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Ecotoxicology (2010) 19:4–23 DOI 10.1007/s10646-009-0397-z

Environmental-endocrine control of reproductive maturation in gastropods: implications for the mechanism of tributyltininduced imposex in prosobranchs Robin M. Sternberg Æ Meredith P. Gooding Æ Andrew K. Hotchkiss Æ Gerald A. LeBlanc

Accepted: 20 July 2009 / Published online: 4 August 2009 Ó Springer Science+Business Media, LLC 2009

Abstract 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

R. M. Sternberg M. P. Gooding A. K. Hotchkiss G. A. LeBlanc (&) Department of Environmental and Molecular Toxicology, North Carolina State University, Campus Box 7633, Raleigh, NC 27695, USA e-mail: [email protected]

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. Keywords Mollusk Endocrinology Tributyltin Imposex Retinoid X-receptor

Present Address: R. M. Sternberg Mid-Continent Ecology Division, U.S. Environmental Protection Agency, ORD, NHEERL, Duluth, MN 55804, USA

Introduction

Present Address: M. P. Gooding U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, USA

In 1970, Blaber reported that a number of female dogwhelks (Nucella lapillus) collected from Plymouth, Millport, and Black Rock, United Kingdom, possessed ‘‘an outgrowth behind the right cephalic tentacle which occupied a similar position to the penis of the male’’ (Blaber 1970). Although dogwhelks are known to be gonochoristic (distinct sexes), the presence of this outgrowth was deemed a normal feature after female spawning (Blaber 1970).

Present Address: A. K. Hotchkiss Reproductive Toxicology Division, U.S. Environmental Protection Agency, ORD, NHEERL, Research Triangle Park, NC 27711, USA

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Environmental-endocrine control of reproductive maturation in gastropods

However, one year later Smith noted a similar penis-like structure as well as a convolution of the ovarian duct to form a ‘‘stunted mimic of a seminal vesicle’’ on female American (eastern) mud snails (Nassarius obsoletus; now Ilyanassa obsoleta) collected from Southport and Westport, Connecticut, USA, and designated the phenomenon as ‘‘imposex’’ because ‘‘it appears as a superimposition of male characters onto un-parasitized and parasitized females’’ (Smith 1971). Imposexed females typically possessed one or more organs associated with the male reproductive tract in addition to the normal female reproductive anatomy. These almost simultaneously-published reports of a similar sexual aberration in two different species of neogastropods collected from locations separated by thousands of miles signaled the possibility of some widespread phenomenon to which the snails were particularly susceptible. By the late 1970’s, imposex had been recognized in at least 34 marine gastropod species (Jenner 1979). One decade later, at least 100 species were known to exhibit imposex (Fioroni et al. 1991). Currently, imposex is recognized as a truly global phenomenon with at least 195 species of prosobranch gastropods known to be affected (Fioroni et al. 1991; Horiguchi et al. 1997; Shi et al. 2005). Imposex gastropods have been documented along the coasts of Scotland (Blaber 1970), England (Gibbs et al. 1987), Brittany (Huet et al. 1995), Italy (Chiavarini et al. 2003), The Netherlands (Ten Hallers-Tjabbes et al. 1994), Portugal (Vasconcelos et al. 2006), Greenland (Strand and Asmund 2003; Strand et al. 2006), Iceland (Svavarsson et al. 2001), South Africa (Marshall and Rajkumar 2003), Japan (Horiguchi et al. 1994), Singapore (Tan 1997), Thailand (Bech 2002), Australia (Wilson et al. 1993), New Zealand (Smith 1996), Canada (Bright and Ellis 1990), United States (Curtis and Barse 1990; Gooding et al. 2003), and Chile (Gooding et al. 1999), among others. Imposex appears to be an irreversible condition in most species suggesting that this sexual aberration could have potential long-term impacts on the fitness of gastropod populations (Foale 1993; Stroben et al. 1992). Indeed, some gastropod populations with a 100% incidence of imposex have been eradicated with only empty shells to indicate previous population ranges (Minchin et al. 1996). The impact of imposex on the fitness of gastropod populations depends on the reproductive strategy of the species. Some species possess a mobile larval stage that allows for repopulation of affected areas whereas other species exhibit direct development from egg to snail. The dogwhelk (N. lapillus) has no larval stage for repopulation resulting in local extinctions of this species at sites with a high degree of imposex (Bryan et al. 1986; Gibbs et al. 1991a; Huet et al. 1996a). Reproductive impairments associated with imposex vary according to the species. In some gastropods such as

