Abstract
The superposition of male sexual characteristics in female marine gastropods (imposex) represents one of the clearest ecological examples of organotin-mediated endocrine disruption. Recent evidences suggest that signaling pathways mediated by members of the nuclear receptor superfamily, RXR and PPARγ, are involved in the development of this pseudohermaphroditic condition. Here, we identified significant differences in RXR expression in two caenogastropod species from Nuevo Gulf, Argentina, Buccinanops globulosus and Trophon geversianus, which present clear contrast in imposex incidence. In addition, B. globulosus males from a polluted and an unpolluted area showed differences in RXR expression. Conversely, PPARγ levels were similar between both analyzed species. These findings indicate specie-specific RXR and PPARγ expression, suggesting a major role of RXR in the induction of imposex.
Similar content being viewed by others
Introduction
Endocrine disrupting chemicals are compounds that alter the normal functioning of the endocrine system of both humans and wildlife1. One of most relevant examples of environmental endocrine disruption in marine gastropods is the phenomenon of imposex2, described as an irreversible syndrome in which a female develops male-type genital organs, such as the penis and vas deferens3,4. Imposex is intimately associated with tributyltin (TBT) marine environmental pollution5. TBT is a common compound in marine antifouling paints, currently used in several countries6 despite having been banned worldwide for more than 10 years7. Although imposex levels appear to be reduced in some regions of the planet8,9 after global ban of organotin antifoulant paints, assessing the incidence of imposex as a biomarker of TBT contamination seems to be a valuable indicator in other regions of the world such as Latin America10,11,12 or South Africa13 where TBT-based antifouling paints are still in use.
The use of TBT, under controlled laboratory conditions, diluted in the water or directly injected to gastropods, was able to induce imposex in several caenogastropods14,15,16,17,18,19.
Different mechanisms have been proposed to explain the induction of imposex by TBT such as an increase in androgen levels, the involvement of penis morphogenetic/retrogressive factor, or even an increase in the neuropeptide alanine-proline-glycine-tryptophan amide20. However, a considerable body of evidence indicates that TBT-induced imposex involves the abnormal modulation of the retinoid X receptor (RXR) signaling pathway20,21,22,23. RXR are members of the nuclear receptor superfamily of transcription factors24. They are activated by retinoic acid, the main derivative of vitamin A, involved in multiple signaling pathways critical for embryonic development, metabolic processes, differentiation and apoptosis in vertebrates25,26. Although retinoid physiology is poorly understood in marine gastropods27, recent findings support their role during gonad maturation28. RXR was cloned in different caenogastropods, namely, Thais clavigera18, Nucella lapillus14, Ilyanassa obsoleta29, Hexaplex trunculus15, Plicopurpura pansa30 and Babylonia japonica31. Interestingly, the DNA and ligand binding domains (DBD and LBD respectively) had almost 100% and 90–80% identities, respectively, between gastropod and mouse/human RXRs32.
It has been shown that TBT binds RXR and activates the RXR-peroxisome proliferator-activated receptor gamma (PPARγ) heterodimer33. Direct administration of 9-cis retinoic acid (9cRA) induced imposex in T. clavigera18,34 and N. lapillus14. However, the application of rosiglitazone (Rosi), a PPARγ ligand, to N. lapillus, induced imposex in the absence of TBT35. All these data suggests the direct association between TBT-RXR-PPARγ with imposex development.
The nassarid gastropod Buccinanops globulosus (Kiener, 1834) inhabits Nuevo Gulf and is distributed along the Argentinean coasts in sandy bottoms of shallow waters36. The genus Buccinanops has been affected by imposex in Argentina37,38. Trophon geversianus (Pallas, 1774) is a muricid gastropod widely distributed along the Southern Atlantic and Pacific coasts39,40,41. The specie is present in subtidal and intertidal habitats from Nuevo Gulf. Imposex incidence is very low in T. geversianus, even in harbor areas with TBT contamination. In contrast, B. globulosus presents high sensitivity to imposex incidence even at low environmental TBT concentrations41.
Working with species that inhabit the same area being exposed to the same environmental conditions but show physiological differences in the development of imposex, will allow us to expand our knowledge on the metabolic pathways altered by organotin compounds. Thus, the specific aim of this study was to evaluate the expression of RXR and PPARγ in two caenogastropods from Nuevo Gulf, Argentina, B. globulosus and T. geversianus, to explain some aspects of its differential sensibility to imposex development.
