 5 are ophn1 and yipf6; this same sequence of three genes (ophn1, yipf6, and ar) is also found on human chromosome X. 3.3. Embryonic mRNA levels of ar increase in response to vinclozolin After ascertaining the evolutionary conservation of the receptor, we examined the message levels of zebrafish ar mRNA to determine if exposure to vinclozolin affects message levels similar to other vertebrates. Analysis of embryonic mRNA indicates that ar levels remain relatively constant between 24 and 72 hpf. At 24 hpf, treatment with 10 μg/L vinclozlin has little effect on ar mRNA levels; however at 48 and 72 hpf, the amount of ar mRNA is approximately 8-fold that of controls at the same timepoint (Fig. 5, p b 0.05). 4. Discussion The zebrafish ar encodes a protein that is 868 aa. This ar has been confirmed by others working concurrently on the receptor (Jorgensen et al., 2007; Hossain et al., 2008). The coding region contains a DNAbinding domain (DBD) of 78 aa and a ligand-binding domain (LBD) of 187 amino acids. These are typical sizes for the DNA- and ligandbinding domains in nuclear hormone receptors (Mangelsdorf et al., 1995). Furthermore, both of these domains have been highly conserved at the amino acid level among teleost fish. Sequence similarities of the DBD and LBD between zebrafish and other fish are very high, ranging from 90% to 100%. The DBDs of zebrafish and human have a 92% identical amino acid sequence, and the LBDs have a 70% identical amino acid sequence. Our data support that the binding domains have been well conserved during the evolutionary history of the two species (Evans, 1988).

Fig. 2. Structural comparison of the zebrafish AR amino acid sequence with other fish species. The numbers above each box refer to the position of amino acids in the DNA-biding domain (DBD) and ligand-binding domain (LBD). The numbers within each box indicate the positive percentage of sequence similarity of each domain relative to that of the zebrafish. Alpha and beta AR are not indicated due to inconsistencies in the literature among fish species.

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Fig. 3. Phylogenetic tree for vertebrate AR proteins. GenBank accession numbers for the different androgen receptors are as follows: eel, Anguilla japonica (BAA75464), goldfish, Carassius auratus (AAM09278), fathead minnow, Pimephales promelas (AAF88138), killifish, Kryptolebias marmoratus (ABC68612), sea bass, Dicentrarchus labrax (AAT76433), atlantic croacker, Micropogonias undulatus (AAU09477), red sea bream, Pagrus major (BAA33451), black sea bream, Acanthopagrus schlegeli (AAO61694), rana, Rana catesbeiana (AAP85538), xenopus, Xenopus laevis (AAC97386), chicken, Gallus gallus (AB193190), finch, Taeniopygia guttata (DAA05742), mouse, Mus musculus (AAA37234), rat, Rattus norvegicus (AAA40733), dog, Canis familiaris (AAF18084), hyena, Crocuta crocuta (AAM96904), pig, Sus domesticus (AAG40566), wild boar, Sus scrofa (AAG37994), lemur, Eulemur fulvus collaris (AAC73049), baboon, Papio hamadryas, (AAC73047), human, Homo sapien (AAA51729), chimpanzee, Pan troglodytes (AAC73048), rhesus monkey, Macaca mulatta (AAS1969), and macaque Macaca fascicularis (AAC73050). The human progesterone receptor (PR) gene (NP_000537) served as an out group to root the tree.

Fig. 4. A comparison between human and zebrafish ar genomic structures. Exon lengths are highly conserved over 400 million years of evolution. Intron lengths are more divergent and are shown below the exons. The 5′UTR, the coding region, and the 3′UTR are indicated.

The receptor protein has been conserved among vertebrates, including the whole genome duplication in the teleost lineage. Zebrafish and other Cypriniformes have lost one of these duplicated copies leaving only ar-alpha present (Douard et al., 2008). The

Table 2 Comparison of exon/intron splice sites between zebrafish (ZF) and human (HU). Intron 1 Intron 2

Table 1 Exon length comparison in basepairs between zebrafish (ZF) and human (HU) ar.

