Environ. Sci. Technol. 2008, 42, 1345–1351
Synchronized Expression of Retinoid X Receptor mRNA with Reproductive Tract Recrudescence in an Imposex-Susceptible Mollusc ROBIN M. STERNBERG, ANDREW K. HOTCHKISS,† AND GERALD A. LEBLANC* Department of Environmental and Molecular Toxicology, North Carolina State University, Campus Box 7633, Raleigh, North Carolina 27695
Received June 25, 2007. Revised manuscript received November 13, 2007. Accepted November 14, 2007.
The biocide tributyltin (TBT) causes the development of male sex characteristics in females of some molluscan species, a phenomenon known as imposex. Recent evidence suggests that the retinoid X receptor (RXR) participates in TBTinduced imposex. Accordingly, we hypothesized that RXR may contribute to the seasonal development of the male reproductive tract in molluscs and would be expressed in concert with this phenomenon. RXR was cloned and sequenced from an imposex-susceptble species, the eastern mud snail Ilyanassa obsoleta. The DNA-binding domain of the receptor protein was 100 and 97% identical to those of the rock shell Thais clavigera and the freshwater snail Biomphalaria glabrata. The ligand-binding domain was 93 and 92% identical to the LBD of these two molluscan species, respectively. Phylogenetic analyses revealed that RXR is an ancient nuclear receptor whose origin predates the emergence of the Bilateria. Interestingly, though inexplicably, the molluscan RXRs were more similar to sequences of vertebrate RXRs than to the RXRs of other lophotrochozoan invertebrates. Next, the expression of RXR mRNA levels in the reproductive tract was determined through the reproductive cycle. RXR mRNA levels increased commensurate with reproductive tract recrudescence in both sexes. However, the timing of coordinate recrudescence-RXR expression differed between sexes. Results demonstrate that RXR expression is associated with reproductive tract recrudescence in both sexes; although, the timing of recrudescence may dictate sex-specific development. Retinoid signaling initiated by TBT during an inappropriate time in females may result in imposex.
Introduction Sex differentiation is a complex process that is regulated by the action of many signaling pathways (1). Retinoid signaling has been increasingly implicated in the regulation of male reproductive differentiation and development. For example, RXRβ (retinoid X receptor, NR2B)-deficient mice produce abnormal spermatogonia (2). Semeniferous tubules of rodents fed a vitamin A (retinol)-deficient diet contained * Corresponding author phone: 919-515-7404; fax: 919-515-7169; e-mail address:
[email protected]. † Current address: U.S. Environmental Protection Agency, ORD, NHEERL, Research Triangle Park, NC 27711. 10.1021/es702381g CCC: $40.75
Published on Web 01/16/2008
2008 American Chemical Society
spermatogonia but were devoid of meiotic cells (3, 4) indicating an arrest of spermatogenesis. Specifically, vitamin A deficiency impeded the differentiation of A1 spermatogonia shortly before the S phase (5), but replenishment of vitamin A to the diet resulted in the restoration (6, 7) and synchronization (5, 8) of spermatogenesis. Thus, retinoids are required for proper spermatogenesis in rodents. The precise mechanism by which retinoic acid regulates spermatogenesis is unknown. However, studies examining the fate of embryonic germ cells in rodents may provide crucial, mechanistic insight. Germ cells in the ovary undergo meiosis and progress into oocytes prenatally whereas germ cells in the testis undergo meiosis and develop into spermatocytes postnatally. Recent studies have implicated retinoic acid as the key determinant for the timing of germ cell meiosis. In particular, retinoic acid stimulated the expression of Stra8 (Stimulated by retinoic acid gene 8), a protein that is necessary for the commencement of meiotic division. Elevated retinoic acid levels in the prenatal ovary induced Stra8 to initiate germ cell meiosis (9). In contrast, prenatal testes expressed high levels of CYP26B1, an enzyme that metabolizes retinoic acid to an inactive form. This high expression in the intact animal was shown to prevent the accumulation of retinoic acid and Stra8 expression in the testis (9, 10). In postnatal testes, CYP26B1 expression is suppressed allowing for the accumulation of retinoic acid levels and the induction of Stra8 (9, 10). Therefore, retinoid signaling determines the ultimate sex-specific fate of rodent germ cells by controlling the timing of meiosis. Sex determination in branchiopod crustaceans is regulated by the terpenoid hormone methyl farnesoate. Exposure of daphnids