Materials and Methods
Collection of animals
Sexually mature males and females of B. globulosus and T. geversianus were collected during July-August 2016 from two places in Nuevo Gulf, with the same physical conditions and separately by 20 km, Cerro Avanzado beach (CA), a zone with scarce maritime traffic and very low imposex incidence, and from Luis Piedra Buena Harbor (LPBH), located in the city of Puerto Madryn42 (Supplementary Fig. S1). LPBH is an area with high maritime traffic characterized by the presence of commercial, fishing and recreational vessels. A total of 895 boats with up to 294 m length, arrived in the city in 2016/2017 season43. Aluminum derivates, porfids and others materials are transported during the year. High levels of trace metals, polycyclic aromatic hydrocarbons (PAHs) and TBT were previously recorded in sediments and organisms41,44,45,46,47,48.
Tissue preparation
After removal of the shell, the percentage of imposex-affected females was calculated in terms of penis and vas deferent presence, and the relative penis length index (RPLI) was calculated49. Male penis and penis/penis-forming area from imposex-affected female were excised out and fixed in formalin solution (4% in PBS) or immediately frozen at −80 °C.
Immunohistochemistry (IHC)
Sample sections of formalin-fixed, paraffin-embedded tissues were stained with hematoxylin-eosin (H&E) or reacted with RXR (sc-774) and PPARγ (sc-7196) antibodies (Santa Cruz Biotechnology) using the avidin-biotin peroxidase complex technique (Vectastain Elite ABC kit; Vector Laboratories). Briefly, 3 penis sections from five independent individuals of both species were rehydrated from xylene to 70% ethanol passing through decreased graded ethanol, before endogenous peroxidase activity inhibition (10% H2O2 in 70% ethanol). Antigen retrieval with HCl 2 N was performed before immunostaining. After PBS washes, sections were blocked (2.5% BSA in PBS) and incubated overnight at 4 °C with the primary antibody. After biotin-conjugated secondary antibody (incubation for 1 h at room temperature, the reaction was developed using the DAKO Liquid DAB + Substrate Chromogen System (K3468, DAKO) according to the manufacturer’s protocol under microscopic control. Specimens were counterstained with hematoxylin, dehydrated and mounted. Positive cells were counted in 10 high-power fields (HPFs) of each section, using 1000× magnification, and expressed as the mean ± SEM of the percentage of the ratios between the number of events and the cell number/HPF.
Tissue extracts and western blot
Frozen tissues from five independent individuals of both species were homogenized in ice-cold TEDGS 10% buffer (50 mM Tris pH = 7.4, 7.5 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol, 0.25 M sucrose), including protease inhibitors. The homogenate was centrifuged (20 min, 12000 rpm at 4 °C) and total protein concentration in each supernatant was determined by Lowry method50. Equivalent amounts of protein (100 μg) from tissue lysates were separated on discontinuous polyacrylamide gels and detected by western blot. Membranes were probed with RXR (sc-774), PPARγ (sc-7273) or β-Actin (sc-47778) antibodies (Santa Cruz Biotechnology) overnight at 4 °C, and then incubated with horse anti-mouse or goat anti-rabbit peroxidase-conjugated secondary antibody (Vector Laboratories). The luminescent signal was generated by enhanced chemiluminescence (ECL) method and the blots were exposed to a medical X-ray film (ortho CP-GU, AGFA). Band intensity was determined by densitometry using ImageJ 1.47 v software (https://imagej.nih.gov/ij/download.html)51.
Statistical analysis
ANOVA and the Tukey multiple post t test were used to analyze the differences of means of multiple samples; the Student’s t test was used to compare the means of two different groups. In all graphs, the mean ± SEM is shown, and experiments were repeated at least three times. Significant differences between groups are indicated with asterisks (*p < 0.05; **p < 0.01; ***p < 0.001).
Results
Imposex incidence in B. globulosus and T. geversianus
B. globulosus females collected from Luis Piedra Buena Harbor (LPBH) presented 100% imposex incidence with different degrees of penis development (between 0.5 to 7.7 mm of length); meanwhile the opposite was observed for T. geversianus specimens (Table 1 and Fig. 1a). No imposex incidence was registered in female gastropods of both species collected in Cerro Avanzado beach (CA).
Histological visualization of imposex
The penis and penis-like structures developed behind the right tentacle in normal male of B. globulosus and T. geversianus and in imposex-affected females of B. globulosus collected from LPBH were histologically examined after H&E staining (Fig. 1b). No morphological differences were observed between penis from male and penis-like structure from imposex-affected females of B. globulosus. An internal vas deferens (VD) was observed in the central area of the penis/penis-like structure. The epithelium of the VD was surrounded by smooth muscle layers. The epidermis of the penis/penis-like structure consisted of epidermal cells with mucous cells (Fig. 1b).
