Elsevier

Aquatic Toxicology

Volume 101, Issue 1, 17 January 2011, Pages 221-227
Aquatic Toxicology

Tributyltin-induced imposex in marine gastropods involves tissue-specific modulation of the retinoid X receptor

https://doi.org/10.1016/j.aquatox.2010.09.022Get rights and content

Abstract

Despite the large number of studies on the phenomenon of imposex, the mechanism underlying the abnormal growth of male sexual characters onto females in numerous gastropod species is yet to be fully elucidated. Although several hypotheses have been raised over the years, a convincing body of evidence indicates that tributyltin-induced imposex involves the abnormal modulation of the retinoid X receptor (RXR). Here, we investigate the RXR gene transcription at different timings and tissues upon exposure to environmentally relevant concentrations of tributyltin (TBT) (100 ng Sn/L TBT) in both genders of the imposex susceptible gastropod Nucella lapillus. RXR gene transcription was determined at two time-points (i.e., before and after imposex initiation) by quantitative Real Time PCR in potential target tissues: the central nervous system (CNS), penis/penis forming area (PFA), gonads and digestive gland. TBT-exposure altered transcription of RXR gene in a tissue and sex specific manner. In the CNS, a significant down-regulation was observed in females both before and after imposex initiation (P  0.01 and P  0.05, respectively). A similar trend was observed in male CNS at the first time-point, although differences between control and the TBT-exposed group were just above significance (P = 0.059). The penis/PFA showed no differences in transcription of RXR gene between control and TBT exposed female snails before imposex induction, or before and after imposex initiation for males. However, male penis showed higher transcription of RXR gene in comparison to the PFA of females. After imposex has been induced, a significant (P  0.001) increase in transcription of RXR gene was observed in penis of females with vas deference sequence index (VDS) levels of 3–4 in comparison with the PFA of both control and imposex females with VDS 1–2. At advanced stages of imposex, females displayed RXR transcription patterns in penis identical to those of males, which points to a functional role of RXR in the penis of both genders. In the other tissues, gonads and digestive gland, RXR gene transcription was not affected by TBT, at any of the analysed time-points. These patterns of RXR gene transcription upon TBT exposure highlight the pivotal involvement of the CNS in the mechanism of imposex induction. We integrate the results in a conceptual model, and discuss the central role of RXR and the retinoic acid signalling pathways in imposex and male genitalia formation in gastropods.

Introduction

Imposex is one of the best documented examples of endocrine disruption in wildlife. It is characterized by the superimposition of male characteristics, such as a penis and a vas deferens, onto females of marine gastropods. Ever since it was first described in Nucella lapillus (Blaber, 1970), numerous studies have been published on the subject. A clear association between exposure to tributyltin (TBT), the active ingredient in antifouling paints, and imposex has been demonstrated for several species. Currently, at least 195 species of prosobranch gastropods are known to be affected, albeit the mechanisms responsible for it are yet to be fully elucidated (Sternberg et al., 2010). A basic understanding on mollusk endocrinology is still today far from achieved, which hinders our comprehension of the imposex process.

Several hypotheses have been raised over the years to explain this condition. A series of in vitro transplantation experiments using the prosobranchs Ocenebra erinacea and Crepidula fornicata are still today particularly informative (Féral and Le Gall, 1983a). This early study indicated that TBT, at environmentally relevant levels, acts on the cerebropleural ganglia, through the action of a “retrogressive factor” (RF), leading to the abnormal release of a “penis morphogenetic factor” (PMF) by the female pedal ganglia. This work highlighted the pivotal role that the central nervous system (CNS) has in female gastropod masculinization by TBT. Other tissues did not intervene directly in imposex induction. Similarly, available evidence indicates that male penis formation in gastropods is under the control of the CNS (Féral and Le Gall, 1983b). In a study reminiscent of the work of Féral and Le Gall (1983a), Oberdörster and McClellan-Green (2000) proposed that the PMF could be the neuropeptide APGWamide, as it was able to induce imposex in Ilyanassa obsoleta and APGWamide immunoreactive neurons were detected in the CNS of several gastropods’ species (de Lange and van Minnen, 1998). However, experiments conducted by Santos et al. (2006) and Castro et al. (2007) showed that APGWamide failed to induce imposex in Bolinus brandaris and N. lapillus.

Since testosterone itself was shown to induce imposex in several gastropod species, some alternative hypothesis postulated that TBT may impact testosterone metabolism (Spooner et al., 1991). It has also been suggested that TBT would competitively inhibit P450 aromatase activity, thereby preventing the conversion of androgens to estrogens (and consequently increasing testosterone levels) (Bettin et al., 1996, Santos et al., 2002); alternatively, TBT would inhibit testosterone excretion (Ronis and Mason, 1996). Recently, the interference of TBT in steroid balance was proposed to be due to a decrease in the esterification of testosterone, thus leading to an increase in free testosterone which could then induce imposex (Gooding et al., 2003). Despite these observations, the existence of a functional androgen signalling pathway in mollusks remains controversial (Castro et al., 2005, Markov et al., 2009, Sternberg et al., 2008a).