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N. lapillus and other Nucella species, the vas deferens grows over the palleal oviduct preventing the deposition of egg capsules; the egg capsules accumulate in the oviduct causing sterilization (Bright and Ellis 1990; Gibbs et al. 1987; Oehlmann et al. 1991). In other species such as Urosalpinx cinerea, the bursa copulatrix and the capsule gland split to prevent copulation and the formation of egg capsules in the oviduct (Gibbs et al. 1991b). In I. obsoleta, the female may possess a penis that approaches the size of the male, but reproduction is seemingly unaffected (Smith 1980). Early investigations into the phenomenon of imposex yielded speculation that this condition simply reflected a transition stage from one sex to another among sequential hermaphrodites previously considered to be dioecious (Smith 1967). However, experiments specifically designed to test this possibility failed to reveal any deviation from gonochorism in these snails (Jenner 1979). Rather, imposex was deemed to be a form of pseudohermaphroditism, similar to that described for some vertebrates (Jenner 1979). Additional experiments and observations suggested that this manifestation of pseudohermaphroditism in snails is environmentally controlled. First, there existed natural variability in the occurrence of the phenomenon among different snail populations (Jenner 1979; Smith 1971, 1981a). Furthermore, imposex could be induced or diminished in experiments involving the transfer of snails from populations with a low incidence of imposex to locations with populations showing a high incidence of imposex and vice versa (Jenner 1979). In turn, a positive correlation between the occurrence of imposex and proximity to yacht basins and marinas was reported (Smith 1981b). Follow-up experiments showed that imposex could be induced in snails transferred from a distant, clean locality to a yacht basin and diminished, but not lost, in snails transported from a marina environment to a clean site (Smith 1981b). Subsequently, snails sampled from a pristine location were exposed to marine antifouling paint under static laboratory conditions for 75 days (Smith 1981c). Imposex significantly increased among these exposed snails whereas no increase in imposex was observed among snails that were sampled from the same initial location but maintained only in seawater for 75 days. Finally, exposure of snails to low concentrations of the antifoulant tributyltin (TBT) was shown to significantly induce imposex (Smith 1981d). TBT has been shown to induce imposex in neogastropods at infinitesimal concentrations below 1 ng/l (as Sn) (Bryan et al. 1987; Gibbs et al. 1988; Gooding et al. 2003). TBT concentrations of this magnitude and greater have been measured in the environment where imposex females occur (LeBlanc and Bain 1997; Svavarsson et al. 2001). Thus, ample evidence implicating TBT as the cause of imposex among prosobranch gastropods exists.

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Tributyltin TBT has been used as wood preservative in industry and agriculture, as a stabilizer in PVC plastics manufacturing, and, most prominently, as an antifouling agent in paints (Fent 1996). As a component of antifouling paints, TBT has been used primarily on ship hulls, docks, buoys, and fishnets in the marine environment where it slowly leaches into the water to create a highly effective barrier against fouling organisms such as barnacles, algae, bacteria, and tubeworms. The fouling of ship hulls by these organisms increases hydrodynamic drag resulting in higher fuel costs. Fuel savings associated with the use of TBT-based paints are estimated to be a hundred million to one billion dollars over a 5–7-year period (Champ 2000). TBT is commonly manufactured as TBT oxide, TBT fluoride, and TBT chloride and is environmentally degraded by debutylation to form dibutyltin (DBT), monobutyltin (MBT), and ultimately inorganic tin. Degradation occurs primarily through biotic processes, and the rate of debutylation in sediments is dependent upon microbial activity (Lee et al. 1989; Yonezawa et al. 1994). The halflife of TBT in sediments has been shown to range from 6 months to 8.7 years (Maguire and Katz 1985; Smith 1996; Stang and Seligman 1986; Stewart and Thompson 1997; Williams et al. 1999). TBT bioaccumulates in gastropods with the highest concentrations typically being measured in the digestive/reproductive complex (Bryan et al. 1986; Mensink et al. 1996; Skarphe´dinsdo´ttir et al. 1996; Stroben et al. 1992) at levels up to 100,000-fold greater than those measured in the aqueous environment (Bryan et al. 1987; Bryan et al. 1989). Gastropods biotransform TBT using debutylation reactions mediated by cytochrome P450 enzymes (Fent 1996). However, P450mediated detoxification/elimination reactions appear to be restricted in molluscs (Gooding and LeBlanc 2001; Livingstone et al. 1989) resulting in limited biotransformation of TBT (Bryan et al. 1993; Lee 1991) and increased accumulation. This propensity to accumulate TBT may contribute to the high sensitivity of gastropods to TBT toxicity. Growing concerns about the decline of neogastropod populations led individual countries to pass legislation limiting the use of TBT and establishing environmental quality standards during the 1980s and early 1990s (Champ 2000). Finally in 1998, the Marine Environmental Protection Committee (MEPC) of the International Maritime Organization (IMO) voted to impose a worldwide prohibition on the application and presence of TBT within 5 (1 January 2003) and 10 years (1 January 2008), respectively (Champ 2000). This total ban of TBT in territorial waters of contracting parties entered into force on the 17th of September 2008. Consensus as to whether bans on the use of TBT over the past