Evaluation of RXR and PPARγ expression
Immunohistochemical stains against RXR and PPARγ illustrate the localization of both proteins in the penis of B. globulosus and T. geversianus male gastropods collected from CA beach (Fig. 2a). RXR and PPARγ were detected in the nuclei of epithelial cells and smooth muscle cells surrounding the epithelium of vas deferens. No immunoreactivity was detected in the absence of primary antibodies (Fig. 2b), confirming binding specificity.
We found a significant lower expression of RXR in penis of T. geversianus males in comparison with the same tissue of B. globulosus (a similar trend was found in epidermal cells of male penis, and in female gonads and digestive glands, Supplementary Fig. S2). In the case of PPARγ, no differences were observed between both analyzed species (Fig. 2c). These results were validated by western blot (Fig. 2d).
To further evaluate the expression of RXR and PPARγ in gastropods from a polluted (LPBH) or unpolluted (CA beach) areas, we used penis tissues from imposex-affected females and males of B. globulosus. We observed RXR and PPARγ expression in males from LPBH and CA beach and in imposex-affected B. globulosus females collected at LPBH (Fig. 3). Significant differences in RXR penis tissues expression were observed among males from CA and LPBH, and males from CA and imposex-affected females from LPBH, while no differences in PPARγ expression was detected (Fig. 3b,c).
Discussion
In this study, we compared for the first time the expression of RXR and PPARγ in two caenogastropod species from South Atlantic, which present clear contrasts in imposex incidence. Most of biochemical or molecular studies related to imposex have been performed under experimental laboratory conditions. Ours, is an entirely field work reflecting what occurs in natural conditions underscoring the relevance of the data. We found that penis of B. globulosus males express higher levels of RXR than the same tissue of T. geversianus males, collected in an unpolluted area (CA) of Nuevo Gulf. These results suggest specie-specific differences in RXR expression that could explain differences registered in penis length between both species (and probably in other neogastropods), and may also be related to their different biological response to environmental contaminants. As was previously mentioned, the hypothesis about the abnormal modulation of RXR as a mechanism by which organotins induce imposex in caenogastropods would turn out to be the most accepted one20,52,53. Therefore, RXR could be a central factor in the molecular mechanism involved in differentiation, proliferation, and morphogenesis of penis in male and imposex-affected female gastropods. Nevertheless, the low expression of RXR in penis from T. geversianus males, relative to that in B. globulosus males, is not an impediment to the normal development of secondary sexual characters as the penis. In addition to the local action that RXR would have in penis development, which would be under the control of the central nervous system (CNS) of gastropods, CNS was described as a tissue with high RXR expression able of being retinoid target21. It was proposed that a “penis morphogenetic factor (PMF)” released by CNS under the control of RXR signaling is induced in males during penis differentiation21. That may explain the normal development of T. geversianus penis even having low levels of RXR expression, relative to B. globulosus males. However, this hypothesis and the identification of a PMF in T. geversianus and other gastropods need to be addressed. Future proteomic strategies, comparing both species under study, will shed light on further important factors or signaling pathways involved in differentiation, growth and penis formation in males.
Interestingly, no differences were observed in PPARγ expression between the species studied, highlighting the role of RXR in normal or abnormal physiological processes. Convergence of 9cRA and PPAR signaling pathways through PPAR/RXRα heterodimerization is well established in mammals54, a crosstalk that is recently starting to be explored in gastropod mollusks affected by imposex. Two PPAR homologues have been already identified in a phylogenetically distant gastropod Biomphalaria glabrata55, indicating that these receptors are conserved in this group. As a whole, these results allow us to assume that PPARγ would function as a heterodimerization partner of RXR in the penis of both B. globulosus and T. geversianus males, and that RXR is the one that triggers imposex onset. However, many aspects of the RXR/PPAR system still remain to be explored.
Our results must be taken into account when performing environmental monitoring studies using imposex as a biomarker, since the studies of morphological structures (i.e. penis or vas deference) could hide physiological responses to marine pollutants. In the case of T. geversianus, this is the only muricid species described that is less sensitive to TBT contamination, developing secondary sexual characters only at high TBT concentrations41.
