Cell Biology and Toxicology

, 24:553 | Cite as

Exposure to 9-cis retinoic acid induces penis and vas deferens development in the female rock shell, Thais clavigera

  • Toshihiro Horiguchi
  • Yasuhiko Ohta
  • Tomohiro Nishikawa
  • Fujio Shiraishi
  • Hiroaki Shiraishi
  • Masatoshi Morita


To clarify how tributyltin (TBT) and triphenyltin (TPT) interact with the retinoid X receptor (RXR) to induce growth of male sex organs in female gastropods, we treated female rock shells (Thais clavigera) with three different concentrations (0.1, 1, or 5 μg/g wet wt) of 9-cis-retinoic acid (9CRA) or with a single concentration (1 μg/g wet wt) of TBT, TPT, or fetal bovine serum (as a control). The effects of each treatment were measured as the incidence of imposex, the length of the penis-like structure, and the vas deferens sequence (VDS) index. 9CRA induced imposex in a dose-dependent manner; imposex incidence was significantly higher in the rock shells that received 1 (P < 0.05) or 5 μg (P < 0.001) 9CRA than in the controls. After 1 month, the rock shells treated with 5 μg 9CRA exhibited substantial growth of the penis-like structure that was not as evident in the other treated shells. The length of the structure differed between the 0.1- and 5-μg 9CRA treatment groups (P < 0.05) but not between the 1- and 5-μg 9CRA treatment groups (P > 0.05). Compared with the control, the VDS index increased significantly in the 1- (P < 0.05) and 5-μg (P < 0.001) 9CRA groups. The penis-like structures behind the right tentacle in female rock shells treated with 5 μg 9CRA were essentially the same as the penises and vasa deferentia of normal males and of TBT-treated or TPT-treated imposexed females. These results further support the hypothesis that imposex in gastropods could be mediated by RXR.


Histological examination Imposex Male genitalia 9-cis-retinoic acid Retinoid X receptor Thais clavigera 



9-cis-retinoic acid


retinoid X receptor






vas deferens sequence



This work was partly supported by grants from the Japanese Ministry of the Environment (to T.H.) and by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (Scientific Research [B] no. 17380121).


