Cloning, expression and functional characterization of carp, Cyprinus carpio, estrogen receptors and their differential activations by estrogens.

Sex-steroid hormones are essential for normal reproductive activity in both sexes. Estrogens are necessary for ovarian differentiation during a critical developmental stage in vertebrates and promote the growth and differentiation of the female reproductive system. Importantly, environmental estrogens can influence the reproductive system and have been shown to disrupt gametogenesis in males. To understand the molecular mechanisms of estrogen actions and to evaluate estrogen receptor ligand interactions in the carp, Cyprinus carpio, a species used widely for both field- and laboratory-based studies, we cloned all three carp estrogen receptors (ER; ERα, ERß1 and ERß2) and applied an estrogen-responsive (ERE)-luciferase reporter assay system to characterize the interactions of these receptors with steroidal and synthetic estrogens. DNA fragments encoding all three ERs in carp, ERα, ERß1 and ERß2, were obtained from the ovary using degenerate primer sets and PCR techniques, and full-length carp ER (cER) cDNAs were then obtained using RACE (rapid amplification of the cDNA end) techniques. Amino acid sequences of cERs showed overall homology of 46% (α vs ß1), 49% (α vs ß2) and 53% (ß1 vs ß2). In the transient transfection ERE-luciferase reporter assay system (using mammalian cells) the cER proteins displayed estrogen-dependent activation of transcription and cERß2 showed a higher sensitivity to the natural steroid oestrogen, 17ß-estradiol, than cERα. The assay system developed is a powerful assay for toxicology and provides a tool for future studies examining the receptor-environmental chemical interactions and estrogen-disrupting mechanisms in carp. The data presented also expand our knowledge of estrogen receptor evolution.

N-terminal domain regulates steroid activation of elephant shark glucocorticoid and mineralocorticoid receptors.

Orthologs of human glucocorticoid receptor (GR) and human mineralocorticoid receptor (MR) first appear in cartilaginous fishes. Subsequently, the MR and GR diverged to respond to different steroids: the MR to aldosterone and the GR to cortisol and corticosterone. We report that cortisol, corticosterone and aldosterone activate full-length elephant shark GR, and progesterone, which activates elephant shark MR, does not activate elephant shark GR. However, progesterone inhibits steroid binding to elephant shark GR, but not to human GR. Together, this indicates partial functional divergence of elephant shark GR from the MR. Deletion of the N-terminal domain (NTD) from elephant shark GR (truncated GR) reduced the response to corticosteroids, while truncated and full-length elephant shark MR had similar responses to corticosteroids. Swapping of NTDs of elephant shark GR and MR yielded an elephant shark MR chimera with full-length GR-like increased activation by corticosteroids and progesterone compared to full-length elephant shark MR. Elephant shark MR NTD fused to GR DBD + LBD had similar activation as full-length MR, indicating that the MR NTD lacked GR-like NTD activity. We propose that NTD activation of human GR evolved early in GR divergence from the MR.

Molecular cloning and characterization of ligand- and species-specificity of amphibian estrogen receptors.

Estrogens are essential for normal reproductive activity in both males and females as well as for ovarian differentiation during a critical developmental stage in most vertebrates. To understand the molecular mechanisms of estrogen action and to evaluate estrogen receptor ligand interactions in amphibians, we isolated cDNAs encoding the estrogen receptors (ERalpha and ERbeta) from the Japanese firebelly newt (Cynops pyrrhogaster), Tokyo salamander (Hynobius tokyoensis), axolotl (Ambystoma mexicanum), and Raucous toad (Bufo rangeri). Full-length amphibian ER cDNAs were obtained using 5' and 3' rapid amplification of cDNA ends. The predicted amino acid sequences of these amphibian ERs showed a high degree of amino acid sequence identity (over 70%) to each other. We analyzed the relationships of these amphibian ER sequences to other vertebrate ER sequences by constructing a phylogenetic tree. We verified that these were bona fide estrogen receptors using receptor dependent reporter gene assays. We analyzed the effects of natural estrogens, ethinylestradiol, and DDT and its metabolites on the transactivation of the four amphibian species listed above, and Xenopus tropicalis ERs and found that there were species-specific differences in the sensitivity of these ERs to hormones and environmental chemicals. These findings will expand our knowledge of endocrine-disrupting events in amphibians.