RXR was previously reported to be expressed at the mRNA level in penis from the marine muricid gastropods T. clavigera56, N. lapillus21 and P. pansa30, and at the protein level in T. clavigera56,57. However, to our knowledge, there is no information about PPARγ protein expression in marine gastropods. RXR and PPARγ interact in heterodimeric complexes after their activation by TBT58 or 9cRA, the biologically active metabolite of vitamin A59. It has been hypothesized that signaling through both proteins has a key role in male and female seasonal reproductive development29,60 and imposex, as previously mentioned. Whereas there is scant information regarding the physiological roles of RA in invertebrates, it is well known that signaling pathways through RA receptors exert a key role in embryo patterning and organogenesis in vertebrates61. 9cRA, is the natural ligand for mammalian RXRs, while the natural ligand of gastropod RXRs is currently unknown62. Vitamin A can either enter in a cascade producing retinal and RA, or undergo esterification to promote retinoid storage63. RA isomers were detected in testis, ovary and CNS of the caenogastropods N. lapillus and Nassarius reticulatus. However, they seem to be unable to store RA27,64. Finally, injection of TBT or 9cRA, into N. lapillus or H. trunculus males induces the outgrowth of reproductive structures14,15. All these data support the close relationship between retinoids and RXR/PPARγ in the development of male reproductive organs in marine gastropod mollusks.
The abnormal modulation of RXR signaling pathway by organotins seems to be the most accepted explanation of imposex development based on imposex induction assays together with the in vitro transcriptional activity of RXR20,31. Our findings, regarding the expression of RXR, contribute to the general understanding of the endocrine system in gastropods, and allow us to hypothesize that the degree of expression of RXR has direct implications on the sensitivity to imposex development.
We have also shown that imposex-affected female of B. globulosus collected from the polluted area (LPBH) of Nuevo Gulf, express RXR and PPARγ. These results are consistent with those obtained in species such as T. clavigera56,65, N. lapillus66, H. trunculus15, P. pansa30. However, our study is the first description of PPARγ protein expression in caenogastropod imposex-affected females. Regarding PPARγ actions, Pascoal et al.67 showed that the PPARγ ligand Rosi elicited the same imposex response in N. lapillus as TBT. However, Giraud-Billoud et al.68 reported that Rosi was not able to induce imposex in a phylogenetically distant gastropod Pomacea canaliculata. This difference indicates that different molecular mechanisms may regulate imposex induction in ampullariid and neogastropod species suggesting that the direct role of PPARγ in imposex deserves further investigation.
Males of B. globulosus that inhabit LPBH area are exposed to TBT41,44, PAHs46, trace metals and products from fishing industries45,69. These populations have increased oxidative stress responses compared to populations in CA beach69, indicating the negative effects of pollutants present at the harbour site on its physiological state. Our previous studies demonstrate the presence of high butyltins (TBT, DBT and MBT) levels in LPBH, both in sediments and in edible gastropods41,44. The sediments from LPBH in 201544 exceeded the TBT limit concentration established by international organizations, this probably continues up to the present time, as occurs in other countries of Latin America10,11,12. This may explain our findings of imposex-affected females in the area. It has been shown using in vitro assays70, that PAHs enhance the effect of natural ligands of retinoid signaling pathway, indicating that these environmental pollutants may influence the differentiation process and the embryonic development mediated by retinoids.
Differences in RXR expression between males from LPBH and CA could be related to the pollutants present in the harbor area. However, this should be tested in controlled experiments exposing normal individuals to TBT, PAHs or trace metals separately. The regulation of RXR expression by TBT seems not to be conclusive. Domínguez-Ojeda and colleagues, reported a down regulation of RXR induced by TBT in different tissues of P. pansa, while no changes were observed in penis of males30. Similar results were obtained in N. lapillus, H. trunculus and T. clavigera where no changes in RXR mRNA expression were observed using penis of males after TBT treatment15,66,71. The ideal correlation between mRNA-protein may be affected by highly dynamic phases, such as cellular differentiation or stress response72. In our case, despite not being able to directly associate RXR protein expression to any specific factor, we found a difference in RXR protein between both sites, but not for PPARγ, probably indicating a major role of RXR in the induction of the imposex phenomenon in these species and probably other around the world.
Overall, our results clearly show that differences in RXR male penises expression between B. globulosus and T. geversianus do not affect the normal development of secondary sexual organs. Future cloning and functional studies with B. globulosus and T. geversianus RXRs might reveal its real contribution to the observed differences in imposex development and penis formation between both species.
References
Matthiessen, P. In Endocrine Disrupters 373–384 (John Wiley & Sons, Inc., 2013).
Gibbs, P. E. & Bryan, G. W. In Oceans ’87. (ed IEEE) 1482–1487 (IEEE).
Smith, B. S. Sexuality in the american mud snail, Nassarius obsoletus say. J. Mollus Stud. 39, 377–378 (1971).
