@misc{sternberg_gooding_hotchkiss_leblanc_2010, title={Environmental-endocrine control of reproductive maturation in gastropods: implications for the mechanism of tributyltin-induced imposex in prosobranchs}, volume={19}, ISSN={["1573-3017"]}, DOI={10.1007/s10646-009-0397-z}, abstractNote={Prosobranch snails have been afflicted globally by a condition whereby females develop male sex characteristics, most notably a penis. This condition, known as imposex, has been causally associated with the ubiquitous environmental contaminant tributyltin (TBT). Deduction of the mechanism by which TBT causes imposex has been hampered by the lack of understanding of the normal endocrine regulation of reproductive tract recrudescence in these organisms. We have reviewed the relevant literature on the environmental and endocrine factors that regulate reproductive tract recrudescence, sexual differentiation, and reproduction in gastropods. We provide a cohesive model for the environmental-endocrine regulation of reproduction in these organisms, and use this information to deduce a most likely mechanism by which TBT causes imposex. Photoperiod appears to be the predominant environmental cue that regulates reproductive tract recrudescence. Secondary cues include temperature and nutrition which control the timing of breeding and egg laying. Several hormone products of the central and peripheral nervous systems have been identified that contribute to recrudescence, reproductive behaviors, oocyte maturation and egg laying. Retinoic acid signaling via the retinoid X-receptor (RXR) has shown promise to be a major regulator of reproductive tract recrudescence. Furthermore, TBT has been shown to be a high affinity ligand for the RXR and the RXR ligand 9-cis retinoic acid causes imposex. We propose that TBT causes imposex through the inappropriate activation of this signaling pathway. However, uncertainties remain in our understanding of the environmental-endocrine regulation of reproduction in gastropods. Definitive elucidation of the mechanism of action of TBT awaits resolution of these uncertainties.}, number={1}, journal={ECOTOXICOLOGY}, author={Sternberg, Robin M. and Gooding, Meredith P. and Hotchkiss, Andrew K. and LeBlanc, Gerald A.}, year={2010}, month={Jan}, pages={4–23} }
 @article{hotchkiss_sternberg_leblanc_2008, title={Environmental cues trigger seasonal regression of primary and accessory sex organs of the mud snail, Ilyanassa obsoleta}, volume={74}, ISSN={["0260-1230"]}, DOI={10.1093/mollus/eyn009}, abstractNote={Animals inhabiting non-tropical environments undergo seasonal changes in reproduction in order to improve their survival and chances of successful reproduction. For example, during certain periods of the year, food shortages and temperature extremes can make survival difficult. Adaptive physiological, behavioral and morphological changes are made to maintain a positive energy balance throughout the year and to ensure that offspring are born during a period when their chances of survival are maximized. Neogastropods display an annual rhythm in the seasonal re-growth of their reproductive tract and secondary sex organs. Although environmental cues likely stimulate neuro-endocrine cascades that orchestrate male and female sex organ differentiation as well as oogenesis, spermatogensis and copulatory behaviour, little is known about the specific seasonal cues or the underlying physiological timekeeping mechanisms responsible for these seasonal changes. Several environmental cues have been identified that initiate or modify reproductive processes in invertebrates. Seasonal adaptations are often the result of changes in day length (photoperiod). As photoperiod is an accurate predictor of changing seasons, many species have evolved the ability to use changes in photoperiod to predict upcoming environmental change. One mediator of photoperiod information, 5-methoxyindole melatonin, has been identified in many vertebrate species as well as in a number of invertebrates (Wayne, 2001; Gorbet & Steel, 2003). The duration of the melatonin signal provides information about day length. The nightly elevation of melatonin varies with the length of night, the long nights (short days) of winter resulting in a relatively long duration of melatonin release. It is this duration that allows animals to decipher the seasons. In molluscs, melatonin has been identified in the ocular tentacles, visceral ganglion, brain, eyes and the cerebral ganglia (Wayne, 2001; Blanc et al., 2003). In most invertebrates studied to date, melatonin is elevated during the night, thus potentially playing a role in daily and annual rhythms. In addition, another 