Function Of Retinoid X Receptor In Development Of Imposex In Small Abalone, Haliotis Diversicolor

The organotin compounds tributyltin(TBT) and triphenyltin(TPT) have been used in antifouling paints for ships and fishing nets and caused serious environmental problems in marine worldwide. One of the most typical toxic effects of TBT and TPT on marine organisms is the induction of imposex in gastropoda. It is characterized by the superimposition of male characteristics, such as a penis and a vas deferens, onto females of marine gastropods. Currently,at least 200 species of prosobranch gastropods are known to be affected. Imposex is one of the best documented examples of endocrine disruption in wildlife. Large amounts of investigation on development and histological examination of imposex have been reported in meso- and neogastropods. Because of less understand of endocrine research about sex differentiation and gonadal development of gastropods, the molecular mechanism of imposex was still controversial.Recently, several research suggested that retinoid X receptor(RXR) signaling may contribute to the regulation of imposex in gastropods. we cloned the RXR c DNA and investigated it’s m RNA expression in small abalone( Haliotis diversicolor) exposed to organotin compounds. The following is the summary of results:1) RXR c DNA has two isoforms: RXRa and RXRb, the encoded protein is 459 and 463 amino acids, predicted molecular weight is 50.56 k Da and 50.93 k Da, respectively.Isoelectric point is 7.36, predicted phosphorylation sites are 10 Ser, 2 Thr and 4 Tyr.RXR contains 107 bp of 5’UTR and 436 bp of 3’UTR, which has a typical polyadenylation signal AATAA. Hd RXR shares the modular structure characteristic of the nuclear receptor superfamily with a divergent N terminal A/B domain, a highly conserved DBD and a less well conserved LBD. Clustal W and phylogenetic analyses revealed that Hd RXR was more similar to sequences of vertebrate RXRs. RXRb has 4additional amino acids in the T-box of the C domain.2) Alignment of the amino acid sequence for the RXR isoforms from mollusc shows that different amino acid residues are inserted in the T-box,which has a consensus amino acid sequence of KREAVQEER. Hd RXRb has 4 additional amino acids(LLAA) in the T-box of the C domain,while five-amino acid insertion exists in the RXR T-box of Nucella lapillus and Thais clavigera. The RXR of Azumapecten Farreri has four different isoforms, which have three types of insertion: 4 amino acids, 20 amino acids and 24 amino acids. T-box is important region in DBD of RXR, which plays a important role in binding with downstream genes. The different insertion in T-box may have relation to downstream regulation.3) Quantitative real-time PCR revealed that level of RXR gene was widely distributed in small abalone. RXR gene expression in hepatopancreas of males was significantly higher than that of in females; level of RXR gene expression in head of females was significantly higher than that of in males. The lowest level of RXR gene expression was observed in hemolymph.4) Histological examination of gonads was performed in normal and TBT(1μg Sn/g wet wt) injected female abalone. The development of ovary consists of dorment stage,recrudescent stage, vegetative stage, mature stage and senescent stage.The results revealed that TBT could slow down or inhibit the development of oocytes, and seminiferous tubule was observed at 56 d of TBT exposure, but no significant spermatogenesis was observed in the abalone ovaries.5) we investigated the RXR gene expression in ovary of adult abalone upon two-month TBT injection,quantitative real-time PCR revealed that RXR gene expression in gonad of abalone treated with TBT, TPT and 9c RA, was significantly higher than BSA group,which indicated that 9c RA and organotins induced the m RNA expression of RXR.Moreover, we also investigated RXR m RNA expression in gonads and hepatopancreas from juvenile female abalone injected with 1μg Sn/g wet wt TBT. In gonads, a significant up-regulation was observed at 12 h, 24 h, 192 h and 8w past injection. In hepatopancreas, a significant up-regulation was also observed after 8 weeks of TBT injection. These results further support the hypothesis that imposex in marine gastropods could be mediated through the modulation of the RXR signaling pathways.6) To further elucidate the candidate target genes of Hd RXR, we examined the m RNA expression of several genes may involved in the development of gonads, including spermatogenesis-associated protein 5(SPATA5), testis-specific expressed gene 10(TEX10), 15-hydroxyprostaglandin dehydrogenase(15-PGDH) and bile-salt-activated lipase(BSAL). The results revealed that, a down-regulation of spata5 was observed in ovary at12 h and 8W past injection; in hepatopancreas, a significant down-regulation was observed at 6h and 12 h past injection, while up-regulation was observed at192 hpast injection(p<0.05). No significant change of m RNA level of tex10 was detected in gonad and hepatopancreas. 15-pgdh was significant up-regulated in gonad at 12 h and 2W past injection, while it was down-regulated in hepatopancreas(p<0.05).There was no change of bsal expression in gonad after TBT injection, but it was significantly down-regulated in hepatopancreas(p<0.001). The possible relationship between these genes and RXR was discussed.