Toshihiro Horiguchi

Retinoid X receptor gene expression and protein content in tissues of the rock shell Thais clavigera

To elucidate the role of retinoid X receptor (RXR) in the development of imposex caused by organotin compounds in gastropod molluscs, we investigated RXR gene expression and RXR protein content in various tissues of male and female wild rock shells (Thais clavigera). Quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry with a commercial antibody against human RXR alpha revealed that RXR gene expression was significantly higher in the penises of males and imposex-exhibiting females than in the penis-forming areas of normal females (P<0.01 and P<0.05, respectively). Western blotting demonstrated that the antibody could detect rock shell RXR and showed that the male penis had the highest content of RXR protein among the analyzed tissues of males and normal females. Immunohistochemical staining revealed nuclear localization of RXR protein in the epithelial and smooth muscle cells of the vas deferens and in the interstitial or connective tissues and epidermis of the penis in males and imposex-exhibiting females. RXR could be involved in the mechanism of induction of male-type genitalia (penis and vas deferens) by organotin compounds in female rock shells.

Cloning and characterization of retinoid X receptor (RXR) isoforms in the rock shell, Thais clavigera

The organotin compounds tributyltin (TBT) and triphenyltin (TPT) belong to a diverse group of widely distributed environmental pollutants that induce imposex in gastropods. These organotins have high affinity for retinoid X receptor (RXR), which is a transcription factor activated by retinoids, such as 9-cis retinoic acid (9cRA), in vertebrates. However, the molecular mechanisms underlying the regulation of RXR by retinoids and organotins have not been clarified in gastropods. We isolated two isoforms of RXR cDNAs, RXR isoform 1 (TcRXR-1) and RXR isoform 2 (TcRXR-2), in the rock shell Thais clavigera. The deduced amino acid sequences of TcRXR-1 and TcRXR-2 are highly homologous with those of other gastropods. These TcRXR isoforms displayed 9cRA-dependent activation of transcription in a reporter gene assay using COS-1 cells. The transcriptional activity of TcRXR-2, the encoded protein of which has five additional amino acids in the T-box of the C domain, was significantly lower than that of TcRXR-1. Decreases of the transcriptional activity by TcRXR-1 were observed when more than equal amount of TcRXR-2 fused expression vector was existed in a co-transfection assay. Immunoblot analysis showed several shifted bands for TcRXR isoforms resulting from phosphorylation. Mutation of potential phosphorylation sites from serine to alanine in the A/B domain of TcRXR-1 showed that, in the S89A/S103A mutant, there was a band shift and significantly higher transcriptional activity than in the controls when stimulated with 9cRA. Our findings could contribute to a better understanding of the role of interactions between RXR and retinoids and organotins, not only in the induction mechanism of imposex in gastropods but also in the endocrinology of mollusks.

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

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 5xa0μg/g wet wt) of 9-cis-retinoic acid (9CRA) or with a single concentration (1xa0μg/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 (Pu2009<u20090.05) or 5xa0μg (Pu2009<u20090.001) 9CRA than in the controls. After 1xa0month, the rock shells treated with 5xa0μg 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 (Pu2009<u20090.05) but not between the 1- and 5-μg 9CRA treatment groups (Pu2009>u20090.05). Compared with the control, the VDS index increased significantly in the 1- (Pu2009<u20090.05) and 5-μg (Pu2009<u20090.001) 9CRA groups. The penis-like structures behind the right tentacle in female rock shells treated with 5xa0μg 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.

Time course of expression of the retinoid X receptor gene and induction of imposex in the rock shell, Thais clavigera, exposed to triphenyltin chloride

AbstractTo examine the role of the retinoid X receptor (RXR) in the development of imposex in gastropods, we investigated the time course of expression of the RXR gene in various tissues (ctenidium, ovary or testis, digestive gland, penis-forming area or penis, and head ganglia) of female and male rock shells (Thais clavigera) exposed to triphenyltin (TPT) in a flow-through exposure system for 3xa0months. Accumulations of TPT in tissues were clearly observed in exposed individuals, whereas no accumulation of TPT was observed in the control groups. In females, 3-month exposure to TPT resulted in the development of imposex, and penis lengths in imposex-exhibiting females were significantly longer in small females (shell height <20xa0mm) than in large females (shell height ≥20xa0mm). RXR gene expression in the ovary, penis-forming area or penis, and head ganglia of females exposed for 3xa0months was significantly higher than expression in control females, and the highest RXR gene expression was found in the penis-forming area or penis. Moreover, RXR gene expression in the penis-forming area or penis of each female exposed to TPT seemed to be associated with an increase in penis length. In males, the ratio of penis length to shell height was significantly larger in the exposed groups than in the controls. Although RXR gene expression in males exposed for 3xa0months was not significantly higher than expression in control males in any tissues, the highest gene expression was observed in the penis of exposed males. These results suggest that RXR plays an important role in the development of male genitalia (i.e., penis and vas deferens) in gastropods, although RXR might also have other physiological functions.n FigureRelationships between average penis length and RXR gene expression in penis-forming area/penis and ovary of female rock shells exposed to 500 ng/L of triphenyltin chloride (TPTCl) for 3 months in a flow-through system. TPT, triphenyltin. Bars in the upper and middle figures represent normalized RXR gene expression in ovary and penis-forming area/penis of females exposed to TPTCl, respectively. Dots or symbols in the bottom figure represent measured values of penis length of each female in the respective composite samples. RXR gene expression in the penis-forming area or penis of each female exposed to TPT seems to be associated with an increase in penis length, however, that in ovary does not.

A Mu-class glutathione S-transferase (GSTM) from the rock shell Thais clavigera.