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two and half decades have improved the viability of neogastropod populations seems mostly positive. The results of field surveys conducted over the past 15 years indicate that although imposex is still widely prevalent, there has been a general decline in the incidence and severity of the pseudohermaphroditic condition (Gibson and Wilson 2003; Huet et al. 2004; Jorunsdottir et al. 2005; Reitsema et al. 2002; Svavarsson 2000) as well as the recolonization of sites where neogastropod populations had been locally extinct (Birchenough et al. 2002; Huet et al. 2004). TBT: an endocrine disruptor? TBT-induced imposex in gastropods is often cited as a prime example of environmental endocrine disruption (LeBlanc et al. 1999; Matthiessen and Gibbs 1998; Morcillo and Porte 1999; 2000; Schulte-Oehlmann et al. 2000), and several endocrine-related hypotheses have been proposed to define possible mechanisms by which TBT causes imposex. However, our general lack of understanding of the endocrinology of sexual differentiation and development in gastropods hampers any meaningful deductions regarding the mechanism of TBT-induced imposex. In this review, we will evaluate what is currently known about the environmental and endocrine signals that regulate sexual differentiation/development in gastropods as well as the known effects of TBT on relevant endocrine parameters in molluscs. This information then will be used to propose a rational model for the mechanism by which TBT causes imposex.

Sexual differentiation and reproduction in gastropods Gastropods utilize a variety of reproductive strategies including gonochorism (unisex), protandritic hermaphroditism (a male phase precedes a female phase), protygynitic hermaphroditism (a female phase precedes a male phase), reversible hermaphroditism (successive changing between male and female phases), simultaneous hermaphroditism, and parthenogenesis. Imposex appears primarily to affect gonochoristic species, specifically neogastropods. However, imposex also has been described in some mesogastropods (Fioroni et al. 1991; Janer et al. 2006a). TBT also has been shown to induce intersex, a disturbance in the phenotypic sex determination of the female gonad and genital tract, in the periwinkle Littorina littorea (Bauer et al. 1995). The apparent unique susceptibility of gonochoristic snails to TBT-induced imposex or intersex may be due to the difficulty in identifying these conditions in hermaphroditic species. The regrowth of the reproductive tract and secondary sex organs after a period of reproductive dormancy, known


as reproductive recrudescence, is seasonally regulated in some gastropods (Hotchkiss et al. 2008; Sternberg et al. 2008a). Environmental cues likely stimulate neuro-endocrine cascades that orchestrate male and female sex organ differentiation as well as oogenesis, spermatogenesis, and copulatory behavior. Environmental factors influencing sexual differentiation and reproduction in gastropods Animals inhabiting non-tropical environments undergo seasonal changes in reproductive function to improve their chances of survival and successful reproduction during periods of increased environmental hardship. For example, food shortage and decreased temperature during the winter can make this season particularly difficult to survive for some species. Therefore, adaptive physiological, behavioral, and morphological changes have evolved that result in a positive energy balance in the organisms throughout the year and help ensure that offspring are born during a time when their chances of survival are maximized. Several environmental cues that initiate or modify reproductive processes in gastropods have been identified (Fig. 1). Photoperiod Seasonal variations of day length can be detected by organisms and lead to associated adaptative changes in individuals. In vertebrates, melatonin has been identified as one mediator of photoperiodic signaling (Goldman 2001). Melatonin is a 5-methoxyindole secreted primarily by the pineal gland of vertebrates (Goldman 2001). Melatonin interacts with membrane-bound G-coupled protein receptors (Reppert 1997). Melatonin levels cycle on a daily basis