to high levels of methyl farnesoate (>300 nM) at the time approximating the first embryonic cleavage caused the embryo to develop a male phenotype whereas exposure to low levels of the hormone ( 0.05). Males: n ) 5 or 6 per month except n ) 3 for Feb; females: n ) 5 or 6 for January, April–June, and September– December, n ) 3 or 4 for March, July, and August. Error bars represent standard errors (SE). Dorm ) dormant; Recrud ) recrudescent; Sen ) senescent end of the sequence was not complete. Therefore, the final alignment (Figure 1) and associated phylogenetic analyses (Figure 2) did not include sequence 5′ to the DBD (i.e., the A/B N-terminal domain). The DBD of the mud snail RXR is 100% identical to that of the rock shell T. clavigera, and 97, 90, 60, and 87% identical to the DBDs of RXRs from the freshwater snail B. glabrata, human (RXRR), the jellyfish Tripedalia cystophora, and the fruitfly Drosophilia melanogaster, respectively. The P-box of the mud snail RXR, the region of the DBD that is important for interacting with DNA response elements, is identical to the P-box of other RXRs (Figure 1). The LBD of the mud snail RXR is 93 and 92% similar to that of the molluscs T. clavigera and B. glabrata, respectively, and 87, 59, and 51% similar to the LBDs of RXRs from human (RXRR), T. cystophora, and D. melanogaster, respectively. The AF-2 sequence of the mud snail RXR, the region of the LBD that is important for ligand-dependent transactivation of the receptor, is identical to the AF-2 sequence of T. clavigera, B. glabrata, and human (RXRR) but differs from that of T. cystophora and D. melanogaster in one and four amino acids, respectively (Figure 1). Twenty residues of human RXRR are predicted to either line the ligand-binding cavity or form hydrogen bonds with 9-cis retinoic acid (28); these residues are identical among I. obsoleta, T. clavigera, B. glabrata, and human (RXRR) (Figure 1). Phylogenetic analyses were performed to establish whether this putative receptor has greatest identity to other molluscan RXRs. Parsimony analyses of the DBD, HD, and LBD of 34 invertebrate and vertebrate RXRs indicated that the mRNA
identified from the mud snail is most identical to the RXRs of other molluscs and that the group containing the mollusc RXRs is a sister group to that of the vertebrate RXRs (Figure 2). Within the mollusc group, the mud snail RXR was most closely related to the rock shell RXR (63.1% parsimony bootstrap) and then to the RXR from freshwater snails. As depicted in Figure 3, RXR is represented in major phyla of all three phylogenetic clades--Deuterstoma, Ecdyzoa, and Lophotrochozoa--indicating that RXR is among the ancestral bilaterian nuclear receptors. Indeed, RXR’s presence in the cnidarian jellyfish (Tripedalia) and sponge (Suberites) indicates that the emergence of RXR predates the emergence of the Bilateria. In general, the results of this cloning effort and subsequent phylogenetic analyses demonstrated that I. obsoleta expresses an RXR that is highly similar to the few RXRs cloned in other molluscs. Inexplicably, phylogenetic analyses revealed that molluscan RXRs are more closely related to vertebrate RXRs than to the RXRs of protostome invertebrate lineages. This relatedness may reflect functional conservation of RXR between vertebrates and molluscs, for example, with respect to ligand-binding characteristics (20). Molluscs also have been shown to express a vertebrate-like estrogen receptor (29–31) which, to our knowledge, has not been identified in any other protostome phyla. The expression of deuterostome-like characteristics in protostome molluscs warrants further investigation with respect to the evolution of this phylum. RXR mRNA Levels. Reproductive development was evaluated over a one-year period in a field population of VOL. 42, NO. 4, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
1349
mud snails. Reproductive status of the population was judged to be dormant, recrudescent, mature, or senescent (Table 1). These data were used to determine whether measured RXR mRNA levels were expressed in coordination with male recrudescence. RXR mRNA levels varied significantly in relation to reproductive status for both males (p < 0.0001; Figure 4B) and females (p < 0.0027; Figure 4D). RXR mRNA levels were low in both sexes during the months of May and June, when both sexes were undergoing senescence, and in July, when both sexes were reproductively dormant (Figure 4A,C). Recrudescence in males commenced during August and was accompanied by a subsequent increase in RXR mRNA levels (Figure 4A). RXR mRNA levels in females also significantly increased