Novel Estrogen Receptor-Related Transcripts in Marisa cornuarietis

Full details of all methodologies (including primer sequences) can be found in the online Supporting ... Extended 5' sequence was attained with the SM...
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Environ. Sci. Technol. 2007, 41, 2643-2650

Novel Estrogen Receptor-Related Transcripts in Marisa cornuarietis; a Freshwater Snail with Reported Sensitivity to Estrogenic Chemicals R I C H A R D B A N N I S T E R , * ,† NICOLA BERESFORD,† DENISE MAY,‡ EDWIN J. ROUTLEDGE,† SUSAN JOBLING,† AND MARIANN RAND-WEAVER‡ Institute for the Environment, Brunel University, Uxbridge, Middlesex, UB8 3PH, UK, and School of Health Sciences and Social Care, Brunel University, Uxbridge, Middlesex, UB8 3PH, UK

We have isolated novel molluskan steroid receptor transcripts orthologous to vertebrate estrogen receptors (ERs) and estrogen receptor-related receptors (ERRs) from the freshwater snail Marisa cornuarietis. Radiolabeled ligand binding analyses showed that neither recombinant receptor protein specifically bound 17β-estradiol over the range applied (0.3-9.6 nM). These novel receptor transcripts have thus been designated mcER-like and mcERR respectively. Quantitative PCR revealed mcER-like to be expressed ubiquitously throughout a range of male and female structures studied, including neural and reproductive tissues. Highest absolute levels were seen in the male penissheath complex. The mcERR mRNA was also expressed ubiquitously throughout all male and female tissues analyzed here, with very low absolute transcript numbers in female accessory sex structures compared to other tissues.

Introduction Currently, there is much scientific debate about the potential for environmentally relevant concentrations of estrogenic chemicals, particularly bisphenol A (BPA), to cause adverse reproductive and developmental effects in the neo-tropical freshwater snail Marisa cornuarietis (1-4). At least some reported responses (1, 2) to xenoestrogens (e.g., enhancement of oocyte production in reproducing adult females, the formation of additional organs in developing juvenile females and reduction in penis length in males) appear to be comparable to effects observed in fish and mammals, suggesting the possible existence of a common mechanism of estrogen action in these taxa. These studies have caused a great deal of argument and have potential political importance as they indicate that BPA may cause effects at doses that are ecologically relevant and lower than those reported to cause effects in all other animals tested. Controversy surrounds the issue (4), largely because the exposure studies currently published concerning the effects of BPA on fecundity (1-3) appear to contradict one another. * Corresponding author phone: +44(0)1895 266267; fax: +44(0)1895 269761; e-mail: [email protected]. † Institute for the Environment. ‡ School of Health Sciences and Social Care. 10.1021/es062565m CCC: $37.00 Published on Web 02/28/2007