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whereby concentrations are typically elevated at night and reduced during the day (Goldman 2001). Thus, the duration of the melatonin signal provides information about day length and allows animals to distinguish the seasons. Melatonin is widespread among organisms (Kumar 1996) and has been identified in invertebrate tissues such as the ocular tentacles, visceral ganglion, brain, eyes, and the cerebral ganglia (Blanc et al. 2003; Vivien-Roels and Pevet 1993; Wayne 2001). In most invertebrates studied to date, melatonin is elevated during the night (Blanc et al. 2003) suggesting a potential role in daily and annual rhythms similar to that observed for vertebrates. In addition, other 5-methoxyindoles may play a role in transducing photoperiodic information in gastropods. One of these, 5-methoxytryptophol (5-ML), has been shown to oscillate rhythmically in gastropods (Blanc et al. 2003). At this time, it is unclear what role 5-ML may play in circadian or circannual rhythms. Many gastropods undergo seasonal reproductive development and breeding. For example, the giant garden slug (Limax maximus) undergoes reproductive maturation in spring and summer. Slugs raised in short photoperiods remain reproductively immature whereas slugs exposed to long photoperiods develop mature reproductive systems (Sokolove et al. 1984). This induced reproductive differentiation appears to be stimulated by long-day expression of some endocrine factor (Melrose et al. 1983). Specifically, the cerebral ganglia receive light input via putative photoreceptors and produce maturation/gonadotropic factors in response to long days (Melrose et al. 1983). In the common garden snail (Helix aspersa), melatonin is elevated at night, but concentrations of 5-ML show a much greater elevation during the dark phase (Blanc et al. 2003). Therefore 5-ML may play an important role in the circadian and seasonal rhythms of this and other gastropod species. Photoperiod modulates the neuroscretory activity of canopy cells of the great pond snail (Lymnaea stagnalis, van Minnen and Reichelt 1980), and the long days of summer stimulate reproductive maturation and breeding (Blanc et al. 2003; Bohlken and Joosse 1982; Vivien-Roels and Pevet 1993; Wayne 2001). Similarly, in the freshwater snail Biomphalaria glabrata reproductive rates are highest under constant illumination and are inhibited under constant darkness (Barbosa et al. 1987). While photoperiod appears to function as the major seasonal regulator of reproductive recrudescence, other environmental cues contribute to the precise timing of breeding and reproduction. Temperature

Fig. 1 Influence of environmental cues on the control of reproduction in gastropods

Temperature is an important environmental cue triggering the onset of breeding in some species of molluscs. For example, changes in water temperature affect egg laying

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of the intertidal sea hare (Aplysia californica), with a drop in water temperature resulting in decreased egg-laying behavior (Wayne and Block 1992). Wayne (2001) hypothesized that this reduction in behavior occurs as the result of signaling either at the level of the bag cells via altered cAMP levels to control egg-laying hormone (ELH) release or at the level of the cerebral ganglia via the transmission of electrical signals to the bag cells. Studies examining the direct effect of temperature on ovotestes have demonstrated that changes in temperature have little or no effect on the responsiveness of the ovotestis to hormones, potentially implicating neuroendocrine mechanisms (Wayne 2001). In the apple snail (Pomacea canaliculata), a seasonal increase in water temperature is critical for the onset of breeding; lower water temperatures cause a later onset of breeding or a cessation of copulation and spawning (Albrecht et al. 1999). Depending on the photoperiod, exposure to cold water (8°C) can reduce or completely inhibit egg laying in L. stagnalis (Wayne 2001). The freshwater species B. glabrata displays the greatest reproductive rates when exposed to temperatures between 20 and 25°C (Barbosa et al. 1987).

Nutrition Food availability has been shown to influence sexual differentiation and breeding in gastropods. For mud snails (I. obsoleta), food consists of periphyton, macroalgae, and detritus (Kelaher et al. 2003; Giannotti and McGlathery 2001). These food sources vary seasonally (Kelaher et al. 2003) and potentially may play a role in the onset of reproductive recrudescence and breeding. Although food availability in many regions co-varies with changes in photoperiod and temperature, experiments have begun to establish which factors are most important for reproductive differentiation and function (Wayne 2001). In certain gastropod species, the quantity of food may trigger the onset of breeding. Although the possibility of seasonal reproduction being stimulated by nutrition exists, most studies have found that nutrition does not act singly on the reproductive axis, but rather, serves as a mediator of breeding (Dogterom et al. 1985; Wayne 2001). Food restriction can slow the onset of breeding in the apple snail (P. canaliculata) (Albrecht et al. 1999). The amount of assimilated food stimulates the onset of the egg-laying season for L. stagnalis (Dogterom et al. 1985). Food availability may affect reproduction by altering the production of a hormone by the neuroendocrine caudodorsal cells that stimulate ovulation (Dogterom et al. 1984). Upon re-feeding, the activity of these cells returns, and the snails are able to resume breeding (Dogterom et al. 1984).