Cloning and functional characterization of Chondrichthyes, cloudy catshark, Scyliorhinus torazame and whale shark, Rhincodon typus estrogen receptors.

Sex-steroid hormones are essential for normal reproductive activity in both sexes in all vertebrates. Estrogens are required for ovarian differentiation during a critical developmental stage and promote the growth and differentiation of the female reproductive system following puberty. Recent studies have shown that environmental estrogens influence the developing reproductive system as well as gametogenesis, especially in males. To understand the molecular mechanisms of estrogen actions and to evaluate estrogen receptor-ligand interactions in Elasmobranchii, we cloned a single estrogen receptor (ESR) from two shark species, the cloudy catshark (Scyliorhinus torazame) and whale shark (Rhincodon typus) and used an ERE-luciferase reporter assay system to characterize the interaction of these receptors with steroidal and other environmental estrogens. In the transient transfection ERE-luciferase reporter assay system, both shark ESR proteins displayed estrogen-dependent activation of transcription, and shark ESRs were more sensitive to 17beta-estradiol compared with other natural and synthetic estrogens. Further, the environmental chemicals, bisphenol A, nonylphenol, octylphenol and DDT could activate both shark ESRs. The assay system provides a tool for future studies examining the receptor-ligand interactions and estrogen disrupting mechanisms in Elasmobranchii.

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.

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.

Two cDNAs of RXR were isolated, for the first time, from the ivory shell, Babylonia japonica, and the transcriptional activities were tested in vitro to compare with other gastropod (Thais clavigera and Nucella lapillus) RXR isoforms. The transcriptional activities of all of these RXR isoforms were significantly induced by mammalian RXR agonist, 9-cis retinoic acid (9cRA). The transcriptional activity of T. clavigera RXR-1 was also examined by using 9cRA and 16 organotin compounds, and significant ligand-dependent transactivations were observed by 9cRA and 5 organotins (tributyltin (TBT), tetrabutyltin (TeBT), tripropyltin (TPrT), tricyclohexyltin (TcHT) and triphenyltin (TPhT)). These 5 organotins also induced significant transcriptional activities in N. lapillus and B. japonica RXR isoforms. These 4 organotins, except for TeBT, have been reported to promote the development of imposex after a month of a single injection each, using female T. clavigera. To investigate the function of gastropod RXR isoforms, the effects of mammalian specific pan-agonist, PA024, and pan-antagonist, HX531, were examined, and significant induction of transcriptional activity by PA024 was demonstrated in these gastropod RXR isoforms. The additions of HX531 significantly suppressed the transcriptional activities of these gastropod RXR isoforms by 9cRA and 5 organotins. Using the mammalian two retinoic acid response elements, the transcriptional activities by 2 agonists, 9cRA and PA024, were different among the RXR isoforms of each gastropod species. With retinoid X response element (RXRE), transcriptional activities of TcRXR-1, BjRXR-1, and NlRXRa were significantly higher than those of TcRXR-2, BjRXR-2, and NlRXRb. Transcriptional activities of TcRXR-2, BjRXR-2, and NlRXRb, however, were significantly higher than those of TcRXR-1, BjRXR-1, and NlRXRa with thyroid hormone response element, TREpal. Thus, induction of imposex in prosobranch gastropods is strongly suggested to be triggered by 9cRA and certain organotins, such as TBT and TPhT through the activation of RXRs. These gastropod RXRs might control the different gene transcription by forming homo- or heterodimer complex with their own isoforms. These findings will contribute to our understanding of the fundamentals of the endocrine system in molluscs, particularly on RXR signaling pathway.

In vitro assessment of transcriptional activation of the estrogen and androgen receptors of mosquitofish, Gambusia affinis affinis.