Exon Exon Exon Exon Exon Exon Exon Exon

1 2 3 4 5 6 7 8

ZF

HU

1834 152 117 279 145 131 152 2469

2140 152 117 288 145 131 158 6934

Intron 3 Intron 4 Intron 5 Intron 6 Intron 7

ZF HU ZF HU ZF HU ZF HU ZF HU ZF HU ZF HU

TGGCAGgtaaga/ttctagGTTTGA CATGCGgtaagt/ttccagTTTGGA CTGAGGgtaata/ctatagGGAAGC CTGAAGgtaaag/tcccagGGAAAC TGGGAGgtaagt/ccacagCCCGCA TGGGAGgtaaga/caatagCCCGGA TTCCAGgtaatt/tctcagGTTTCC TGCCTGgtaagg/ctccagGCTTCC CAATGAgtcagt/tgtcagTCGTAG CAATGAgtaagt/tcccagGTACCG GCGTCAgtgagt/ctgcagTTCCAG GCATTAgtaagt/catcagTTCCAG CAACCAgttagt/acacagGTTGTT CAGCCTgtaagc/ctacagATTGCG

Exon sequences are capitalized and intron sequences are lowercase.

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Fig. 5. Embryonic mRNA levels of zebrafish ar during the first 72 h post fertilization (hpf). Each sample was analyzed in biological and technological triplicates using competitive PCR. All mRNA measurements were divided by the 24 hpf control amount to present information in the fold change from 24 hpf. Standard error of the mean is indicated. Vinclozolin induction at 48 and 72 hpf is significantly greater than controls using a Student's t test (p b 0.05).

phylogenetic tree constructed using the coding region indicates that the zebrafish AR is most closely related to the fathead minnow (P. promelas) AR. This relationship has been confirmed by others using a tree constructed with only the ligand-binding domain sequence (Hossain et al., 2008). Our tree also differs from the work of Hossain et al. (2008) by using a human progesterone receptor instead of an eel progesterone receptor as an outgroup, yet the trees are rather similar among teleost species. The tree presented here also compares the zebrafish sequence to a broader diversity of tetrapod species showing AR conservation throughout vertebrates. The genomic structure of the androgen receptor has been retained for 400 million years, since teleosts split from tetrapods in the vertebrate linage. There are eight coding exons in both the zebrafish and human ar. Hossain et al. (2008) reported the full-length ar gene as having 13 exons, including a 3′UTR of more than 2.3 kb. We focus here upon the eight exons that include the coding region as these are most likely to be conserved. The lengths of four of these exons (2, 3, 5, and 6) are identical. These regions are the location of the highly conserved ligand-binding and DNA-binding domains. Exons 4 and 7, which code for less conserved domains, are still within 9 bp of their human homolog. The exon/intron splice sites have also been well conserved. In contrast, the intron lengths, which are not involved in coding for the AR protein, have not been under stringent selective pressure and have diverged noticeably. The microsynteny, conservation of a chromosomal region, extends beyond the ar locus. ar, ophn1 and yipf6 are adjacent on zebrafish chromosome 5 and human chromosome X. Chromosomal microsynteny and splice site conservation have been described for other zebrafish genes (Lassiter et al., 2002). Our data indicate that vinclozolin has an effect on gene expression in embryonic zebrafish. The gene is expressed at this time in zebrafish from our own work and others (Jorgensen et al., 2007; Gorelick et al., 2008; Hossain et al., 2008). Vinclozolin affects a variety of male traits in fish, including a reduced sperm count in adult guppies (Bayley et al., 2003) and medaka (Kiparissis et al., 2003). Vinclozolin treatment also affects androgen-regulated proteins, such as spiggin (Jolly et al., 2009). The ar mRNA levels in zebrafish at 48 and 72 hpf described here indicate a change from normal expression when treated with vinclozolin; the difference between control and treated embryos is approximately 8-fold. This profile suggests that the ar vinclozolin response does not occur at 24 hpf, and that the response plateaus between 48 and 72 hpf. An increase in ar transcript abundance has been detected in the adult fathead minnow after exposure to vinclozolin (Martinovic et al., 2008) as well as in rats (Loutchanwoot et al., 2008). Our data lends support to the hypothesis that ar upregulation in response to vinclozolin is evolutionarily conserved.