Bryan, G. W., Gibbs, P. E., Hummerstone, L. G. & Burt, G. R. The decline of the gastropod Nucella lapillus around South-West England: Evidence for the effect of Tributyltin from antifouling paints. J. Mar. Biol. Assoc. U K. 66, 611–640 (1986).
Goldberg, E. D. TBT: An Environmental Dilemma. Environment: Sci. Policy Sustain. Dev. 28, 17–44, https://doi.org/10.1080/00139157.1986.9928814 (1986).
Laranjeiro, F., Sánchez-Marín, P., Oliveira, I. B., Galante-Oliveira, S. & Barroso, C. Fifteen years of imposex and tributyltin pollution monitoring along the Portuguese coast. Environ. Pollut. 232, 411–421, https://doi.org/10.1016/j.envpol.2017.09.056 (2018).
International Maritime Organization. & International Maritime Organization. Marine Environment Protection Committee. Anti-fouling systems: International Convention on the Control of Harmful Anti-fouling Systems on Ships, 2001 (AFS 2001) and Guidelines for survey and certification of anti-fouling systems on ships (resolution MEPC.102(48)). (International Maritime Organization, 2003).
Schøyen, M. et al. Levels and trends of tributyltin (TBT) and imposex in dogwhelk (Nucella lapillus) along the Norwegian coastline from 1991 to 2017. Mar. Environ. Res. 144, 1–8, https://doi.org/10.1016/j.marenvres.2018.11.011 (2019).
Lahbib, Y., Abidli, S. & Trigui-El Menif, N. First assessment of the effectiveness of the international convention on the control of harmful anti-fouling systems on ships in Tunisia using imposex in Hexaplex trunculus as biomarker. Mar. Pollut. Bull. 128, 17–23, https://doi.org/10.1016/j.marpolbul.2018.01.012 (2018).
Batista, R. M., Castro, I. B. & Fillmann, G. Imposex and butyltin contamination still evident in Chile after TBT global ban. Sci. Total. Environ. 566–567, 446–453, https://doi.org/10.1016/j.scitotenv.2016.05.039 (2016).
Maciel, D. C. et al. Assessment of organotins and imposex in two estuaries of the northeastern Brazilian coast. Mar. Pollut. Bull. 126, 473–478, https://doi.org/10.1016/j.marpolbul.2017.11.061 (2018).
Castro, Í. B., Iannacone, J., Santos, S. & Fillmann, G. TBT is still a matter of concern in Peru. Chemosphere 205, 253–259, https://doi.org/10.1016/j.chemosphere.2018.04.097 (2018).
van Gessellen, N., Bouwman, H. & Averbuj, A. Imposex assessment and tributyltin levels in sediments along the Atlantic coast of South Africa. Mar. Environ. Res. 142, 32–39, https://doi.org/10.1016/j.marenvres.2018.09.016 (2018).
Castro, L. F. C. et al. Imposex induction is mediated through the Retinoid X Receptor signalling pathway in the neogastropod Nucella lapillus. Aquat. Toxicol. 85, 57–66 (2007).
Abidli, S. et al. Imposex development in Hexaplex trunculus (Gastropoda: Caenogastropoda) involves changes in the transcription levels of the retinoid X receptor (RXR). Chemosphere 93, 1161–1167, https://doi.org/10.1016/j.chemosphere.2013.06.054 (2013).
Bettin, C., Oehlmann, J. & Stroben, E. TBT-induced imposex in marine neogastropods is mediated by an increasing androgen level. Helgol. Mar. Res. 50, 299–317 (1996).
Gibbs, P. E., Bryan, G. W. & Pascoe, P. L. TBT-induced imposex in the dogwhelk, Nucella lapillus: Geographical uniformity of the response and effects. Mar. Env. Res. 32, 79–87 (1991).
Nishikawa, J. et al. Involvement of the retinoid x receptor in the development of imposex caused by Organotins in gastropods. Env. Sci. Technol. 38, 6271–6276 (2004).
Rossato, M., Costa, M. B., de Castro, Í. B. & Pinho, G. L. L. Size, season and origin of gastropods matter in imposex assessments. Ecotoxicol. Environ. Saf. 159, 324–331, https://doi.org/10.1016/j.ecoenv.2018.05.013 (2018).
Horiguchi, T. Mode of Action of Organotins to Induce the Development of Imposex in Gastropods, Focusing on Steroid and the Retinoid X Receptor Activation Hypotheses 199–219, https://doi.org/10.1007/978-4-431-56451-5_9 (2017).