during recrudescence (Figure 4C and D). However, female recrudescence began two months after recrudescence in males (Figure 4A and C). Accordingly, the recrudescence-associated increase in RXR mRNA levels in males occurred earlier in the year than that of females. Both sexes also expressed significantly higher levels of RXR mRNA during reproductive maturity. Thus, RXR mRNA levels increased appreciably in both sexes commensurate with recrudescence. However, the timing of recrudescence and commensurate expression of RXR mRNA differed between males and females. For example, RXR mRNA levels were high in the male during the months of September and October, but were low during these months in the female. RXR may be a regulator of recrudescence in both sexes with the timing of expression being critical to sex-specific differentiation. This phenomenon is analogous to retinoid signaling controlling the timing of sex differentiation in rodent germ cells. Retinoid induction of Stra8 prenatally resulted in germ cells differentiating into oocytes whereas retinoid induction of Stra8 postnatally caused germ cells to differentiate into spermatocytes (9, 10). Stra8 or similar genes that are susceptible to regulation by retinoid signaling and control sex differentiation may prove to be targets of retinoid signaling in molluscs. Elucidating the cascade of biochemical events that leads to the induction of RXR expression during recrudescence was beyond the purview of this study. However, these results do provide a foundation upon which the mechanism of reproductive tract recrudescence can be postulated. We propose that, during the nonreproductive months, RXR is under the suppressive control of a repressor element such as SMRT or NCoR. Such elements have been shown to negatively regulate the transcriptional activity of RXR in other species through the recruitment of histone deacetylases (32). At the onset of the period of reproductive tract recrudescence, an environmental cue such as photoperiod signals the loss of the repressor element, and RXR is no longer refractory to activation by ligand. Then during the window of recrudescence, early retinoid signaling (e.g., August–October) stimulates male reproductive tract development while late signaling (e.g., November–December) stimulates female reproductive tract development. The timing of the accumulation of RXR ligand (e.g., retinoic acid) is likely controlled by a second environmental cue such as temperature. Both photoperiod and temperature have been shown to be important in the timing of reproduction in molluscs (33). The early elevation of RXR mRNA levels in males during male reproductive tract recrudescence and the late elevation in females may reflect autoinduction of the receptor by its ligand, a common response of nuclear receptors (34). This proposed model provides a possible explanation for the ability of TBT to cause imposex. Exposure of females to TBT during the period that is permissive of male of reproductive tract recrudescence (August-October) may cause aberrant activation of the RXR signaling pathway with commensurate development of male sex characteristics. Exposure of females to TBT outside of the period of male reproductive tract 1350
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 4, 2008
recrudescence may have no effect either because the pathway is refractory (May–July) or is primed to stimulate female reproductive tract development (November–December). Female mud snails are insensitive to TBT-induced imposex when exposed outside of the period of male reproductive tract recrudescence (McClellan-Green, http://es.epa.gov/ncer/science/endocrine/pdf/wildlife/r827401_mcclellan-green-030405final.pdf; personal observations). Overall, the results of this study provide support for the hypothesis that RXR signaling regulates male reproductive differentiation in neogastropods. The results also provide insight into how TBT may manipulate the retinoid signaling pathway to cause imposex in female neogastropods. The importance of retinoid signaling in sex-specific gametogenesis of vertebrates, sex determination in some crustaceans, and recrudescence of neogastropods highlights the highly conserved nature of this signaling pathway among metazoans.
Acknowledgments We thank Re Bai for his technical contributions. This work was supported by NSF grant no. IBN 0234676.
Supporting Information Available Methods for quantification of mRNA levels, PCR quality control parameters, oligonucleotide primers used in all PCR reactions, and statistical methods. This material is available free of charge via the Internet at http://pubs.acs.org.