The discovery of the intriguing ability of TBT to bind and activate the human retinoid X receptor (RXR) at the same levels of its natural ligand 9-cis retinoic acid (9-cis RA) has expanded our understanding of the process (Nishikawa et al., 2004). In the wild rock shell Thais clavigera, injection of 9-cis RA into females was able to induce the development of imposex (Nishikawa et al., 2004). This finding was further confirmed with the dogwhelk N. lapillus, where injections of 9-cis RA and a selective RXR agonist, methoprene acid, induced imposex (Castro et al., 2007), thus reinforcing the hypothesis of an RXR-mediated induction of imposex by TBT.

In vertebrates, the retinoic acid signalling pathways regulate genes involved in many biological processes, such as cell proliferation, differentiation and apoptosis during embryonic development and other physiological processes in adults (Albalat and Cañestro, 2009, MacLean et al., 2007). The male reproductive differentiation also seems to be under control of retinoid action, as 50% of RXRβ disrupted mice die before or at birth, and those that survive become sterile (Kastner et al., 1996). RXR isoforms are also differentially expressed during mice external genitalia formation (Ogino et al., 2001). In invertebrates, retinoic acid signalling is less known, although RXR has been identified in a wide range of metazoans, i.e., sponges (Wiens et al., 2003), arthropods, nematodes, platyhelminthes and mollusks (Simões-costa et al., 2008).

So far, RXR has been characterized in various mollusks species, namely, T. clavigera, Biomphalaria glabrata, N. lapillus and I. obsoleta (Bouton et al., 2005, Castro et al., 2007, Nishikawa et al., 2004, Sternberg et al., 2008b). Previously, we have isolated the ortholog of RXR in N. lapillus and demonstrated that NlRXR effectively binds to 9-cis RA in vitro (Castro et al., 2007). NlRXR gene transcription levels were determined through qPCR, showing RXR gene to be transcribed ubiquitously. In addition, RXRs proposed ligand 9-cis RA and an RXR agonist metophrene acid induced imposex in female N. lapillus and impacted male penis outgrowth (Castro et al., 2007). Here, we test whether TBT exposure would alter RXR mRNA levels in various target tissues. We hypothesize that the patterns of RXR expression upon TBT exposure may provide significant insights into the involvement of RXR in normal male penis development and imposex induction, elucidating namely the timings and tissues targeted by organotins. Thus, in the present study, N. lapillus specimens were exposed to an environmentally relevant concentration of TBT (100 ng Sn/L TBT) (Berto et al., 2007), and RXR transcription determined before and after imposex initiation. Transcription of RXR gene was evaluated in potential target tissues: the CNS, penis/penis forming area (PFA), gonads, a tissue which previously indicated a role of RXR in reproductive recrudescence (Sternberg et al., 2008b) and digestive gland, a metabolic tissue. The biological relevance of the findings is discussed with respect to imposex and male penis development in gastropod mollusks.

Section snippets

Chemicals

TBTCl (96%) and DMSO were purchased from Sigma–Aldrich.

Experimental conditions and dosing

Adult N. lapillus were collected in January 2009 at praia da Apúlia, an area in the North of Portugal known to display an imposex incidence below 5% (Santos et al., unpublished data). Animals were allowed to acclimatize to laboratory conditions for one week before the onset of the experiment.

After this period, about 35 specimens per replicate (three replicates per treatment) were placed in 30 L aquaria filled with artificial salt water

Imposex and experimental parameters

Fig. 1A–C displays imposex indexes (VDSI, imposex frequency and female penis length, respectively) in N. lapillus during the course of the experiment. No differences were observed in any of the analyses indexes after one month. However, exposure to TBT significantly induced imposex in female N. lapillus after two months, if compared with the control group. Similarly, male penis lengths from the TBT exposed groups were significantly increased at the end of the experiment in comparison with the

Discussion

It has recently been shown that TBT causes ligand dependent transactivation of the human RXRα and that it binds with high affinity to both human and rock shell RXR (Kanayama et al., 2005). Injection of females with its suspected natural ligand, 9-cis RA, demonstrated that 9-cis RA induces imposex both in T. clavigera (Nishikawa et al., 2004) and N. lapillus (Castro et al., 2007), to the same extent of TBT and within the same range of concentrations (Castro et al., 2007). Hence, compiling

Acknowledgements

We acknowledge Hugo Santos for his help with the aquarium maintenance. Daniela Lima was funded by Fundação para a Ciência e a Tecnologia (SFRH/BD/41561/2007). This research has been supported by project PTDC/MAR/68106/2006 and POCI/MAR/59462/2004 (Portugal). We would like to acknowledge the comments of two anonymous reviewers that help us improve the manuscript.

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These authors contributed equally to the study.

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