 2007 American Chemical Society

Moreover, fundamental evidence of a plausible signaling pathway via which estrogenic chemicals might exert their effects in snails has not been described. One very recent scientific study concludes there is little evidence for effects of BPA in M. cornuarietis (3). Other earlier papers report strong evidence for effects and for an estrogen-receptor mediated mechanism of action for BPA and other estrogens (1, 2). The differences between the effects of BPA on fecundity presented in the various studies are not surprising given the vast differences in study design (e.g., number of replicates, origin of snails, temperature, and density) between those conducted by Forbes et al. (3) and the earlier studies conducted by Oehlmann et al. (1, 2). Furthermore, the displacement of tritiated 17β-estradiol from cytosolic extracts of M. cornuarietis gonadal tissues by non-labeled 17β-estradiol and by BPA (2) do not demonstrate conclusively the existence of functional steroid hormone receptors in M. cornuarietis as has been pointed out by Deitrich et al. (4). These findings could indicate binding of 17β-estradiol and BPA to other targets, such as steroid hormone binding globulins (5). Nevertheless, the fact that the in-vivo responses were also antagonized by classical antiestrogens (Tamoxifen and Faslodex) lends further support to the contention that Marisa cornuarietis has functioning estrogen-signaling pathways. In vertebrates, estrogens are known to exert their effects via nuclear hormone receptors such as the estrogen receptors (ERR and ERβ), which are members of the nuclear receptor (NR) subfamily 3 (6-8). Also included within NR subfamily 3 are proteins that are highly similar in terms of structure to the estrogen receptors, but for which no endogenous ligand is known. These estrogen receptor-related receptors (ERRs) are termed “orphan receptors” (9), and increasing attention is being paid to their possible role in estrogen related diseases, such as breast cancer (10). Mammalian ERs are found in a wide range of reproductive and non-reproductive tissues and are known to perform key roles in both male and female reproductive systems (11), as well as playing important roles in a diverse range of other functions, including bone development (12). ERs are also known to be important in reproduction in non-mammalian vertebrates. For example, fish possess three ER subtypes (R, β1, and β2), all of which are expressed in male and female gonads (13) and in the brain, indicating a role for these factors in the control of reproduction and behavior (14). In addition, several fish ERRs have recently been characterized (15). Relatively recently, estrogen receptor orthologues have been reported in molluskan species such as the sea hare Aplysia californica (16), the common octopus Octopus vulgaris (17), and the rockshell Thais clavigera (18). To date, however, no evidence for ligand binding by any of these receptors has been demonstrated despite the fact that a range of potential ligands have been tested in both radio-ligand binding and mammalian-cell reporter-gene assays. When compared to the vertebrates, very little is known about molluskan endocrinology, and it is unclear whether estrogens and their mimics may also exert reproductive effects upon M. cornuarietis via nuclear hormone receptors. The aim of this paper is to investigate the existence and distribution of estrogen-receptor-related transcripts in adult M. cornuarietis as a first step to further understanding a possible mode of action of estrogenic chemicals in this organism. We report here the first evidence for estrogen receptor and estrogen-receptor-related receptor transcripts in adult M. cornuarietis and their differential expression in a range of tissue types. Although the results described herein VOL. 41, NO. 7, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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do not explain or confirm the findings reported by Oehlmann et al. (1, 2), they provide new molecular insights into the mode of estrogen action in M. cornuarietis.

Materials and Methods Full details of all methodologies (including primer sequences) can be found in the online Supporting Information. Animals. Marisa cornuarietis used in this study were obtained from stocks bred at Brunel University, UK, which were originally derived from a stock maintained by Prof. Dr. Jo¨rg Oehlmann, Johann Wolfgang Goethe University Frankfurt am Main, Department of Ecology & Evolution, Frankfurt am Main, Germany. PCR Amplification and cDNA Cloning. A degenerate primer RT-PCR approach was employed to identify cDNAs encoding putative steroid receptors derived from M. cornuarietis total tissue extracts. Total RNA was isolated from reproductively mature female snails using the guanidinium thiocyanate-phenol-chloroform extraction method described by Chomczynski and Sacchi (19), including a further precipitation step with 4 M lithium chloride. Messenger RNA was isolated using the Nucleotrap mRNA purification kit (Macherey-Nagel). First strand cDNA synthesis was performed using Superscript III (Invitrogen) according to the manufacturer’s instructions. All degenerate PCR was performed using Platinum Taq (Invitrogen). PCR products were resolved on agarose/TBE gels, and fragments of the expected size were excised, purified, cloned and sequenced. A gene-specific sense primer together with a degenerate antisense primer were used to obtain further 3′ sequence of a degenerate fragment encoding a putative ER orthologue transcript. Complete 3′ sequence (including stop-codons and 3′ untranslated regions) of the putative ER and ERR orthologue transcripts were elucidated by 3′ RACE (rapid amplification of cDNA ends). Extended 5′ sequence was attained with the SMART RACE cDNA amplification kit (Clontech) using gene-specific primers. The full translated region of an ER orthologue coding sequence was verified by PCR-cloning using specific primers. Full ERR coding sequence was also verified by PCR and cloning. Specific primer PCRs were performed using Pwo Superyield polymerase (Roche). Phylogenetic Analyses. Non-M. cornuarietis amino acid sequences included in phylogenetic analyses conducted were obtained via Entrez on the National Center for Biotechnology Information (NCBI) server (http://www.ncbi.nlm.nih.gov). Multiple alignments were performed using Clustal W (20). Phylogenetic tree construction was performed by maximum likelihood using PhyML (21, 22). Expression Plasmids. The full coding regions of mcERlike (short protein-coding isoform) and mcERR were directionally cloned into pSG5 expression plasmid (Stratagene). Ligand-Binding. Binding of [2, 4, 6, 7-3H] 17β-estradiol to mcER-like and mcERR was assessed in triplicate by radioligand binding assay using the method described by Routledge et al. (23). The transcription/translation product from reaction containing pSG5-mouseERR (a gift from Prof. Malcolm Parker) was used as a positive control. Quantitative and Semiquantitative PCR. Whole albumen gland (female), capsule gland (female), cephalic tentacle, cerebral ganglia, gonad-digestive complex, and penis sheath complex (male) were dissected from individual snails along with sections of foot and lung tissue. All tissue was snapfrozen in liquid nitrogen immediately following dissection. Total RNA was isolated from individual organs from five replicate snails, using Tri-Reagent (Sigma), according to the manufacturers recommended protocol. All total RNA samples were treated with DNase I (Invitrogen) to remove traces of genomic DNA. To determine absolute amounts of transcripts, RNA standards were synthesized in-vitro from DNA tem2644