Literature Cited (1) Wilhelm, D.; Palmer, S.; Koopman, P. Sex determination and gonadal development in mammals. Physiol. Rev. 2007, 87 (1), 1–28. (2) Kastner, P.; Mark, M.; Leid, M.; Gansmuller, A.; Chin, W.; Grondona, J. M.; Decimo, D.; W., K.; Dierich, A.; Chambon, P. Abnormal spermatogenesis in RXRβ mutant mice. Genes Dev. 1996, 10, 80–92. (3) Mitranond, V.; Sobhon, P.; Tosukhowong, P.; Chindaduangrat, W. Cytological changes in the testes of vitamin-A-deficient rats. 1. Quantitation of germinal cells in the seminiferous tubules. Acta Anat. (Basel) 1979, 103 (2), 159–168. (4) Unni, E.; Rao, M. R. S.; Banguly, J. Histological and ultrastructural studies on the effect of vitamin-A depletion and subsequent repletion with vitamin-A on germ cells and Sertoli cells in rat testis. Indian J. Exp. Biol. 1983, 21 (4), 180–192. (5) VanPelt, A. M. M.; Derooij, D. G. The origin of the synchronization of the seminiferous epithelium in vitamin-A-deficient rats after vitamin-A replacement. Biol. Reprod. 1990, 42 (4), 677– 682. (6) Thompson, J. N.; Pitt, G. A.; Howell, J. M. Vitamin A and reproduction in rats. Proc. R. Soc. London, Ser. B 1964, 159 (976), 510–535. (7) Huang, H. F. S.; Hembree, W. C. Spermatogenic response to viatmin-A in vitamin-A deficient rats. Biol. Reprod. 1979, 21 (4), 891–904. (8) Morales, C.; Griswold, M. D. Retinol-induced stage synchronization in seminiferous tubules of the rat. Endocinology 1987, 121 (1), 432–434. (9) Koubova, J.; Menke, D. B.; Zhou, Q.; Capel, B.; Griswold, M. D.; Page, D. C. Retinoic acid regulates sex-specific timing of meiotic initiation in mice. Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (8), 2474–2479. (10) Bowles, J.; Knight, D.; Smith, C.; Wilhelm, D.; Richman, J.; Mamiya, S.; Yashiro, K.; Chawengsaksophak, K.; Wilson, M. J.; Rossant, J.; Hamada, H.; Koopman, P. Retinoid signaling determines germ cell fate in mice. Science 2006, 312, 596–600. (11) Olmstead, A. W.; LeBlanc, G. A. The juvenoid hormone methyl farnesoate is a sex determinant in the crustacean Daphnia magna. J. Exp. Zool. 2002, 293, 736–739. (12) Olmstead, A. W.; LeBlanc, G. A. The environmental-endocrine basis of gynandromorphism in a crustacean. Int. J. Biol. Sci. 2007, 3, 77–84. (13) Jones, G.; Jones, D.; Teal, P.; Sapa, A.; Wozniak, M. The retinoid-X receptor ortholog, ultraspiracle, binds with nanomolar affinity to an endogenous morphogenetic ligand. FEBS J. 2006, 273, 1–14.
(14) Wang, Y. H.; Wang, G.; LeBlanc, G. A. Cloning and characterization of the retinoid X receptor from a primitive crustacean Daphnia magna. Gen. Comp. Endrocrinol. 2007, 150, 309–318. (15) Smith, B. S. Sexuality of the American mud snail Nassarius obsoletus (Say). Proc. Malac. Soc. London 1971, 39, 377–78. (16) Grun, F.; Watanabe, H.; Zamanian, Z.; Maeda, L.; Arima, K.; Cubacha, R.; Gardiner, D. M.; Kanno, J.; Iguchi, T.; Blumberg, B. Endocrine-disrupting organotin compounds are potent inducers of adipogenesis in vertebrates. Mol. Endocrinol. 2006, 20, 2141–2155. (17) Kanayama, T.; Kobayashi, N.; Mamiya, S. Organotin compounds promote adipocyte differentiation as agonists of the peroxisome proliferator-activated receptor/retinoid X receptor pathway. Mol. Pharmacol. 2005, 67, 766–774. (18) Nishikawa, J.-I.; Mamiya, S.; Kanayama, T.; Nishikawa, T.; Shiraishi, F.; Horiguchi, T. Involvement of the retinoid X receptor in the development of imposex caused by organotins in gastropods. Environ. Sci. Technol. 