Sex-steroid hormones are essential for normal reproductive activity in both sexes. Estrogens are necessary for ovarian differentiation during a critical developmental stage in many vertebrates and promote the growth and differentiation of the female reproductive system. Androgens play essential roles in the development and functioning of the vertebrate male reproductive system as well as actively supporting spermatogenesis. Importantly, recent studies suggest that androgens and estrogens have important reproductive roles in both males and females. To understand the molecular mechanisms of estrogen and androgen actions and to evaluate estrogen and androgen receptor-ligand interactions in the mosquitofish, Gambusia affinis affinis, we used degenerate primer sets and PCR techniques to isolated DNA fragments encoding estrogen receptor alpha (ERalpha; ESR1), ERbeta1 (ERbeta1) and ERbeta2 from the ovary. Full-length mosquitofish ER (mfER) cDNAs were obtained using cDNA library screening and RACE techniques. Amino acid sequences of mfERs showed over-all homology of 46% (alpha versus beta1), 43% (alpha versus beta2), and 52% (beta1 versus beta2). We applied the ERE-luciferase reporter assay system to characterize these receptors. In this transient transfection assay system using mammalian cells, the mfER proteins displayed estrogen-dependent activation of transcription. In addition to ERs, the transactivation of mosquitofish ARs (mfARs) previously isolated by our group, were examined using an androgen-responsive MMTV-luciferase assay system. Mosquitofish ARs showed androgen-dependent activation of transcription from the MMTV promoter. These data provide a basic tool allowing future studies examining the receptor-ligand interactions and endocrine disrupting mechanisms in mosquitofish and also expands our knowledge of estrogen and androgen receptor evolution.

Gene expression change in the Müllerian duct of the mouse fetus exposed to diethylstilbestrol in utero.

In utero exposure to diethylstilbestrol (DES) induces various abnormalities in the Müllerian duct of the mouse. In order to understand the underlying molecular mechanisms associated with DES-induced abnormalities of the Müllerian duct, gene expression was examined on Gestation Day (GD) 19 in mouse fetuses exposed to DES (67 microg/kg body weight) from GDs 10 to 18. Microarray analysis revealed that 387, 387, and 225 genes were upregulated and 177, 172, and 75 genes were downregulated by DES in the oviduct, uterus, and vagina, respectively. DES exposure in utero commonly upregulated 72 genes and downregulated 15 genes in these three organs. The present study demonstrated that organ-specific gene expression patterns in the mouse Müllerian duct were altered by in utero DES exposure. DES-induced changes in expression of genes such as Dkk2, Nkd2, and sFRP1 as well as changes in genes of the Hox, Wnt, and Eph families in the female mouse fetal reproductive tract could be the basis for various abnormalities in reproductive tracts following exposure to this estrogenic drug.

Comparison of estrogen responsive genes in the mouse uterus, vagina and mammary gland.

Female reproductive organs are mainly regulated by estrogen and progesterone. Specifically, the uterus, vagina and mammary gland show organ-specific mitosis and morphological changes during proliferative events, such as estrous cycle, gestation and lactation. The mechanism underlying these organ-specific estrogen-dependent events is still unknown. We examined, therefore, global gene expression in the mature uterus, vagina and mammary gland of ovariectomized adult mice 6 hr after an injection of 5 microg/kg 17beta-estradiol (E2) using a microarray method in order to identify primary E2-responsive genes. Half of the E2 up-regulated genes in the uterus were similar to those in the vagina. E2 up-regulated the expression of Insulin-like growth factor 1 (Igf-1) genes in the uterus and vagina. In the vagina, E2 up-regulated the expression of IGF binding proteins (Igfbp2 and Igfbp5). In the mammary gland, unlike the uterus and vagina, no gene showed altered expression 6 hr after the E2 exposure. These results suggest that expression of Igf-1 and morphogenesis genes is regulated by E2 in an organ-specific manner, and it is supported by the results of BrdU labeling showing E2-induced mitosis in the uterus and vagina except the mammary gland. The differences in organ specificity in response to E2 may be attributed by differences in gene expression regulated by E2 in female reproductive organs. The candidate estrogen-responsive genes in the uterus and vagina identified by profiling provide an important foundation understanding functional mechanisms of estrogen regulating morphogenesis and maintenance of each reproductive organ.

Medaka (Oryzias latipes) for use in evaluating developmental effects of endocrine active chemicals with special reference to gonadal intersex (testis-ova).