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plates. Real-time PCR was carried out using the one-step QuantiTect SYBR Green RT-PCR kit (Qiagen) with an ABI PRISM 7900 sequence detection system (Applied Biosystems). Each experiment included absolute negative controls (no template), no reverse transcriptase controls and negative tissue controls (RNA isolated from fathead minnow testis). All determinations were carried out in triplicate. Semiquantitative RT-PCR was performed on equal volumes of first strand cDNA synthesized from 500 ng DNase I-treated RNA using mcER-like and mcERR-specific primers with Platinum Taq (Invitrogen). Statistical Analyses. Data sets were tested for normality by Kolmogorov-Smirnov test and for homogeneity of variances by Levenes test. Inter-tissue differences in mean mcER-like and mcERR copy number per ng total RNA in males were tested using 1-way ANOVA followed by Tukeys test. Female tissue data sets for mcER-like and mcERR were found to have non-homogeneous variances, and therefore, differences were tested for by Kruskal-Wallis test followed by pairwise Mann-Whitney tests. All statistical analyses were conducted using SPSS 13.0.

Results Isolation of Novel ER-Related cDNAs from Marisa cornuarietis. Degenerate Primer RT-PCR. Degenerate primer RT-PCR produced a DNA band around 450 bases in size (data not shown) and sequencing of multiple clones revealed that DNA of distinct nuclear receptors had been isolated. Initial analyses conducted on these sequences revealed a 448-base fragment (excluding sequence derived from degenerate primers) of a novel member of the nuclear receptor superfamily. A second 433-base sequence of a novel nuclear receptor, clearly distinct from the first, was also identified. Characterization of a Novel ER Orthologue from Marisa cornuarietis. Use of further RT-PCR and RACE using primers designed to the 448-base sequence led to the isolation and characterization of the full open-reading frame of a NR with high sequence similarity to ERs from various species (Figure 1), which was named “Marisa conuarietis estrogen-receptorlike (mcER-like)”. Three isoforms differing in sequence in the 5′ untranslated region and 5′-most end of the coding region were identified, including one variant with a putative start codon upstream of that present in other isoforms. Total isoform transcript lengths varied between 1713 and 1857 nucleotides; the theoretically translated protein was 470 amino acids long (527 amino acids for the identified putative isoform variant longer protein form). Accession numbers for these isoforms are DQ923062, DQ923063 (shorter proteincoding isoforms) and DQ923064 (longer protein-coding isoform). The closest known NRs to this novel receptor in terms of sequence are molluskan estrogen receptor orthologues, the first of which was isolated from the sea hare Aplysia californica (16). Like the A. californica molecule, the NR characterized here shares various features with vertebrate ERs including six identifiable domains (A-F) characteristic of NRs. These domains include a DNA binding domain (DBD) containing the eight conserved cysteine residues characteristic of the 2-zinc finger motif of this domain, and a putative ligand binding domain (LBD) with regions of high amino acid conservation between the mollusk and vertebrate ERs. There is high sequence identity of the mcER-like DBD to the molluscan (95% to A. californica) and vertebrate ERs (88 and 84% to human ERR and ERβ, respectively). This novel sequence has a proximal (P)-box identical to that of vertebrate ERs, and highly similar distal (D)-box sequence (the P- and D-box are small amino acid regions of the DBD important in target DNA recognition). The LBD is somewhat less well conserved between mollusk and human ERs (37 and 35% identity of mcER-like with ERR and ERβ respectively),