5-methoxyindole, 5-methoxytryptophol (5-ML), has also been shown to oscillate in a number of vertebrate species as well as gastropods (Blanc et al., 2003). At present, it is unclear what role 5-ML may play in daily circadian or seasonal rhythms. In addition to photoperiod, several other factors have been implicated in the seasonal regulation of reproductive differentiation and breeding. Water temperature is the predominant environmental cue triggering the onset of breeding in some species of molluscs, whereas the seasonal availability of food has been shown to influence sex differentiation and breeding in other species (review by Wayne, 2001). While various environmental cues have been investigated as triggers for either reproductive differentiation or breeding in molluscs, few studies have examined the potential for an underlying seasonal timer. In this paper, we investigate whether the seasonal onset of regression in mud snails is controlled by two environmental cues, photoperiod or temperature. Mud snails (Ilyanassa obsoleta ) were collected from a field population on Oak Island, North Carolina, USA, in January 2006. Imposex females have never been detected in this field population. Snails were rinsed with seawater to remove mud and sand, and transported to the laboratory in seawater from the sample location. Once in the laboratory, snails were held in 8-l glass aquaria containing 1 l of reconstituted seawater (35 + 1 ppt) (Instant Ocean; Aquarium Systems, Mentor, OH). Solutions were changed every other day. Seasonal control (SC) snails were held in photoperiods and water temperatures that changed commensurate with the field site according to the meteorological websites http://www.weather.com (Oak Island, NC) and http://storms.nos.noaa.gov (Wilmington, NC), respectively. Peak water temperature for this population generally occurs approximately 1.5 months after the summer solstice, while water temperatures are lowest approximately 1.5 months after the winter solstice (Sternberg et al., 2007). Phasedelayed (PD) snails were held for 4.5 months at a constant photoperiod and temperature consistent with that of early February at the field site. After 4.5 months, snails were released from these conditions and maintained in seasonally progressing conditions that mimicked the sample site conditions but with a 4.5 month delay. Starting in February 2006, a sub-sample of the snails from each treatment was collected monthly, except as noted, for evaluation of reproductive status. The November collection}, journal={JOURNAL OF MOLLUSCAN STUDIES}, author={Hotchkiss, Andrew K. and Sternberg, Robin M. and LeBlanc, Gerald A.}, year={2008}, month={Aug}, pages={301–303} }
 @article{sternberg_hotchkiss_leblanc_2008, title={Synchronized expression of retinoid X receptor mRNA with reproductive tract recrudescence in an imposex-susceptible mollusc}, volume={42}, ISSN={["1520-5851"]}, DOI={10.1021/es702381g}, abstractNote={The biocide tributyltin (TBT) causes the development of male sex characteristics in females of some molluscan species, a phenomenon known as imposex. Recent evidence suggests that the retinoid X receptor (RXR) participates in TBT-induced imposex. Accordingly, we hypothesized that RXR may contribute to the seasonal development of the male reproductive tract in molluscs and would be expressed in concert with this phenomenon. RXR was cloned and sequenced from an imposex-susceptble species, the eastern mud snail Ilyanassa obsoleta. The DNA-binding domain of the receptor protein was 100 and 97% identical to those of the rock shell Thais clavigera and the freshwater snail Biomphalaria glabrata. The ligand-binding domain was 93 and 92% identicalto the LBD of these two molluscan species, respectively. Phylogenetic analyses revealed that RXR is an ancient nuclear receptor whose origin predates the emergence of the Bilateria. Interestingly, though inexplicably, the molluscan RXRs were more similar to sequences of vertebrate RXRs than to the RXRs of other lophotrochozoan invertebrates. Next, the expression of RXR mRNA levels in the reproductive tract was determined through the reproductive cycle. RXR mRNA levels increased commensurate with reproductive tract recrudescence in both sexes. However, the timing of coordinate recrudescence-RXR expression differed between sexes. Results demonstrate that RXR expression is associated with reproductive tract recrudescence in both sexes; although, the timing of recrudescence may dictate sex-specific development. Retinoid signaling initiated by TBT during an inappropriate time in females may result in imposex.}, number={4}, journal={ENVIRONMENTAL SCIENCE & TECHNOLOGY}, author={Sternberg, Robin M. and Hotchkiss, Andrew K. and Leblanc, Gerald A.}, year={2008}, month={Feb}, pages={1345–1351} }