2004, 38, 6271–6276. (19) Nishikawa, J. Imposex in marine gastropods may be caused by binding of organotins to retinoid X receptor. Mar. Biol. 2006, 149 (1), 117–124. (20) Bouton, D.; Escriva, H.; de Mendonca, R. L.; Glineur, C.; Bertin, B.; Noel, C.; Robinson-Rechavi, M.; de Groot, A.; Cornette, J.; Laudet, V.; Pierce, R. J. A conserved retinoid X receptor (RXR) from the mollusk Biomphalaria glabrata transactivates transcription in the presence of retinoids. J. Mol. Endocrinol. 2005, 34, 567–582. (21) Filipe, L.; Castro, C.; Lima, D.; Machado, A.; Melo, C.; Hiromori, Y.; Nishikawa, J.; Nakanishi, T.; Reis-Henriques, M. A.; Santos, M. M. Imposex induction is mediated through the retinoid X receptor signalling pathway in the neogastropod Nucella lapillus. Aquat. Toxicol. 2007, 85, 57–66. (22) Grahame, J. Shedding of the penis in Littorina littorea. Nature 1969, 221, 976. (23) Jenner, C. E.; Chamberlain, N. A. Seasonal resorption and restoration of copulatory organs in the mud snailNassa obsoleta. Biol. Bull. 1955, 109, 347. (24) Abdu, U.; Takac, P.; Yehezkel, G.; Chayoth, R.; Sagi, A. Administration of methyl farnesoate through the Artemia vector, and its effect on Macrobrachium rosenbergiilarvae. Isr. J. Aquacult. 1998, 50, 73–81.
(25) Chen, Y. N.; Fan, H. F.; Hsieh, S. L.; Kuo, C. M. Physiological involvement of DA in ovarian development of the freshwater giant prawnMacrobrachium rosenbergii. Aquaculture 2003, 228, 383–395. (26) Adoutte, A.; Balavione, G.; Lartillot, N.; Lespinet, O.; Prod’homme, B.; deRosa, R. The new animal phylogeny: Reliability and implications. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 4453–4456. (27) Livak, K. J.; Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2–∆∆CT method. Methods 2001, 25 (4), 402–408. (28) Egea, P. F.; Mitschler, A.; Rochel, N.; Ruff, M.; Chambon, P.; Moras, D. Crystal structure of the human RXR alpha ligandbinding domain bound to its natural ligand:9-cis retinoic acid. EMBO J. 2000, 19 (11), 2592–2601. (29) Thornton, J. W.; Need, E.; Crews, D. Resurrecting the ancestral steroid receptor: Ancient origin of estrogen signaling. Science 2003, 301, 1714–1717. (30) Kajiwara, M.; Kuraku, S.; Kurokawa, T.; Kato, K.; Toda, S.; Hirose, H.; Takahashi, S.; Shibata, Y.; Iguchi, T.; Matsumoto, T.; Miyata, T.; Takahashi, Y. Tissue preferential expression of estrogen receptor gene in the marine snail. Thais clavigera. Gen. Comp. Endocrinol. 2006, 148, 315–326. (31) Keay, J.; Bridgham, J. T.; Thornton, J. W. The Octopus vulgaris estrogen receptor is a constitutive transcriptional activator and functional implications. Endocinology 2006, 147, 3861–3869. (32) Cohen, R. N.; Putney, A.; Wondisford, F. E.; Hollenberg, A. N. The nuclear corepressors recognize distinct nuclear receptor complexes. Mol. Endocrinol. 2000, 14, 900–914. (33) Wayne, N. L. Regulation of seasonal reproduction in mollusks. J. Biol. Rhythms 2001, 16, 391–402. (34) Tata, J. R. Autoinduction of nuclear hormone receptors during metamorphosis and its significance. Insect Biochem. 2000, 30 (8–9), 645–651. (35) Jenner, M. C. Pseudohermaphroditism: A newly recognized sexual phenomena in Ilyanassa obsoleta and other neogastropods, Dissertation, University of North Carolina, Chapel Hill, 1978. (36) Shea, C.; Hough, D.; Xiano, J.; Tzertzinis, G.; Maina, C. V. An rxr/usp homolog from the parasitic nematode Dirofilaria immitis. Gene 2004, 324, 171–182.
ES702381G
VOL. 42, NO. 4, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
1351