FIGURE 1. mcER-like. (A) Schematic representation of mcER-like domains (theoretically translated from the isoform encoding the shorter A/B domain) and their similarity to selected steroid hormone receptors. Percentage amino acid identities of each domain to the corresponding mcER-like domain are shown. Amino acid sequence alignments of mcER-like, (B) DNA-binding domain, and (C) ligand-binding domain (AF2 region) with selected comparable NR sequences. The positions of the eight conserved cysteines of the DBD are asterisked, and the P-box and D-box are enclosed in boxes. (.) Indicates amino acid identity to mcER-like. All sequences are human unless otherwise stated, ac ) Aplysia californica, dm ) Drosophila melanogaster, mc ) Marisa cornuarietis. Genbank accession numbers: A. californica ER (AY327135); D. melanogaster EcR (M74078); Human AR (M20132), ERr (X03635), ERβ (AB006590), ERRr (X51416), GR (X03225), MR (M16801), PR (M15716), RXRr (X52773). although there are regions of extremely high similarity, including activation function 2 (AF2). The AF2 region is important in ligand-dependent transactivation of downstream target DNA by enabling the recruitment of co-activator proteins. Characterization of a Novel ERR Orthologue from Marisa cornuarietis. RACE performed using sequence information from the 433-base fragment cloned from the initial RT-PCR led to the isolation of a full-length novel nuclear receptor with high sequence identity to known vertebrate and invertebrate estrogen receptor-related receptors (Figure 2; Genbank accession number DQ923065). The DBD of this novel nuclear receptor has higher sequence identity to vertebrate and invertebrate ERRs than to any other receptor type, including the ERs (g86% amino acid identity with all ERR DBDs shown here, with 70% identity to the 2 vertebrate ER subtype DBDs). The P-box sequence is identical to other ERRs and the D-box is similar to that of other ERRs, particularly ERRR, to which there is only one amino acid difference. There is relatively weak conservation of sequence between the LBD sequence of this novel