Japanese medaka (Oryzias latipes) has been widely used for the evaluation of the toxicity of endocrine active chemicals (EACs) and other chemicals as well as for monitoring the adverse effects of effluent discharges in relation to sexual development and function. It is useful for these evaluations for many reasons including the following: 1) it has a short life cycle facilitating studies extending over long phases of development and over multigenerations, 2) it is easy to rear, 3) male and female phenotypes can easily be distinguished on the basis of secondary sex characteristics, and 4) a genetic marker (DMY) is available for identifying the true genotypic sex. Several biomarkers have been found to be useful for identifying the effects of exposure to estrogenic and androgenic chemicals in medaka and they include increased levels of hepatic vitellogenin (VTG) and testis-ova induction in males for exposure to estrogenic chemicals, and decreased levels of hepatic VTG in females and an altered morphology of dorsal and anal fins and formation of papillae for androgenic chemicals. In this paper, we present a critical analysis of the use of medaka as a test species for studies of endocrine disruption and report on the use of sex-related genetic markers and alterations in gonadal development, including the induction of testis-ova formation, for assessing the disruptive effects of EACs. In this paper, we focus on some of the more recent studies and findings.

Molecular cloning of estrogen receptor alpha (ERalpha; ESR1) of the Japanese giant salamander, Andrias japonicus.

Estrogens are essential for normal reproductive activity in females and males and for ovarian differentiation during a critical developmental stage in many vertebrates. To understand the molecular mechanisms of estrogen action and to evaluate estrogen receptor ligand interactions in the Japanese giant salamander (Andrias japonicus), we isolated cDNA encoding the estrogen receptor (ER) from the liver. A full-length Japanese giant salamander ER cDNA (jgsER) was obtained using 5' and 3' rapid amplification cDNA ends (RACE). The deduced amino acid sequence of the jgsER showed high identity to the Xenopus ERalpha (ESR1) (77.7%). We have applied both the conventional ERE-luciferase reporter assay system and the GAL4-transactivation system to characterize this receptor. In two different transient transfection assay systems using mammalian cells, the jgsER protein displayed estrogen-dependent activation of transcription. The GAL4-transactivation system showed about 10-fold greater activity of the estrogen receptor by hormone when compared to the conventional ERE-luciferase reporter assay system. Tissue distribution of ERalpha mRNA was examined and kidney, ovary and liver exhibited expression. This is the first isolation of an estrogen receptor from a salamander and also is the first functional cDNA obtained from the Japanese giant salamander, an endangered species considered a special natural monument of Japan.

Availability of in vitro vitellogenin assay for screening of estrogenic and anti-estrogenic activities of environmental chemicals.

Vitellogenin (VTG) protein, VTG mRNA, other egg yolk proteins, vitelline envelope proteins and their mRNAs are produced in the liver of oviparous species by stimulation of endogenous estrogen and exogenous estrogenic chemicals. The VTG assay based on enzyme-linked immunosorbent assay (ELISA) has been widely used for many fish species to screen estrogenic and anti-estrogenic activities of chemicals and sewage effluents using immature fish and/or male fish. In order to reduce the number of fish for screening of estrogenicity and anti-estrogenicity of chemicals, primary cultured fish hepatocytes can be used. In fact, primary cultured hepatocytes have been successfully used for the detection of estrogenic and anti-estrogenic activities of environmental chemicals in selected OECD fish species, e.g., medaka (Oryzias latipes) and rainbow trout (Oncorhynchys mykiss) together with other fish species such as Atlantic salmon (Salmo salar L.), Siberian sturgeon (Acipenser baeri), tilapia (Oreochromis mossambicus), carp (Cyprinus carpio), bream (Abramis brama), Carassius auratus, silver eel (Anguilla anguilla L.), and channel catfish (Ictalurus punctanus). In terms of hepatocyte assays relating to other taxa, these include frogs such as Xenopus laevis and the common green frog (Rana esculenta), chickens (Gallus domesticus) and herring gulls (Larus argentatus). VTG mRNA measurement by quantitative reverse transcription-polymerase chain reaction has also been successfully applied in the primary cultured hepatocytes of various species.

Cloning and characterization of estrogen receptor alpha in mummichog, Fundulus heteroclitus.