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Parasitism Trematode parasites (e.g., Trichobilharzia, Prosorhynchus, Neophasis) modulate neuroendocrine pathways in host molluscs resulting in the diminution of sexual differentiation and function. In at least some species, parasites typically occupy the gonads (Tetreault et al. 2000) and stimulate the release of an anti-gonadotropic neuroendocrine substance called schistosomin (Schallig et al. 1991). In L. stagnalis, schistosomin consists of a 79-amino acid, single chain peptide that is stored by neurosecretory light green cells located in the cerebral ganglia of the central nervous system (Hordijk et al. 1991a). This peptide acts on cells of both the central and peripheral nervous systems (de Jong-Brink et al. 1992) to cause reduced sexual differentiation (Tetreault et al. 2000), reduced function of the reproductive organs (Hordijk et al. 1991b), and enhanced somatic growth (de Jong-Brink et al. 1992). Schistosomin functions at least in part by inhibiting the binding of gonadotropins to their cell surface receptors (Hordijk et al. 1991a). In the aquatic snail, Lymnaea elodes, infection by the parasitic worm, Echinostoma revolutum, as well as diet, can have adverse effects on both reproduction and survival (Sandland and Minchella 2003). Hormonal control of sexual differentiation and reproduction in gastropods Anatomy of the reproductive neuro-endocrine cascade Neogastropods are evolutionarily recent prosobranch gastropods in which individuals are of distinct sexes (dioecious), and the sex of an individual is irreversible (Gibbs and Bryan 1996). The male reproductive tract of neogastropods consists primarily of a testis that produces sperm, a prostate gland that produces seminal fluid, a vas deferens that transports sperm from the testis, and a penis that delivers sperm to the female (Fig. 2) (Gibbs et al. 1991a; Gibbs et al. 1987). The female reproductive system includes an ovary that produces eggs, an oviduct that transports eggs from the ovary, and an albumen/capsule gland complex that packages the eggs for release into the environment (Fig. 2) (Fretter and Graham 1976; Gibbs et al. 1991a). Most components of the reproductive system of neogastropods develop in preparation for reproduction, i.e., recrudescence, and regress upon completion of reproduction, i.e., senescence (Fretter and Graham 1976). Elements of the endocrine cascade that are responsible for recrudescence and senescence of the reproductive system of neogastropods remain poorly understood. Neurosecretory loci of the central nervous system form the core of the molluscan endocrine system (Geraerts et al. 1988; Nassel 1996). Consequently, hormonal control of

Environmental-endocrine control of reproductive maturation in gastropods Fig. 2 Reproductive tract of the female (a) and male (b) neogastropod Ilyanassa obsoleta

A posterior oviduct

anterior oviduct

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B mantle skirt mantle skirt

vagina

ovary albumen gland

capsule gland

reproductive physiology in molluscs seems to be heavily dependent upon neurohormones, specifically neuropeptides (LeBlanc et al. 1999). Indeed, evaluations of molluscan reproductive endocrinology have focused largely on the neuropeptides that control egg production, oviposition, male copulatory behavior, and protandric sex reversal in gastropods (Table 1; Geraerts et al. 1988; Joosse 1988; Lagadic et al. 2007; Linacre et al. 1990; Saunders et al. 1992; Vreugdenhil et al. 1988). The dorsal bodies appear to have a major role in seasonally-regulated neuro-endocrine cascades in female pulmonate gastropods. Dorsal bodies are endocrine glands that regulate various aspects of female reproduction such as yolk protein synthesis by the albumen gland and oocyte maturation (Joosse 1988). Cells of the dorsal bodies are organized in groups on both sides of the perineurium of the cerebral ganglia allowing for direct innervation by neurosecretory cells of these ganglia (Saleuddin et al. 1997; Saleuddin and Ashton 1996). Physiological characteristics of dorsal bodies suggest that these glands are inactive in virgin snails, are activated by mating, and remain active during the reproductive phase of the organisms (Joosse and Geraerts 1983; Khan et al. 1990; Saleuddin et al. 1997). Ultrastructural analyses have suggested that the juxtaganglionar organ of prosobranch snails may be analogous to the dorsal bodies (Switzer-Dunlap 1987). Bag cells are secretory cells of the neuro-endocrine system that function in the regulation of egg laying in prosobranch gastropods (Kupfermann and Kandel 1970). In response to neurological stimulation, bag cells secrete the peptide egg laying hormone (ELH). ELH is actually a prohormone that is processed into a suite of hormones that coordinately regulate aspects of egg-laying activities and behaviors (Hatcher and Sweedler 2008). Hormones of the reproductive neuro-endocrine cascade Neuro-endocrine hormones A putative neurohormone secreted by the cerebral ganglia of common limpets (Patella vulgata) was shown to cause male differentiation