 @article{sternberg_hotchkiss_leblanc_2008, title={The contribution of steroidal androgens and estrogens to reproductive maturation of the eastern mud snail Ilyanassa obsoleta}, volume={156}, ISSN={["1095-6840"]}, DOI={10.1016/j.ygcen.2007.12.002}, abstractNote={Molluscs exposed to endocrine-disrupting chemicals (EDCs) have exhibited changes in reproductive tract development that are typically associated with androgen or estrogen signaling in vertebrates. However, a role for androgens and estrogens in molluscan reproductive endocrinology has yet to be established. In this study, we investigated putative roles for steroidal androgens and estrogens in recrudescence of the eastern mud snail Ilyanassa obsoleta. Our objectives were to: (1) identify associations among concentrations of testosterone and 17beta-estradiol, sex, and reproductive status in mud snails that suggest these hormones are involved in recrudescence; and (2) determine whether mud snails express NR3C4-like (androgen receptor) and NR3A-like (estrogen receptor) mRNAs in a manner indicative of a role in recrudescence. Temporal changes in testosterone levels in males were consistent with a positive role in recrudescence. Such a trend was not evident in females or for 17beta-estradiol in either sex. Efforts to identify an androgen receptor from the mud snail using targeted, degenerate RT-PCR were unsuccessful. However, an estrogen receptor (ER) cDNA was identified that is highly similar to known ERs of other molluscs. Studies with the ER of other molluscs have shown that this protein does not actually bind estrogens. We therefore considered the possibility that the mud snail ER may regulate reproductive maturation as a ligand-independent transcription factor based upon its tissue abundance. Males expressed greater levels of ER mRNA than did females over the entire reproductive cycle, and this difference was most evident during recrudescence. ER mRNA levels were significantly elevated during recrudescence in males but not females. In conclusion, testosterone may have a role in male reproductive tract recrudescence; however, this putative activity is independent of a NR3C4-type androgen receptor. The ER also may function in male recrudescence, though apparently independent of 17beta-estradiol. The retinoid signaling pathway is discussed as a possible alternative hormone/receptor-mediated signaling pathway that regulates male recrudescence.}, number={1}, journal={GENERAL AND COMPARATIVE ENDOCRINOLOGY}, author={Sternberg, Robin M. and Hotchkiss, Andrew K. and LeBlanc, Gerald A.}, year={2008}, month={Mar}, pages={15–26} }
 @article{blystone_lambright_howdeshell_furr_sternberg_butterworth_durhan_makynen_ankley_wilson_et al._2007, title={Sensitivity of fetal rat testicular steroidogenesis to maternal prochloraz exposure and the underlying mechanism of inhibition}, volume={97}, ISSN={["1096-6080"]}, DOI={10.1093/toxsci/kfm055}, abstractNote={The fungicide prochloraz (PCZ) induces malformations in androgen-dependent tissues in male rats when administered during sex differentiation. The sensitivity of fetal testicular steroidogenesis to PCZ was investigated to test the hypothesis that the reported morphological effects from maternal exposure were associated with reduced testosterone synthesis. Pregnant Sprague-Dawley rats were dosed by gavage with 0, 7.8, 15.6, 31.3, 62.5, and 125 mg PCZ/kg/day (n = 8) from gestational day (GD) 14 to 18. On GD 18, the effects of PCZ on fetal steroidogenesis were assessed by measuring hormone production from ex vivo fetal testes after a 3-h incubation. Lastly, PCZ levels in amniotic fluid and maternal serum were measured using liquid chromatography/mass spectroscopy and correlated to the inhibition of steroidogenesis. Fetal progesterone and 17alpha-hydroxyprogesterone production levels were increased significantly at every PCZ dose, whereas testosterone levels were significantly decreased only at the two high doses. These results suggest that PCZ inhibits the conversion of progesterone to testosterone through the inhibition of CYP17. To test this hypothesis, PCZ effects on CYP17 gene expression and in vitro CYP17 hydroxylase activity were evaluated. PCZ had no effect on testicular CYP17 mRNA levels as measured by quantitative real-time polymersase chain reaction. However, microsomal CYP17 hydroxylase activity was significantly inhibited by the fungicide (K(i) = 865nM). Amniotic fluid PCZ concentrations ranged from 78 to 1512 ppb (207-4014nM) and testosterone production was reduced when PCZ reached approximately 500 ppb, which compares favorably with the determined CYP17 hydroxylase K(i) (326 ppb). These results demonstrate that PCZ lowers testicular testosterone synthesis by inhibiting CYP17 activity which likely contributes to the induced malformations in androgen-dependent tissues of male offspring.}, number={2}, journal={TOXICOLOGICAL SCIENCES}, author={Blystone, Chad R. and Lambright, Christy S. and Howdeshell, Kembra L. and Furr, Johnathan and Sternberg, Robin M. and Butterworth, Brian C. and Durhan, Elizabeth J. and Makynen, Elizabeth A. and Ankley, Gerald T. and Wilson, Vickie S. and et al.}, year={2007}, month={Jun}, pages={512–519} }