Lima, D. et al. Tributyltin-induced imposex in marine gastropods involves tissue-specific modulation of the retinoid X receptor. Aquat. Toxicol. 101, 221–227, https://doi.org/10.1016/j.aquatox.2010.09.022 (2011).
Nishikawa, J. Imposex in marine gastropods may be caused by binding of organotins to retinoid X receptor. Mar. Biol. 149, 117–124 (2006).
Urushitani, H. et al. Cloning and characterization of retinoid X receptor (RXR) isoforms in the rock shell, Thais clavigera. Aquat. Toxicol. 103, 101–111, https://doi.org/10.1016/j.aquatox.2011.02.012 (2011).
Helsen, C. et al. Structural basis for nuclear hormone receptor DNA binding. Mol. Cell Endocrinol. 348, 411–417, https://doi.org/10.1016/j.mce.2011.07.025 (2012).
Das, B. C. et al. Retinoic acid signaling pathways in development and diseases. Bioorg Med. Chem. 22, 673–683, https://doi.org/10.1016/j.bmc.2013.11.025 (2014).
Osz, J. et al. Structural basis of natural promoter recognition by the retinoid X nuclear receptor. Scientific Reports 5, 1–10, https://doi.org/10.1038/srep08216, https://www.nature.com/articles/srep08216#supplementary-information (2015).
Gesto, M., Castro, L. F. & Santos, M. M. Differences in retinoid levels and metabolism among gastropod lineages: imposex-susceptible gastropods lack the ability to store retinoids in the form of retinyl esters. Aquat. Toxicol. 142–143, 96–103, https://doi.org/10.1016/j.aquatox.2013.08.001 (2013).
Gesto, M. et al. Retinoid level dynamics during gonad recycling in the limpet Patella vulgata. Gen. Comp. Endocrinol. 225, 142–148, https://doi.org/10.1016/j.ygcen.2015.10.017 (2016).
Sternberg, R. M., Hotchkiss, A. K. & LeBlanc, G. A. Synchronized expression of retinoid X receptor mRNA with reproductive tract recrudescence in an imposex-susceptible mollusc. Env. Sci. Technol. 42, 1345–1351 (2008).
Domínguez-Ojeda, D., Rojas-García, E. A., Robledo-Marenco, M. L., Barrón-Vivanco, B. S. & Medina-Díaz, I. M. Exposure to tributyltin chloride induces penis and vas deferens development and increases RXR expression in females of the purple snail (Plicopurpura pansa) Invertebrate Survival. Journal 11, 204–2011 (2014).
Urushitani, H. et al. Characterization and comparison of transcriptional activities of the retinoid X receptors by various organotin compounds in three prosobranch gastropods; Thais clavigera, Nucella lapillus and Babylonia japonica. Aquat. Toxicol. 199, 103–115, https://doi.org/10.1016/j.aquatox.2018.03.029 (2018).
Urushitani, H. et al. Cloning and characterization of retinoid X receptor (RXR) isoforms in the rock shell, Thais clavigera. Aquat. Toxicol. 103, 101–111, https://doi.org/10.1016/j.aquatox.2011.02.012 (2011).
le Maire, A., Bourguet, W. & Balaguer, P. A structural view of nuclear hormone receptor: endocrine disruptor interactions. Cell Mol. Life Sci. 67, 1219–1237 (2010).
Horiguchi, T. et al. Exposure to 9- retinoic acid induces penis and vas deferens development in the female rock shell, Thais clavigera. Cell Biol. Toxicol. 24, 553–562 (2008).
Pascoal, S. et al. Transcriptomics and in vivo tests reveal novel mechanisms underlying endocrine disruption in an ecological sentinel, Nucella lapillus. Mol. Ecol. 22, 1589–1608, https://doi.org/10.1111/mec.12137 (2013).
Pastorino, G. The taxonomic status of Buccinanops d’Orbigny, 1841 (Gastropoda: Nassariidae). Veliger 36, 160–165 (1993).
Averbuj, A. & Penchaszadeh, P. E. On the reproductive biology and impact of imposex in a population of Buccinanops monilifer from Mar del Plata, Argentina. J. Mar. Biol. Assoc. U K. 90, 729–734 (2010).
Penchaszadeh, P. E., Averbuj, A. & Cledón, M. Imposex in gastropods from Argentina (South-Western Atlantic). Mar. Pollut. Bull. 42, 790–791 (2001).
Griffin, M. & Pastorino, G. The genus Trophon Monfort, 1810 (Gastropoda: Muricidae) in the Tertiary of Patagonia. J. Paleontol. 79, 296–311 (2005).