testis

vas deferens

prostate

penis

(Choquet 1971). Furthermore, a product of the pedal ganglia of slipper limpets (Crepidula fornicata) was shown to stimulate penis differentiation near the right tentacle of prosobranch gastropods (Le Gall and Le Gall 1975; Le Gall 1981). This neurohormone from the pedal ganglia appeared to accumulate in the right tentacle. The right tentacle, under the stimulatory control of the pedal ganglia, then initiated penis differentiation, presumably through the secretion of the ultimate hormone (Joosse and Geraerts 1983). The cerebral ganglia and pedal ganglia may provide two sources for the same penultimate masculinizing hormone. Alternatively, each tissue might produce a distinct hormone that contributes to a common endocrine cascade. Although these observations were made two to three decades ago, little additional information has accrued regarding the nature of these masculinizing hormones. The neuropeptide APGWamide is secreted by the anteromedial region of the right cerebral ganglion, the right pedal ganglion, the copulatory duct, and penial complex of males (de Lange and van Minnen 1998; Fan et al. 1997). APGWamide contributes to male mating behavior (Koene et al. 2000) as well as the activity of the penis retractor muscle and muscles involved in ejaculation (Li et al. 1992; Vangolen et al. 1995). In addition, APGWamide reportedly stimulated penis growth in female I. obsoleta (Oberdorster and McClellan-Green 2000). However, Castro et al. (2007) were unable to reproduce this effect in N. lapillus. The observations that both APGWamide and the masculinizing neurohormone described above are secreted by the cerebral and pedal ganglia provide enticing evidence for APGW amide being the neuro-endocrine, masculinizing hormone of neogastropods. Conopressin is a peptide hormone that stimulates contraction of the vas deferens during ejaculation (van Kesteren et al. 1995). APGWamide inhibits this action of conopression. Peptide hormones that originate from the central nervous system and bag cells have been implicated in the regulation of egg laying. These include caudodorsal cell hormone, FMRFamide, and egg laying hormone (Hatcher and Sweedler 2008; Saunders et al. 1992; Vreugdenhil et al. 1988). A gonadotropin releasing

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Table 1 Purported reproductive hormones in gastropods Hormone

Source

Function

Melatonin, 5methoxytryptophol

Cerebral ganglia

Transduce photoperiodic signals

APGW amide

Cerebral and pedal ganglia, male reproductive tract

Control of male reproductive behavior and ejaculation

Conopressin Caudodorsal cell hormone

Vas deferens Central nervous system (CNS)

Control of ejaculation Control of egg laying

FMRF amide

CNS

Control of egg laying

GNRH

CNS; cerebral, pedal, and abdominal ganglia; gonads

Unknown, possibly egg laying

Egg-laying hormone

Bag cells

Control of egg laying

20-hydroxyecdysone

Dorsal bodies

Stimulation of oogenesis and oocyte maturation

Progesterone

Unknown

Stimulation of oogenesis

Testosterone

Unknown

Maturation of the male reproductive tract and spermatogenesis; development of male secondary sex characteristics; stimulation of oocyte maturation

17b-estradiol

Unknown

Stimulation of oogenesis

9-cis retinoic acid

Unknown

Male reproductive tract development

References to the information are provided in the text

hormone (GNRH) gene has been cloned from Aplysia that bears significant molecular architectural similarity to the octopus GNRH (Zhang et al. 2008). This gene is expressed throughout the central and peripheral nervous systems and in the gonads of Aplysia. Though presumed to function in the control of aspects of reproduction, the precise role of this hormone remains unknown. Endocrine hormones Ecdysteroids. Mukai et al. (2001) isolated a heat and protease resistant secretory product of the dorsal bodies of Helisoma duryi that shared immunochemical and chromatographic characteristics with ecdysteroids. These investigators further established that exogenously-administered 20-hydroxyecdysone stimulated egg mass production, oocyte maturation, and polysaccharide synthesis by the albumen gland in H. duryi. The stimulation of polysaccharide synthesis by 20-hydroxyecdysone also was demonstrated in H. aspersa (Bride et al. 1991). Thus, limited evidence suggests a role for ecdysteroids secreted by the dorsal bodies in female reproduction. Evidence for a role of ecdysteroids in male reproduction is lacking. Sex steroids. Vertebrate-type sex steroids including estrogens (estrone, 17b-estradiol, estriol) and androgens (androstenedione, androsterone, dehydroepiandrosterone) have been definitively identified in gastropods using gas chromatography-mass spectroscopy (GC-MS) (Le Guellec et al. 1987). In addition, sex steroids have been quantified via less-specific immunoassays (e.g., radioimmunoassay) in multiple tissues of gastropods (Bose et al. 1997;