  1. Berrodin TJ, Marks MS, Ozato K, Linney E, Lazar MA. Heterodimerization among thyroid hormone receptor, retinoic acid receptor, retinoid X receptor, chicken ovalbumin upstream promoter transcription factor, and an endogenous liver protein. Mol Endocrinol 1992;6:1468–78.PubMedCrossRefGoogle Scholar
  2. Bettin C, Oehlmann J, Stroben E. TBT-induced imposex in marine neogastropods is mediated by an increasing androgen level. Helgol Meeresunters 1996;50:299–317.CrossRefGoogle Scholar
  3. Bouton D, Escriva H, de Mendonca RL, Glineur C, Bertin B, Noël C, Robinson-Rechavi M, de Groot A, Cornette J, Laudet V, Pierce RJ. A conserved retinoid X receptor (RXR) from the mollusk Biomphalaria glabrata transactivates transcription in the presence of retinoids. J Mol Endocrinol 2005;34:567–82.PubMedCrossRefGoogle Scholar
  4. Bryan GW, Gibbs PE, Hummerstone LG, Burt GR. The decline of the gastropod Nucella lapillus around south-west England: evidence for the effect of tributyltin from antifouling paints. J Mar Biol Assoc UK 1986;66:611–40.Google Scholar
  5. Bryan GW, Gibbs PE, Burt GR, Hummerstone LG. The effects of tributyltin (TBT) accumulation on adult dog-whelk, Nucella lapillus: long-term field and laboratory experiments. J Mar Biol Assoc UK 1987;67:525–44.Google Scholar
  6. Bryan GW, Gibbs PE, Burt GR. A comparison of the effectiveness of tri-n-butyltin chloride and five other organotin compounds in promoting the development of imposex in the dog-whelk, Nucella lapillus. J Mar Biol Assoc UK 1988;68:733–44.CrossRefGoogle Scholar
  7. de Groot A, de Rosny E, Juillan-Binard C, Ferrer JL, Laudet V, Pierce RJ, et al. Crystal structure of a novel tetrameric complex of agonist-bound ligand-binding domain of Biomphalaria glabrata retinoid X receptor. J Mol Biol 2005;354:841–53.PubMedCrossRefGoogle Scholar
  8. de Urquiza AM, Liu S, Sjoberg M, Zetterstrom RH, Griffiths W, Sjovall J, Perlmann T. Docosahexaenoic acid, a ligand for the retinoid X receptor in mouse brain. Science 2000;290:2140–4.PubMedCrossRefGoogle Scholar
  9. Devine C, Hinman VF, Degnan BM. Evolution and developmental expression of nuclear receptor genes in the ascidian Herdmania. Int J Biol 2002;46:687–92.Google Scholar
  10. Escriva H, Safi R, Hanni C, Langlois MC, Saumitou-Laprade P, Stehelin D, et al. Ligand binding was acquired during evolution of nuclear receptors. Proc Natl Acad Sci USA 1997;94:6803–8.PubMedCrossRefGoogle Scholar
  11. Fent K. Ecotoxicology of organotin compounds. Crit Rev Toxicol 1996;26:1–117.PubMedCrossRefGoogle Scholar
  12. Féral C, Le Gall S. The influence of a pollutant factor (TBT) on the neurosecretory mechanism responsible for the occurrence of a penis in the females of Ocenebra erinacea. In: Lever J, Boer HH, editors. Molluscan neuro-endocrinology. Amsterdam: North-Holland Publishing; 1983. p. 173–5.Google Scholar
  13. Fioroni P, Oehlmann J, Stroben E. The pseudohermaphroditism of prosobranch; morphological aspects. Zool Anz 1991;226:1–26.Google Scholar
  14. Freebern WJ, Osman A, Niles EG, Christen L, LoVerde PT. Identification of a cDNA encoding a retinoid X receptor homologue from Schistosoma mansoni. Evidence for a role in female-specific gene expression. J Biol Chem 1999;247:4577–85.CrossRefGoogle Scholar
  15. Gearing KL, Gottlicher M, Teboul M, Widmark E, Gustafsson JA. Interaction of the peroxisome-proliferator-activated receptor and retinoid X receptor. Proc Natl Acad Sci USA 1993;90:1440–4.PubMedCrossRefGoogle Scholar
  16. Gibbs PE, Bryan GW. Reproductive failure in populations of the dog-whelk, Nucella lapillus, caused by imposex induced by tributyltin from antifouling paints. J Mar Biol Assoc UK 1986;66:767–77.Google Scholar
  17. Gibbs PE, Bryan GW, Pascoe PL, Burt GR. The use of the dog-whelk, Nucella lapillus, as an indicator of tributyltin (TBT) contamination. J Mar Biol Assoc UK 1987;67:507–23.CrossRefGoogle Scholar