Pastorino, G. A revision of the genus Trophon Montfort, 1810 (Gastropoda: Muricidae) from southern South America. Nautilus 119, 55–82 (2005).
Bigatti, G. et al. Biomonitoring of TBT contamination and imposex incidence along 4700 km of Argentinean shoreline (SW Atlantic: from 38S to 54S). Mar. Pollut. Bull. 58, 695–701, https://doi.org/10.1016/j.marpolbul.2009.01.001 (2009).
Primost, M. A., Bigatti, G. & Márquez, F. Shell shape as indicator of pollution in marine gastropods affected by imposex. Mar. Freshw. Res. 67, 1948, https://doi.org/10.1071/mf15233 (2016).
APPM. Administración Portuaria de Puerto Madryn. Available at, http://www.appm.com.ar/estadisticas, (2017).
Del Brio, F. et al. Distribution and bioaccumulation of butyltins in the edible gastropod Odontocymbiola magellanica. Mar. Biol. Res. 12, 608–620, https://doi.org/10.1080/17451000.2016.1169296 (2016).
Primost, M. A., Gil, M. N. & Bigatti, G. High bioaccumulation of cadmium and other metals in Patagonian edible gastropods. Mar. Biol. Res. 13, 774–781, https://doi.org/10.1080/17451000.2017.1296163 (2017).
Primost, M. A., Commendatore, M., Torres, P. J. & Bigatti, G. PAHs contamination in edible gastropods from north Patagonian harbor areas. Mar. Pollut. Bull. 135, 828–831, https://doi.org/10.1016/j.marpolbul.2018.08.021 (2018).
Commendatore, M. G. & Esteves, J. L. An Assessment of Oil Pollution in the Coastal Zone of Patagonia, Argentina. Environ. Manag. 40, 814–821, https://doi.org/10.1007/s00267-005-0221-3 (2007).
Commendatore, M. G. et al. BTs, PAHs, OCPs and PCBs in sediments and bivalve mollusks in a mid-latitude environment from the Patagonian coastal zone. Environmental toxicology and chemistry/SETAC, https://doi.org/10.1002/etc.3134 (2015).
Gibbs, P. E. & Bryan, G. W. Biomonitoring of Tributyltin (TBT) Pollution using the Imposex Response of Neogastropod Molluscs. Biomonitoring of Coastal Waters and Estuaries. Biomonitoring of Coastal Waters and Estuaries, 205–226 (1994).
Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275 (1951).
Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Meth 9, 671–675 (2012).
Castro, L. F. et al. Imposex induction is mediated through the Retinoid X Receptor signalling pathway in the neogastropod Nucella lapillus. Aquat. Toxicol. 85, 57–66, https://doi.org/10.1016/j.aquatox.2007.07.016 (2007).
Sternberg, R. M., Hotchkiss, A. K. & LeBlanc, G. A. Synchronized expression of retinoid X receptor mRNA with reproductive tract recrudescence in an imposexsusceptible mollusc. Environ. Sci. Technol. 42, 1345–1351 (2008).
Kliewer, S. A., Umesono, K., Noonan, D. J., Heyman, R. A. & Evans, R. M. Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors. Nature 358, 771–774, https://doi.org/10.1038/358771a0 (1992).
Fong, P. P. et al. The Nuclear Receptors of Biomphalaria glabrata and Lottia gigantea: Implications for Developing New Model Organisms. PLoS One 10, e0121259, https://doi.org/10.1371/journal.pone.0121259 (2015).
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. 84, 379–388 (2007).
Horiguchi, T., Urushitani, H., Ohta, Y., Iguchi, T. & Shiraishi, H. Establishment of a polyclonal antibody against the retinoid X receptor of the rock shell Thais clavigera and its application to rock shell tissues for imposex research. Ecotoxicology 9, 571–576 (2010).
le Maire, A. et al. Activation of RXR-PPAR heterodimers by organotin environmental endocrine disruptors. EMBO Rep. 10, 367–373, https://doi.org/10.1038/embor.2009.8 (2009).
Hiromori, Y., Aoki, A., Nishikawa, J., Nagase, H. & Nakanishi, T. Transactivation of the human retinoid X receptor by organotins: use of site-directed mutagenesis to identify critical amino acid residues for organotin-induced transactivation. Metallomics: Integr. biometal Sci. 7, 1180–1188, https://doi.org/10.1039/c5mt00086f (2015).