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Vinclozolin disrupts the signaling pathways of androgenic hormones, causing effects in many vertebrates. In mammals, vinclozolin exposure causes physical defects related to demasculization including retained nipples, atrophic testes, reduced prostate weight, and vaginal pouching (Gray et al., 2001; Wolf et al., 2004). Other health problems include Leydig cell tumors, delayed puberty, reduced sperm count, and altered DNA methylation patterns in male and female germ cells, which result in adult-onset disease and cancer in subsequent generations (Monosson et al., 1999; Kavlock and Cummings, 2005; Anway et al., 2006a,b). Psychological effects in rats, such as alterations in play behaviors (Hotchkiss et al., 2003) and learning disorders (Andre and Markowski, 2006) have also been observed. Vinclozolin may even affect mate preference through an epigenetic, transgenerational mechanism (Crews et al., 2007). Studies with dark-eyed juncos demonstrate that social behavior in songbirds can be affected by vinclozolin (Satre et al., 2009). These numerous effects in both aquatic and terrestrial model vertebrates suggest that pesticide exposure could be a health issue; exposure may be impacting human fertility (Roeleveld and Bretveld, 2008). Vinclozolin is being partially phased out of use in the United States, but is still used in that country and much of the world. By the year 2000, the Environmental Protection Agency (EPA) has banned residential use on turf in the U.S.A., but allowed industrial and golf course applications. In addition all crop use was phased out except for canola, and an important tolerance level was established for wine grapes (EPA, 2000). Nevertheless, in 2009 the EPA listed vinclozolin in its Contaminant Candidate List for products that may be assessed for contamination levels in drinking water (EPA, 2009). In conclusion, we have found the sequence homology and genomic structure of ar to be highly conserved during evolution. Developmental expression of ar indicates the gene is up-regulated in response to the anti-androgen vinclozolin. This up-regulation also appears to be evolutionarily conserved. The increase in ar expression level is consistent with our hypothesis that embryos would increase ar production in order to compensate with the presence of the anti-androgen, vinclozolin. While this up-regulation of the gene could provide some amelioration to vinclozolin effects, high doses of the fungicide would likely overwhelm the embryo's response. Vinclozolin or its metabolites may be directly interacting with a repressing transcription factor that binds the promoter region of the ar gene itself. However, the regulation may be indirect if vinclozolin metabolites interfere with transcription of genes that normally repress transcription of ar. Further research is required to identify the vinclozolin-responsive elements in the promoter region of the ar gene. In addition, our laboratory plans to investigate the effect of vinclozolin on androgen-regulated genes. Future studies on vinclozolin and other EDCs should elucidate the process by which environmental hormones affect human health and the health of other organisms. Acknowledgements The authors are grateful to E. Taylor and C. Waterstraut for assistance in cloning the receptor. B. Ford and G. Schaperjahn aided in zebrafish husbandry and embryo collection. Funding was provided by the Roanoke College Summer Scholars Program to L.-T. Kratzke, A. Smolinsky, and J. Doughman. Funding was provided by a Roanoke College Faculty Development Grant to C. Lassiter. The sequence for zebrafish ar indicated is found in GenBank under accession number EU708622. References Andre, S.M., Markowski, V.P., 2006. Learning deficits expressed as delayed extinction of a conditioned running response following perinatal exposure to vinclozolin. Neurotoxicol. Teratol. 28, 482–488. Anway, M.D., Leathers, C., Skinner, M.K., 2006a. Endocrine disruptor vinclozolin induced epigenetic transgenerational adult-onset disease. Endocrinology 147, 5515–5523.

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