Developmental exposure to 17 beta-estradiol (E(2)) induced the death of embryos and fry, malformations, sex reversal, and incomplete ossification of vertebrae and cranial bones in the cyprinodont fish, the mummichog (Fundulus heteroclitus). To clarify the mechanism by which exogenous estrogens caused these developmental effects, we determined the sequence of an estrogen receptor (ER) coding region, encoded by 620 amino acid residues. This region shared 80% identity to that of ER alpha of medaka (Oryzias latipes). Northern blot analysis showed that two ER alpha mRNAs with 5.5 and 4 kb were expressed in the liver. These mRNAs were strongly induced by E(2) stimulation. The 4 kb mRNA was expressed 8 h after treatment, whereas the 5.5 kb mRNA was not induced until 12 h after E(2) stimulation. Vitellogenin (VTG) was expressed 8 h after E(2) stimulation in the male liver. Receptor binding assays using the protein of F. heteroclitus ER alpha (fh ER alpha) ligand binding domain showed that alkylphenols bind to fh ER alpha with a higher affinity (50 times or more) as compared with the human ER alpha. The present results demonstrate that the fh ER alpha has a sequence very similar to that of medaka, and the mRNA for this receptor was induced by E(2)-stimulation, followed subsequently by VTG expression. Furthermore, alkylphenols bind to fh ER alpha more efficiently than to human ER alpha.

Development of reproductive organs in the ivory shell Babylonia japonica: observations from wild populations and laboratory-reared juveniles.

We histologically examined normal differentiation and development of genital tract and gonad in the ivory shell Babylonia japonica (Buccinidae) to determine whether the formation of male-type genitalia in imposex-exhibiting females mimics the normal development of male genitalia in prosobranch gastropods. We used a wild-caught 2-year-old specimen and laboratory-reared juveniles aged 0-24 months. Gonad differentiation was unclear before age 14 months, but progressed after 16 months. Both sexes had complete genital tract and mature gonad at 20 months. However, differentiation and development occurred earlier in females than in males. Development of genital tract preceded gonad differentiation. Vas deferens morphogenesis in males resembled that in imposex-exhibiting females. These findings help to understand the morphogenesis of genital tract and gonad in prosobranch gastropods and will contribute to more in-depth studies of the mode of action of organotin compounds such as TBT and TPhT in imposex development in female prosobranch gastropods.

Molecular cloning and characterization of the corticoid receptors from the American alligator.

Steroid hormones are essential for health in vertebrates. Corticosteroids, for example, have a regulatory role in many physiological functions, such as osmoregulation, respiration, immune responses, stress responses, reproduction, growth, and metabolism. Although extensively studied in mammals and some non-mammalian species, the molecular mechanisms of corticosteroid hormone (glucocorticoids and mineralocorticoids) action are poorly understood in reptiles. Here, we have evaluated hormone receptor-ligand interactions in the American alligator (Alligator mississippiensis), following the isolation of cDNAs encoding a glucocorticoid receptor (GR) and a mineralocorticoid receptor (MR). The full-length alligator GR (aGR) and aMR cDNAs were obtained using 5' and 3' rapid amplification cDNA ends (RACE). The deduced amino acid sequences exhibited high identity to the chicken orthologs (aGR: 83%; aMR: 90%). Using transient transfection assays of mammalian cells, both aGR and aMR proteins displayed corticosteroid-dependent activation of transcription from keto-steroid hormone responsive, murine mammary tumor virus promoters. We further compared the ligand-specifity of human, chicken, Xenopus, and zebrafish GR and MR. We found that the alligator and chicken GR/MR have very similar amino acid sequences, and this translates to very similar ligand specificity. This is the first report of the full-coding regions of a reptilian GR and MR, and the examination of their transactivation by steroid hormones.

Cloning and characterization of the retinoic acid receptor-like protein in the rock shell, Thais clavigera.