 @article{sternberg_leblanc_2006, title={Kinetic characterization of the inhibition of acyl coenzyme A: Steroid acyltransferases by tributyltin in the eastern mud snail (Ilyanassa obsoleta)}, volume={78}, ISSN={["1879-1514"]}, DOI={10.1016/j.aquatox.2006.03.004}, abstractNote={Exposure to tributyltin (TBT) has been causally associated with the global occurrence of a pseudohermaphroditic condition called imposex in neogastropod species. TBT elevates free testosterone levels in these organisms, and this upsurge in testosterone may be involved in the development of imposex. We investigated the ability of TBT to inhibit acyl coenzyme A:testosterone acyltransferase (ATAT) activity as well as microsomal acyl-coenzyme A:17β-estradiol acyltransferase (AEAT) in a neogastropod, the eastern mud snail Ilyanassa obsoleta as a mechanism by which TBT elevates free testosterone. TBT significantly inhibited both ATAT and AEAT activities in vitro at toxicologically relevant in vivo concentrations. Kinetic analyses revealed that TBT is a competitive inhibitor of ATAT (Ki = ∼9 μM) and is a weaker, noncompetitive inhibitor of AEAT (Ki = ∼31 μM). ATAT and AEAT activities associated with different microsome preparations were significantly correlated, and 17β-estradiol competitively inhibited the fatty acid esterification of testosterone suggesting that one enzyme is responsible for biotransforming both testosterone and 17β-estradiol to their corresponding fatty acid esters. Overall, the results of this study supply the much-needed mechanistic support for the hypothesis that TBT elevates free testosterone in neogastropods by inhibiting their major regulatory process for maintaining free testosterone homeostasis—the fatty acid esterification of testosterone.}, number={3}, journal={AQUATIC TOXICOLOGY}, author={Sternberg, Robin M. and LeBlanc, Gerald A.}, year={2006}, month={Jun}, pages={233–242} }
 @article{leblanc_gooding_sternberg_2005, title={Testosterone-fatty acid esterification: A unique target for the endocrine toxicity of tributyltin to gastropods}, volume={45}, ISSN={["1557-7023"]}, DOI={10.1093/icb/45.1.81}, abstractNote={Abstract Over the past thirty years, a global occurrence of sexual aberration has occurred whereby females among populations of prosobranch snails exhibit male sex characteristics. This condition, called imposex, has been causally associated with exposure to the biocide tributyltin. Tributyltin-exposed, imposex snails typically have elevated levels of testosterone which have led to the postulate that this endocrine dysfunction is responsible for imposex. This overview describes recent evidence that supports this postulate. Gastropods maintain circulating testosterone levels and administration of testosterone to females or castrates stimulates male sex differentiation in several snail species. Studies in the mud snail (Ilyanassa obsoleta) have shown that gastropods utilize a unique strategy for regulating free testosterone levels. Excess testosterone is converted to fatty acid esters by the action of a testosterone-inducible, high capacity/low affinity enzyme, acyl-CoA:testosterone acyl transferase, and stored within the organisms. Free testosterone levels are regulated during the reproductive cycle apparently due to changes in esterification/desterification suggesting that testosterone functions in the reproductive cycle of the organisms. Testosterone esterification provides a unique target in the testosterone regulatory machinery of snails that is altered by tributyltin. Indeed, imposex and free testosterone levels were elevated in field collected snails containing high tin levels, while testosterone-fatty acid ester pools were reduced in these organisms. These observations indicate that tributyltin elevates free testosterone by reducing the retention of testosterone as fatty acid-esters. This endocrine effect of tributyltin may be responsible for imposex.}, number={1}, journal={INTEGRATIVE AND COMPARATIVE BIOLOGY}, author={LeBlanc, GA and Gooding, MP and Sternberg, RM}, year={2005}, month={Feb}, pages={81–87} }