  18. Gibbs PE, Pascoe PL, Burt GR. Sex change in the female dog-whelk, Nucella lapillus, induced by tributyltin from antifouling paints. J Mar Biol Assoc UK 1988;68:715–31.CrossRefGoogle Scholar
  19. Gibbs PE, Bryan GW, Pascoe PL, Burt GR. Reproductive abnormalities in female Ocenebra erinacea (Gastropoda) resulting from tributyltin-induced imposex. J Mar Biol Assoc UK 1990;70:639–56.Google Scholar
  20. Goldberg ED. TBT: an environmental dilemma. Environment 1986;28:17–20, 42–4.Google Scholar
  21. Golub M, Doherty J. Triphenyltin as a potential human endocrine disruptor. J Toxicol Environ Health B Crit Rev 2004;7:281–95.PubMedCrossRefGoogle Scholar
  22. Gooding MP, LeBlanc GA. Biotransformation and disposition of testosterone in the eastern mud snail Ilyanassa obsoleta. Gen Comp Endocrinol 2001;122:172–80.PubMedCrossRefGoogle Scholar
  23. Heyman RA, Mangelsdorf DJ, Dyck JA, Stein RB, Eichele G, Evans RM, Thaller C. 9-cis-Retinoic acid is a high affinity ligand for the retinoid X receptor. Cell 1992;68:397–406.PubMedCrossRefGoogle Scholar
  24. Horiguchi T. Masculinization of female gastropod molluscs induced by organotin compounds, focusing on mechanism of actions of tributyltin and triphenyltin for development of imposex. Environ Sci 2006;13:77–87.PubMedGoogle Scholar
  25. Horiguchi T, Shiraishi H, Shimizu M, Morita M. Imposex and organotin compounds in Thais clavigera and T. bronni in Japan. J Mar Biol Assoc UK 1994;74:651–69.Google Scholar
  26. Horiguchi T, Shiraishi H, Shimizu M, Morita M. Effects of triphenyltin chloride and five other organotin compounds on the development of imposex in the rock shell, Thais clavigera. Environ Pollut 1997a;95:85–91.CrossRefGoogle Scholar
  27. Horiguchi T, Shiraishi H, Shimizu M, Morita M. Imposex in sea snails, caused by organotin (tributyltin and triphenyltin) pollution in Japan: a survey. Appl Organomet Chem 1997b;11:451–55.CrossRefGoogle Scholar
  28. Horiguchi T, Kojima M, Hamada F, Kajikawa A, Shiraishi H, Morita M, et al. Impact of tributyltin and triphenyltin on ivory shell (Babylonia japonica) populations. Environ Health Perspect 2006;114(Suppl 1):13–9.PubMedGoogle Scholar
  29. Horiguchi T, Nishikawa T, Ohta Y, Shiraishi H, Morita M. Retinoid X receptor gene expression and protein content in tissues of the rock shell Thais clavigera. Aquat Toxicol 2007;84:379–88.PubMedCrossRefGoogle Scholar
  30. Horton C, Maden M. Endogenous distribution of retinoids during normal development and teratogenesis in the mouse embryo. Dev Dyn 1995;202:312–23.PubMedGoogle Scholar
  31. Iguchi T, Katsu Y, Horiguchi T, Watanabe H, Blumberg B, Ohta Y. Endocrine disrupting organotin compounds are potent inducers of imposex in gastropods and adipogenesis in vertebrates. Mol Cell Toxicol 2007;3:1–10.Google Scholar
  32. Kajiwara M, Kuraku S, Kurokawa T, Kato K, Toda S, Hirose H, et al. Tissue preferential expression of estrogen receptor gene in the marine snail, Thais clavigera. Gen Comp Endocrinol 2006;148:315–26.PubMedCrossRefGoogle Scholar
  33. Kamimura M, Fujiwara S, Kawamura K, Yubisui T. Functional retinoid receptors in budding ascidians. Dev Growth Differ 2000;42:1–8.PubMedCrossRefGoogle Scholar
  34. Kostrouch Z, Kostrouchova M, Love W, Jannini E, Piatigorsky J, Rall JE. Retinoic acid X receptor in the diploblast, Tripedalia cystophora. Proc Natl Acad Sci USA 1998;95:13442–7.PubMedCrossRefGoogle Scholar
  35. Levin AA, Sturzenbecker LJ, Kazmer S, Bosakowski T, Huselton C, Allenby G, et al. 9-cis-Retinoic acid stereoisomer binds and activates the nuclear receptor RXR alpha. Nature 1992;355:359–61.PubMedCrossRefGoogle Scholar
  36. Makishima M, Okamoto AY, Repa JJ, Tu H, Learned RM, Luk A, et al. Identification of a nuclear receptor for bile acids. Science 1999;284:1362–5.PubMedCrossRefGoogle Scholar
  37. Mangelsdorf DJ, Evans RM. The RXR heterodimers and orphan receptors. Cell 1995;83:841–50.PubMedCrossRefGoogle Scholar
  38. Mangelsdorf DJ, Borgmeyer U, Heyman RA, Zhou JY, Ong ES, Oro AE, et al. Characterization of three RXR genes that mediate the action of 9-cis-retinoic acid. Genes Dev 1992;6:329–44.PubMedCrossRefGoogle Scholar