The organotin compounds have a high affinity for the retinoid X receptor (RXR), which is a transcriptional factor activated by retinoids that induce imposex in gastropods. However, the molecular mechanisms underlying the regulation of RXR and its related genes in gastropods remain unclear. We isolated a retinoic acid receptor (RAR)-like cDNA (TcRAR) in the rock shell, Thais clavigera, and examined the transcriptional activity of the TcRAR protein by using all-trans retinoic acid (ATRA). However, we did not observe any ligand-dependent transactivation by this protein. We also examined the transcriptional activity of the TcRAR-ligand binding domain fused with the GAL4-DNA binding domain by using retinoic acids, retinol, and organotins and again saw no noteworthy transcriptional induction by these chemicals. Use of a mammalian two-hybrid assay to assess the interaction of the TcRAR protein with the TcRXR isoforms suggested that TcRAR might form a heterodimer with the RXR isoforms. The transcriptional activity of domain-swapped TcRAR chimeric proteins (the A/B domain of TcRAR combined with the D-F domain of human RARα) was also examined and found to be ATRA-dependent. These results suggest that TcRAR is not activated by retinoic acids, but can form a heterodimer with TcRXR isoforms. These data contribute to our understanding of the mechanism by which RXR functions in gastropods.

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. clavigera, 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. clavigera was proposed as VDS 1-6.

Comparative responsiveness to natural and synthetic estrogens of fish species commonly used in the laboratory and field monitoring.

Exposure to estrogenic chemicals discharged into the aquatic environment has been shown to induce feminization in wild freshwater fish and although fish species have been reported to differ in their susceptibility for these effects, empirical studies that directly address this hypothesis are lacking. In this study, in vitro ERα activation assays were applied in a range of fish species used widely in chemical testing (including, zebrafish, fathead minnow, medaka) and/or as environmental monitoring species (including, roach, stickleback, carp) to assess their comparative responsiveness to natural (estrone, estradiol, estriol) and synthetic (17α-ethinylestradiol (EE2), diethylstilbestrol (DES)) estrogens. In vivo exposures to EE2 via the water (nominal 2 and 10 ng/L for 7 days) were also conducted for seven fish species to compare their responsiveness for hepatic vitellogenin (VTG) mRNA induction (an ER mediated response). Of the fish species tested, zebrafish ERα was found to be the most responsive and carp and stickleback ERα the least responsive to natural steroid estrogens. This was also the case for exposure to EE2 with an ERα-mediated response sensitivity order of zebrafish > medaka > roach > fathead minnow > carp > stickleback. For VTG mRNA induction in vivo, the order of species responsiveness was: rainbow trout (not tested in the ERα activation assays) > zebrafish > fathead minnow > medaka > roach > stickleback > carp. Overall, the responses to steroid estrogens in vitro via ERα compared well with those seen in vivo (VTG induction for exposure to EE2) showing in vitro screening of chemicals using fish ERα-mediated responses indicative of estrogenic responses (VTG induction) in vivo.

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.

Molecular cloning of anti-Müllerian hormone from the American alligator, Alligator mississippiensis.

Anti-Müllerian hormone (AMH) plays an important role in male sex differentiation in vertebrates. AMH produced by Sertoli cells of the fetal testis induces regression of the Müllerian duct in mammalian species. In alligators, sexual differentiation is controlled by the temperature during egg incubation, termed temperature-dependent sex determination (TSD). The TSD mechanism inducing sex differentiation is thought to be unique and different from that of genetic sex determination as no gene such as the SRY of mammals has been identified. However, many of the genes associated with gonadal differentiation in mammals also are expressed in the developing gonads of species exhibiting TSD. To clarify the molecular mechanisms associated with gonad formation during the temperature-sensitive period (TSP), we have cloned the full length AMH gene in the alligator, and quantitatively compared mRNA expression patterns in the gonad-adrenal-mesonephros (GAM) complex isolated from alligator embryos incubated at male and female producing temperatures. The deduced amino acid sequence of the alligator AMH cDNA showed high identity (59-53%) to avian AMH genes. AMH mRNA expression was high in the GAM of male alligator embryos at stage 24 (immediately after sex determination) and hatchlings, but suppressed in the GAM of estrogen-exposed hatchlings incubated at the male-producing temperature. In the alligator AMH proximal promoter, a number of transcriptional factors (for SF-1. GATA, WT-1 and SOX9) binding elements were also identified and they exhibit a conserved pattern seen in other species. SOX9 up-regulates transcriptional activity through the amAMH promoter region. These results suggested that AMH and SOX9 play important roles in TSD of the American alligator.