Sternberg, R. M., Gooding, M. P., Hotchkiss, A. K. & LeBlanc, G. A. Environmental-endocrine control of reproductive maturation in gastropods: implications for the mechanism of tributyltininduced imposex in prosobranchs. Ecotoxicology 19, 4–23 (2010).
Cunningham, T. J. & Duester, G. Mechanisms of retinoic acid signalling and its roles in organ and limb development. Nat. Rev. Mol. Cell Biol. 16, 110–123, https://doi.org/10.1038/nrm3932 (2015).
Horiguchi, T. In Biological Effects by Organotins (ed Toshihiro Horiguchi) 199-219 (Springer Japan, 2017).
Theodosiou, M., Laudet, V. & Schubert, M. From carrot to clinic: an overview of the retinoic acid signaling pathway. Cell Mol. Life Sci. 67, 1423–1445, https://doi.org/10.1007/s00018-010-0268-z (2010).
André, A., Ruivo, R., Gesto, M., Castro, L. F. C. & Santos, M. M. Retinoid metabolism in invertebrates: When evolution meets endocrine disruption. Gen. Comp. Endocrinol. 208, 134–145, https://doi.org/10.1016/j.ygcen.2014.08.005 (2014).
Ip, J. C. H., Leung, P. T. Y., Ho, K. K. Y., Qiu, J. W. & Leung, K. M. Y. De novo transcriptome assembly of the marine gastropod Reishia clavigera for supporting toxic mechanism studies. Aquat. Toxicol. 178, 39–48, https://doi.org/10.1016/j.aquatox.2016.07.006 (2016).
Lima, D. et al. Tributyltin-induced imposex in marine gastropods involves tissue-specific modulation of the retinoid X receptor. Aquat. Toxicol. 101, 221–227 (2011).
Pascoal, S. et al. Transcriptomics and in vivo tests reveal novel mechanisms underlying endocrine disruption in an ecological sentinel, Nucella lapillus. Mol. Ecol. 22, 1589–1608, https://doi.org/10.1111/mec.12137 (2013).
Giraud-Billoud, M. & Castro-Vazquez, A. Aging and retinoid X receptor agonists on masculinization of female Pomacea canaliculata, with a critical appraisal of imposex evaluation in the Ampullariidae. Ecotoxicol. Environ. Saf. 169, 573–582, https://doi.org/10.1016/j.ecoenv.2018.10.096 (2019).
Primost, M. A., Sabatini, S. E., Di Salvatore, P., Ríos De Molina, M. C. & Bigatti, G. Oxidative stress indicators in populations of the gastropod Buccinanops globulosus affected by imposex. J. Mar. Biol. Assoc. U K. 97, 35–42, https://doi.org/10.1017/S0025315415002155 (2015).
Beníšek, M., Bláha, L. & Hilscherová, K. Interference of PAHs and their N-heterocyclic analogs with signaling of retinoids in vitro. Toxicol. Vitro 22, 1909–1917, https://doi.org/10.1016/j.tiv.2008.09.009 (2008).
Horiguchi, T., Nishikawa, T., Ohta, Y., Shiraishi, H. & Morita, M. Time course of expression of the retinoid X receptor gene and induction of imposex in the rock shell, Thais clavigera, exposed to triphenyltin chloride. Anal. Bioanal. Chem. 396, 597–607 (2010).
Liu, Y., Beyer, A. & Aebersold, R. On the Dependency of Cellular Protein Levels on mRNA Abundance. Cell 165, 535–550, https://doi.org/10.1016/j.cell.2016.03.014 (2016).
Acknowledgements
This work was supported by the Agencia Nacional de Promoción de Ciencia y Tecnología (ANAPCYT: PICT 1491-2012 and 2709-2015), CONICET: PIP 11220120100067CO, Waitt Foundation and the PADI Foundation. SG, MAP, CL and GB are members of the Research Career, CONICET. This is publication #130 of the Laboratorio de Reproducción y Biología Integrativa de Invertebrados Marinos (LARBIM).
Author information
Authors and Affiliations
Contributions
Authors’ individual contributions Sebastián Giulianelli: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Writing - Original Draft. Mónica Primost: Investigation, Resources, Writing - Review & Editing. Claudia Lanari: Resources, Writing - Review & Editing, Funding acquisition. Gregorio Bigatti: Resources, Writing - Review & Editing, Funding acquisition. All authors reviewed the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Giulianelli, S., Primost, M.A., Lanari, C. et al. RXR Expression in Marine Gastropods with Different Sensitivity to Imposex Development. Sci Rep 10, 9507 (2020). https://doi.org/10.1038/s41598-020-66402-1
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-020-66402-1
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.