The rock shell (Thais clavigera) has attracted interest due to high frequency of imposex induced by endocrine disrupting chemicals (EDCs) in its natural populations. Oxidative stress is one of the mechanisms of action of EDCs. Glutathione S-transferases (GSTs) play an important role in antioxidant defense protecting the cells from oxidative stress. So far, there is no information on antioxidant defense or detoxification genes from T. clavigera. We cloned the full length cDNA sequence for a Mu-class of GST gene from T. clavigera (Tc-GSTM) and purified recombinant Tc-GSTM protein by bacterial expression. The deduced amino acid sequence of Tc-GSTM exhibited 45 to 66% identity with other Mu-class GSTs. Real-time RT-PCR analysis showed highest expression of Tc-GSTM in gill, while reproductive organs showed low expression. The biochemical characteristics of purified recombinant Tc-GSTM were typical, and thus Tc-GSTM showed highest specific activity for the universal GST substrate, 1-chloro-2,4-dinitrobenzene (CDNB). After exposure to prooxidant H(2)O(2), transformed Escherichia coli containing Tc-GSTM showed higher survival rate compared to control bacteria without expressed Tc-GSTM. The present study reveals a conserved antioxidant role for GSTM in rock shells, and the tissue-specific differences in Tc-GSTM transcripts would partly reflect vulnerability of reproductive organs to chemically induced oxidative stress.

Specific accumulation of organotin compounds in tissues of the rock shell, Thais clavigera.

Concentrations of organotin compounds (butyltins and phenyltins) were determined in gonad, accessory sex organs, penis, digestive gland, kidney, radula with sac, oesophagus with crop, stomach, hypobranchial gland, rectum, mantle, osphradium, ctenidium, heart, salivary gland, head ganglia and muscle of imposex-exhibiting female and male rock shells (Thais clavigera), by gas chromatography with flame photometric detection (GC-FPD). Different tissue distributions were observed between butyltin and phenyltin compounds. More than 1000 ng TBT/g wet wt. were observed in ovary, digestive gland, kidney, heart, ctenidium, osphradium, stomach, head ganglia and penis, of both imposex-exhibiting females and males. More than 1000 ng TPT/g wet wt. were found in almost all tissues of both sexes. Approximately one-third or more of total TBT was accumulated in the digestive glands of both females and males, respectively. Meanwhile, approximately 40-50% and one-half of total TPT accumulated in the digestive glands of females and males, respectively.

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

In the chain of study to further elucidate the role of retinoid X receptor (RXR) in the development of imposex caused by organotin compounds in gastropod mollusks, we established a polyclonal antibody against RXR of the rock shell Thais clavigera. Immunoblotting demonstrated that this antibody could recognize T. clavigera RXR. In males and imposex-exhibiting females, immunohistochemical staining with the antibody revealed nuclear localization of RXR protein in the epithelial and smooth muscle cells of the vas deferens and in the interstitial and epidermal cells of the penis. These results suggest that the polyclonal antibody against T. clavigera RXR can specifically recognize RXR protein in tissues of T. clavigera and therefore is useful for evaluating RXR protein localization. Furthermore, RXR may be involved in the induction of male-type genitalia (penis and vas deferens) in normal male and organotin-exposed female rock shells.

Mitochondrial genome of Thais clavigera (Mollusca: Gastropoda): affirmation of the conserved, ancestral gene pattern within the mollusks.

Class Gastropoda includes a large number of described species, many with extensively rearranged mitochondrial genomes. We sequenced the mitogenome of the rock shell, Thais clavigera (Gastropoda: Muricidae), an intertidal snail, using long PCR with primers designed on the basis of expressed sequence tags. The mitogenome of T. clavigera consists of 2 rRNAs, 22 tRNAs, and 13 protein-coding genes, but no control region. Structural comparisons revealed that the order Sorbeoconcha, including T. clavigera, have nearly identical mitochondrial gene patterns. However, they have an inversion between a tRNA(Phe)-tRNA(Glu) cluster that comprises 21 genes, but most of the remaining structure is similar to the putative mollusk ground pattern. These findings will provide a better insight into mitochondrial gene rearrangement over the course of gastropod evolution.

The Endocrine-Disrupting Effect of Organotin Compounds for Aquatic Organisms

Abbreviations CRM: Certified Reference Material; DBT: Dibutyltin; DPT: Diphenyltin; GC-FPD: Gas chromatography with flame photometric detection; AFS Convention: International Convention on the Control of Harmful Anti-fouling System on Ships; IMO: International Maritime Organization; MBT: Monobutyltin; MPT: Monophenyltin; RPL: Relative penis length; ΣBTs: Sum of butyltins; ΣPTs: Sum of phenyltins; TBT: Tributyltin; TPT: Triphenyltin; VDS: Vas deferens sequence; wet wt: Wet weight

Vas deferens and penis development in the imposex-exhibiting female rock shell, Thais clavigera.

The characteristics of the development of male genitalia (penis and vas deferens) in imposex-exhibiting female rock shells, Thais clavigera, were histologically examined using specimens from a wild population and tributyltin (TBT)-exposed females in the laboratory. A variety of vas deferens morphogenesis patterns were observed in wild female T.xa0clavigera, and the characteristics were summarized. The immature vas deferens at an initial stage, however, was only observed beneath or behind the penis, and no vas deferens was observed close to the vaginal opening (i.e., vulva) of the capsule gland in TBT-exposed females, which was different from the characteristics of vas deferens formation observed in wild females. Taking into consideration both the observed results from wild female specimens and from TBT-exposed females in the laboratory, the vas deferens sequence (VDS) index for T.xa0clavigera was proposed as VDS 1-6.