molluskan ERR, and other vertebrate and invertebrate ERRs (32-41%). The AF-2 region of the mcERR LBD is, however, much more similar to that of vertebrate (human, 75%) and invertebrate (Drosophila, 67%) ERRs than to other nuclear receptors (e50%). In terms of amino acid sequence the mcERR DBD is less similar to the mcER-like DBD (68% identity) than to vertebrate ERRs and ERs. Amino acid identity between the mcERR and mcER-like E/F domain is 25%. Note that no isoform variants of the mcERR mRNA were discovered here. Phylogenetic Analyses. Construction of a phylogenetic tree using maximum likelihood on DBD, hinge and LBD sequences of selected nuclear receptors shows that mcERlike is closely related to other identified molluskan ER orthologues, and that these receptors form a secondary clade with vertebrate ERs within the NR subfamily 3 that is strongly supported by a boot strap value of 97% (Figure 3). Furthermore, the analyses conducted here show that this novel NR is more similar to known ERs than to other steroid hormone receptors or to the ERRs. The M. cornuarietis ERR orthologue forms a primary clade with Drosophila ERR, which, in turn, VOL. 41, NO. 7, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. mcERR. (A) Schematic representation of mcERR domains and their similarity to selected steroid hormone receptors. Percentage amino acid identities of each domain to the corresponding mcERR domain are shown. Due to the structure of the ERR, the E and F domains were grouped for comparison here. Amino acid sequence alignments of mcERR, (B) DNA-binding domain, and (C) ligand-binding domain (AF2 region) with selected comparable NR sequences. The positions of the eight conserved cysteines of the DBD are asterisked, and the P-box and D-box are enclosed in boxes. (.) Indicates amino acid identity to mcER. All sequences are human unless otherwise stated, dm ) D. melanogaster, mc ) Marisa cornuarietis. Accession numbers not given previously; Drosophila melanogaster ERR (AF359420); Human ERRβ (AF094517), ERRγ (AF058291). cluster secondarily with vertebrate ERRs. mcERR is, therefore, phylogenetically more closely related to vertebrate and invertebrate ERRs than to the vertebrate steroid receptors or molluskan ER orthologues. Ligand-Binding Analyses. No evidence of specific binding of [2, 4, 6, 7-3H] 17β-estradiol to either mcER-like or mcERR recombinant proteins was found (Figure 4). Assay validation was performed using recombinant mouse ERR protein, which specifically bound [2, 4, 6, 7-3H] 17β-estradiol. The novel snail proteins were also challenged with radiolabeled testosterone and progesterone, with no specific binding observed (data not shown). Expression Analyses. Quantitative real-time RT-PCR was performed to elucidate the expression profiles of mcER-like 2646

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and mcERR in adult male and female snails across a range of tissues types. Males. Both mcER-like and mcERR were ubiquitously expressed in all male tissues analyzed (Figure 5). High mcERlike expression was seen in the penis and sheath. Indeed, the mean absolute transcript number in this accessory reproductive structure was significantly greater than in any other male tissue studied here (Figure 5A). Copy numbers of mcERR in the penis and sheath were not as markedly elevated when compared with other tissues and indeed were only significantly higher than those levels measured in the lung (Figure 5B). The close proximity and structural “interlocking” of the digestive and gonadal tissues of the visceral coil of the snail

FIGURE 3. Phylogram constructed based on DNA, hinge and ligand-binding domains of nuclear receptor subfamily 3 receptors using maximum likelihood. Numbers on branches indicate percentage of 100 bootstrap replicates supporting topology shown. Human RXRr was included as an outgroup for tree construction. All sequences are human unless otherwise stated, dm ) Drosophila melanogaster, mc ) Marisa cornuarietis, ov ) Octopus vulgaris, tc ) Thais clavigera. Accession numbers not given previously; Octopus vulgaris ER (DQ533956), Thais clavigera ER (AB077032). tissues analyzed (Figure 6). There were no significant differences between absolute mcER-like transcript number measured in any female tissues. The most striking aspect of mcERR expression in the female snail was the low transcript levels detected in reproductive tissues (gonad-digestive complex, albumen gland, and capsule gland) compared to non-reproductive tissues (Figure 6B). Absolute mcERR copy numbers in these reproductive tissues were significantly lower than in all other tissues studied here. Expression was particularly low in the albumen gland; indeed copy number in this tissue was significantly lower than even the gonaddigestive complex. Subsequent semiquantitative RT-PCR carried out on dissected gonadal and digestive components of the female visceral coil revealed expression of both transcripts in digestive and ovarian tissue (data not shown).