  39. Matthiessen P, Gibbs PE. Critical appraisal of the evidence for tributyltin-mediated endocrine disruption in molluscs. Environ Toxicol Chem 1998;17:37–43.CrossRefGoogle Scholar
  40. Matthiessen P, Reynoldson T, Billinghurst Z, Brassard DW, Cameron P, Chandler GT, et al. Field assessment for endocrine disruption in invertebrates. In: de Fur PL, Ingersoll C, Tattersfield L, editors. Endocrine disruption in invertebrates: endocrinology, testing, and assessment. Pensacola, FL: SETAC; 1999. p. 199–270.Google Scholar
  41. Morris-Kay GM. Retinoids in mammalian embryonic development. In: Sherbet GV, editor. Retinoids: their physiological function and therapeutic potential. Greenwich, CT: JAI; 1997. p. 79–93.Google Scholar
  42. Nagatomo K, Fujiwara S. Expression of Raldh2, Cyp26 and Hox-1 in normal and retinoic acid-treated Ciona intestinalis embryos. Gene Expr Patterns 2003;3:273–7.PubMedCrossRefGoogle Scholar
  43. Nagatomo K, Ishibashi T, Satou Y, Satoh N, Fujiwara S. Retinoic acid affects gene expression and morphogenesis without upregulating the retinoic acid receptor in the ascidian Ciona intestinalis. Mech Dev 2003;120:363–72.PubMedCrossRefGoogle Scholar
  44. Nishikawa J, 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;38:6271–6.PubMedCrossRefGoogle Scholar
  45. Oberdörster E, McClellan-Green P. The neuropeptide APGWamide induces imposex in the mud snail, Ilyanassa obsoleta. Peptides 2000;21:1323–30.PubMedCrossRefGoogle Scholar
  46. Redfern CPF. Molecular mechanisms of the retinoid function. In: Sherbet GV, editor. Retinoids: their physiological function and therapeutic potential. Greenwich, CT: JAI; 1997. p. 35–77.Google Scholar
  47. Ronis MJJ, Mason AZ. The metabolism of testosterone by the periwinkle (Littorina littorea) in vitro and in vivo: effects of tributyltin. Mar Environ Res 1996;42:161–6.CrossRefGoogle Scholar
  48. Schrader M, Bendik I, Becker-Andre M, Carlberg C. Interaction between retinoic acid and vitamin D signaling pathways. J Biol Chem 1993;268:17830–6.PubMedGoogle Scholar
  49. Smith BS. Sexuality in the American mud snail, Nassarius obsoletus Say. Proc Malacol Soc Lond 1971;39:377–8.Google Scholar
  50. Spooner N, Gibbs PE, Bryan GW, Goad LJ. The effect of tributyltin upon steroid titres in the female dogwhelk, Nucella lapillus, and the development of imposex. Mar Environ Res 1991;32:37–49.CrossRefGoogle Scholar
  51. Thornton JW, Need E, Crews D. Resurrecting the ancestral steroid receptor: ancient origin of estrogen signaling. Science 2003;301:1714–7.PubMedCrossRefGoogle Scholar
  52. Waxman DJ. P450 gene induction by structurally diverse xenochemicals: central role of nuclear receptors CAR, PXR, and PPAR. Arch Biochem Biophys 1999;369:11–23.PubMedCrossRefGoogle Scholar
  53. Wiens M, Batel R, Korzhev M, Muller WE. Retinoid X receptor and retinoic acid response in the marine sponge Suberites domuncula. J Exp Biol 2003;206:3261–71.PubMedCrossRefGoogle Scholar
  54. Willy PJ, Umesono K, Ong ES, Evans RM, Heyman RA, Mangelsdorf DJ. LXR, a nuclear receptor that defines a distinct retinoid response pathway. Genes Dev 1995;9:1033–45.PubMedCrossRefGoogle Scholar
  55. Zhang XK, Hoffmann B, Tran PB, Graupner G, Pfahl M. Retinoid X receptor is an auxiliary protein for thyroid hormone and retinoic acid receptors. Nature 1992;355:441–6.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Toshihiro Horiguchi
    • 1
  • Yasuhiko Ohta
    • 2
  • Tomohiro Nishikawa
    • 1
  • Fujio Shiraishi
    • 1
  • Hiroaki Shiraishi
    • 1
  • Masatoshi Morita
    • 1
    • 3
  1. 1.Research Center for Environmental RiskNational Institute for Environmental StudiesTsukubaJapan
  2. 2.Department of Veterinary Science, Faculty of AgricultureTottori UniversityTottoriJapan
  3. 3.Department of Environmental Conservation, Faculty of AgricultureEhime UniversityMatsuyamaJapan

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