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Gooding and LeBlanc 2004; Janer et al. 2006b; Kavaliers et al. 2000; Le Guellec et al. 1987; Sternberg et al. 2008a; Takeda 1980). However, the mere detection of sex steroids in a gastropod provides no insight as to whether these biomolecules actually function as hormones in these organisms. A few studies have attempted to supply evidence for a physiological role of sex steroids in molluscan reproduction by measuring sex steroid levels in a gastropod species over the reproductive cycle and examining the resulting profiles for relationships among sex steroid concentrations, sex, and reproductive status. In both sexes of I. obsoleta, free testosterone concentrations as well as the percentage of total testosterone found in the free, non-esterified form followed the same trajectories over the reproductive cycle and spiked during November and April, months that corresponded to the onset and end of the reproductive period, respectively (Gooding and LeBlanc 2004). In a more detailed study with the same species, Sternberg et al. (Sternberg et al. 2008a) reported that total and free testosterone and 17b-estradiol levels were comparable between females and males throughout the year. When testosterone levels were grouped according to reproductive status, females had higher free testosterone levels than males during reproductive dormancy, and a decreasing trend in both total and free testosterone was apparent from recrudescence to dormancy in males but not females (Sternberg et al. 2008a). This latter trend suggests that testosterone may have a role in male reproductive tract recrudescence. Somewhat differently, when 17b-estradiol


levels were grouped according to reproductive status, a depression in both total and free 17b-estradiol from dormancy to recrudescence was apparent in females but not males (Sternberg et al. 2008a). Nevertheless, the emergence of a sex difference in testosterone and 17b-estradiol levels when analyzed by reproductive status was an artifact of the differences in the duration of the recrudescent and dormant periods between females and males (Sternberg et al. 2008a). Therefore, the investigators concluded that sex-specific differences in testosterone and 17b-estradiol levels through reproductive development provided no strong evidence for a role of these hormones in gastropod recrudescence. Numerous studies have examined the ability of gastropods to convert various precursors to progestagens, androgens, and estrogens in vitro and in vivo (Bose et al. 1997; de Jong-Brink et al. 1981; Gottfried 1970; Gottfried and Dorfman 1969; Gottfried and Dorfman 1970; Gottfried and Lusis 1966; Janer et al. 2005a, b, 2006a; Krusch et al. 1979; Le Guellec et al. 1987; Lehoux and Williams 1971; Lupo Di Prisco and Dessi’ Fulgheri 1975; Lupo Di Prisco et al. 1973; Lyssimachou et al. 2009; Oberdorster et al. 1998; Ronis and Mason 1996; Teshima and Kanazawa 1971). Almost all of the enzyme activities in vertebrate steroidogenesis have been detected in at least one gastropod species indicating that gastropods have the enzymatic machinery for synthesizing sex steroids. Gastropods exhibit a unique means of regulating free sex steroid levels. Gooding and LeBlanc (2001) first showed that mud snails (I. obsoleta) primarily biotransform exogenously-administered testosterone in vivo to apolar conjugates that are retained by the organism as opposed to polar metabolites that are excretable. Enzymatic hydrolysis of the most prevalent apolar metabolite indicated that the snails conjugate testosterone to free fatty acids to form a testosterone-fatty acid ester (Gooding and LeBlanc 2001). The reaction was confirmed in vitro by incubating microsomes prepared from mud snail tissue with free testosterone and oleoyl or palmitoyl coenzyme A (Gooding and LeBlanc 2001). Additional experiments and analyses by these researchers revealed that the fatty acid esterification/ de-esterification of testosterone is the major regulatory process by which I. obsoleta maintains free testosterone homeostasis (Gooding and LeBlanc 2004). The microsomal enzyme that conjugates testosterone to fatty acids has been designated as acyl coenzyme A:testosterone acyltransferase (ATAT). Since these initial observations, this same process for regulating free hormone levels has been documented in other gastropods for both testosterone and 17b-estradiol (Janer et al. 2005c, 2006b; Santos et al. 2005; Sternberg et al. 2008a). Collectively, these metabolic studies indicate that gastropods are capable of