Discussion FIGURE 4. Radio-ligand binding assays. The potential of recombinant mcER-like and mcERR proteins to bind [2, 4, 6, 7-3H] 17β-estradiol was investigated. Specific binding was calculated after incubation for 16 h at 4 °C and subsequent removal of unbound radio-labeled ligand. Nonspecific counts were subtracted from specific bound counts and normalized to the maximum binding observed of [2, 4, 6, 7-3H] 17β-estradiol to recombinant mouse ERr (% maximum specific binding). All work was conducted in triplicate. (9) mcERlike, ([) mcERR, and (2) mouse ERr. make achieving perfect separation of these tissues extremely difficult. In a subsequent experiment, however, we were able to dissect out portions of each tissue that were not contaminated by the neighboring tissue. Semiquantitative RTPCR performed on cDNA derived from these dissected tissues showed that both mcER-like and mcERR were expressed in both the testes and digestive gland (data not shown). Females. The M. cornuarietis ER and ERR orthologue transcripts were ubiquitously expressed throughout all female

We report here the isolation and characterization of two novel NR superfamily members from the freshwater gastropod Marisa cornuarietis. Phylogenetic analyses show that both these molluskan NRs lay within nuclear receptor (NR) subfamily 3. One is an orthologue of vertebrate estrogen receptors and reported molluskan estrogen receptors (ERs). The other is structurally highly similar to vertebrate and invertebrate estrogen receptor-related receptors (ERRs). Multiple isoform variants of mcER-like were isolated here, including one encoding a theoretical protein with a 57 amino acid N-terminal extension. ER N-terminal variants have also been reported from vertebrate species including rainbow trout (Oncorhynchus mykiss) (24). Comparisons of the structure, organization and sequence (particularly in the DBD) of the molluskan and vertebrate ERs indicate that these are true orthologues. Unlike vertebrate estrogen receptors, no molluskan ER orthologue has been shown to bind 17β-estradiol, despite VOL. 41, NO. 7, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 5. Expression of estrogen receptor-related transcripts in adult male Marisa cornuarietis tissues. Absolute levels of mcERlike (A) and mcERR (B) were measured in tissues dissected from male snails by real-time RT-PCR using the SYBR green I method. Data is presented as box and whisker plots. Black bars ) medians, white bars ) means. N ) 5 unless stated otherwise. Superscripts denote significant differences in mean transcript number. the use of both radio-ligand binding assays and cell-based reporter gene assays (16-18). The radio-ligand binding assay used here shows that like other molluskan ER orthologues, recombinant mcER-like does not specifically bind 17βestradiol over the range studied (0.3-9.6 nM). Although it is possible that mcER-like may bind 17β-estradiol at higher concentrations, our results suggest that this compound is not the endogenous ligand to the M. cornuarietis ER orthologue. There is, therefore, no evidence that mcER-like is a likely candidate binding site for 17β-estradiol in cytosolic extracts used to show displacement of radiolabeled 17βestradiol by unlabeled compound (2). It cannot be discounted that mcER-like may bind 17β-estradiol with low affinity and be responsible for at least some of the cytosolic binding; however, further study is required to elucidate the true nature of these binding sites. mcER-like was so named to reflect the fact that, while this is indeed a vertebrate ER orthologue, there are evidently important functional differences between these receptors in different phyla. To our knowledge, the present study contains the first complete published report of a molluskan ERR. Unlike the ERs, ERRs have now been discovered in a relatively wide range of animal groups, including vertebrates, the cepaholchordate invertebrate amphioxus (25), the urochordate Ciona intestinalis (26), Drosophila (27), and now mollusks, 2648

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FIGURE 6. Expression of estrogen receptor-related transcripts in adult female Marisa cornuarietis tissues. Absolute levels of mcERlike (A) and mcERR (B) were measured in tissues dissected from female snails by real-time RT-PCR using the SYBR green I method. Data is presented as box and whisker plots. Black bars ) medians, white bars ) means. N ) 5. Superscripts denote significant differences in median transcript number. suggesting that ERRs arose in an ancestor of the bilateral metazoans (28). A greater understanding of the functions of ERRs from different phylogenetic groups will provide insights into why they appear to have been retained by a relatively wide spectrum of taxa compared to the ERs. mcERR did not bind [2, 4, 6, 7-3H] 17β-estradiol in radio-ligand binding assays here, which is consistent with other vertebrate and invertebrate ERRs. Despite this, various phytoestrogens (29) and synthetic compounds (30) have been shown to affect downstream transcriptional regulation by ERRs. Therefore, the identity of the natural endogenous ligand for ERRs may await discovery, and the notion that ERRs are indeed true orphans remains unresolved (31). Expression of mcER-like and mcERR was observed throughout all male and female tissues studied here and was not confined to the reproductive tissues. Universal expression of a molluskan ER orthologue in a wide range of tissues has been previously reported from Octopus vulgaris (17), indicating that this nuclear receptor may play a key role in general cell growth or maintenance. Alternatively, molluskan ERs, like their vertebrate orthologues, may perform diverse roles in a number of processes in a tissue-specific manner. In Octopus (17) and Thais clavigera (18), the ER transcripts were quantified relative to “housekeeping” transcripts. Here, quantification of mcER-like was performed by measuring absolute levels of RNA by real-time PCR. When taken together,

the results obtained using these different methods provide some interesting insights. For example molluskan ER levels relative to housekeeping transcripts are high in the ovaries when compared with other tissues, whereas we show here that absolute levels of mcER-like in female reproductive tissues are lower (although not significantly) than in other female tissues sampled. It is suggested that further functional studies are required to elucidate the true biological significance of all these data. Perhaps the most intriguing finding in this expression study is the high absolute levels of mcER-like seen in the penis sheath complex. Here, mean copy number per ng total RNA is significantly greater than in any other male tissue studied. Although mRNA expression levels do not necessarily relate to protein expression, this finding may indicate a putative important role for this novel nuclear receptor in male reproductive physiology. Vertebrate ERs are also known to be associated with the male reproductive system. Indeed, ERβ was first cloned from rat prostate (7) and both human ER subtypes are expressed in penile tissues including the corpus cavernosum, corpus spongiosum and urethra (32). In-vivo exposure studies conducted using M. cornuarietis reported a reduction in penis length in individuals exposed to the xeno-estrogen Bisphenol A (2). The imposition of male sex organs (including a penis) onto female mollusks in response to organotins is also well-documented (33, 34). Assuming putative roles for mcER-like in growth, development and maintenance of male accessory reproductive structures, it may transpire that this factor is involved in one or more pathways that may be affected by these chemicals; however, further studies are needed to test this hypothesis. Expression profiling of mcERR in female tissues shows that this factor is weakly expressed in reproductive tissues (gonad-digestive complex, capsule gland, and most notably in the albumen gland). This may indicate that this factor may not play an important role in M. cornuarietis female reproductive biology, although it must be considered that a low-level effect of this receptor may be vital. It must also be considered that reproductive cycling of this factor cannot be discounted, and that the ERR might modulate ER-like activity in some tissues, as it does in some cancer cell lines (35). In the males, mcERR expression is highest in the penis and sheath, although the difference between transcript levels in this and other tissues is not as marked as is the case for mcER-like. Despite the fact that relatively little is known about the functions of ERRs in chordates, roles that have been suggested for these factors include control of cell proliferation and differentiation in the brain, lipid storage and consumption, and mitochondrial biogenesis (28). The ubiquitous and differential expression of mcERR in adult snail tissues seen here indicates that this factor may play multiple tissuespecific roles, although it is difficult to speculate what these roles may be. The aforementioned roles for ERRs in chordates are likely to be conserved through evolution (28). It is, therefore, plausible that mcERR may be involved in one or more of these processes, along with other, as yet unidentified functions. The presence of ER and ERR receptor orthologues in mollusks, together with sex-specific differences in their expression in reproductive tissues, suggests a possible role of these receptors in invertebrate reproduction. However, binding studies suggest that their ligands (if any) will be somewhat different to their vertebrate counterparts.

Acknowledgments We acknowledge Professor Yuji Takehashi for advice on degenerate primer design and PCR procedures. This work was funded by BBSRC grant 100/S183300, the European Union (COMPRENDO EVK1-CT-2002-00129) and by the U.K. Environment Agency.

Supporting Information Available Full details of all methodologies (including primer sequences). This material is available free of charge via the Internet at http://pubs.acs.org.

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Received for review October 26, 2006. Revised manuscript received January 21, 2007. Accepted January 26, 2007. ES062565M