11

biosynthesizing and biotransforming the sex steroids that are present in their tissues. Some studies have attempted to provide support for the hypothesis that vertebrate-like sex steroids are functional in gastropods by evaluating the physiological responses of these organisms to treatment with these hormones. The effects of steroidal progestagens have been examined in a few of these studies. In vivo injection of progesterone stimulated oogenesis but not spermatogenesis in the snail Helix pomatia (Csaba and Bierbauer 1979). A greater number of studies have examined the effects of steroidal androgens on gastropods. In vivo administration of various androgens via aqueous exposure or injection has been shown to stimulate C-reactive protein synthesis in the snail Achatina fulica (Bose and Bhattacharya 2000); induce maturation of the testicular elements of the gonad and precocious spermatogenesis in the banana slug Ariolimax californicus (Gottfried 1970); increase the rate of egg development in the slugs Deroceras reticulatum and Limax flavus (Takeda 1979); induce head-wart development and recovery from castration in the snail Euhadra peliomphala (Takeda 1980); stimulate oogenesis but not spermatogenesis in H. pomatia (Csaba and Bierbauer 1979); induce male phenotypic characteristics in I. obsoleta (Oberdorster and McClellan-Green 2000), the ramshorn snail Marisa cornuarietis (Tillmann et al. 2001), and the dogwhelks N. lapillus and Nassarius reticulatus (Bettin et al. 1996; Spooner et al. 1991); and accelerate spermatogenesis in the snail Theba pisana (Sakr et al. 1992). Additional studies have examined the effects of steroidal estrogens on gastropods. In vivo aqueous exposure to or injection of estrogens stimulated egg laying but decreased the rate of egg development in D. reticulatum and L. flavus (Takeda 1979); stimulated oogenesis but not spermatogenesis in H. pomatia (Csaba and Bierbauer 1979); and stimulated egg/embryo production in the snails Potamopyrgus antipodarum and M. cornuarietis (Jobling et al. 2004; Oehlmann et al. 2006). In some cases, the same physiological response was observed after treatment with progestagens, androgens, and estrogens. For example, testosterone, progesterone, and estrone acetate all stimulated oogenesis but not spermatogenesis in H. pomatia (Csaba and Bierbauer 1979). Methyltestosterone and ethinylestradiol both increased the development of imposex in M. cornuarietis (Tillmann et al. 2001). The similarity in outcome generated by treatment with different sex steroids may indicate that: the response itself was non-specific or the steroids, at high exposure levels, all activated a common receptor target. Indeed, responses suggesting a possible function for sex steroids were typically evident at concentrations that exceed physiological relevance.

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If sex steroids do have a function in the reproductive physiology of gastropods, then the missing link becomes how androgens, estrogens, and progestagens function at the cellular and molecular levels to play a role in reproductive signaling. These hormones could use classical steroid hormone signaling that requires the involvement of nuclear receptor proteins. Alternatively, sex steroids in gastropods could use membrane-bound receptors to elicit the observed effects. Efforts to identify an NR3C4-type androgen receptor in I. obsoleta using targeted, degenerate RT-PCR were unsuccessful (Sternberg et al. 2008a). In addition, phylogenetic analysis has established that the NR3C subfamily of nuclear receptors (including androgen and progesterone receptors) emerged during vertebrate evolution (Laudet and Gronemeyer 2002; Thornton 2001). In contrast, an NR3A-like estrogen receptor (ER) that bears a high degree of homology to vertebrate ERs has been cloned, sequenced, expressed, and experimentally characterized in molluscs including the sea hare Aplysia californica (Thornton et al. 2003), the rock shell Thais clavigera (Kajiwara et al. 2006), the ramshorn snail M. cornuarietis (Bannister et al. 2007) and the mud snail I. obsoleta (Sternberg et al. 2008a). Interestingly, the molluscan ER does not appear capable of binding estrogens (Bannister et al. 2007; Kajiwara et al. 2006; Keay et al. 2006; Thornton 2001) but may be a constitutive transcriptional activator via estrogen response elements (Kajiwara et al. 2006; Keay et al. 2006; Thornton 2001). Thornton (2001) reconstructed, synthesized, and experimentally characterized the ancestral protein from which all existent nuclear steroid receptors may have evolved and found it to have estrogen receptor-like functionality, i.e., the ligand-binding domain of the ancestral steroid receptor bound estradiol specifically, albeit with lower affinity than that of human ERa, and activated transcription in the presence of estrogens. Thus, the identified molluscan ER probably lost the ability to bind estradiol and gained constitutive activity deep in the molluscan lineage and probably does not interact directly with estrogens. Collectively, the absence of androgen and progesterone receptors and the presence of an estrogen-unresponsive estrogen receptor suggest that the sex steroids detected in gastropods, if functional, must act via some other signaling pathway. Retinoids. A retinoid X receptor (RXR; NR2B subfamily) that bears significant homology to other invertebrate and vertebrate RXRs has been cloned and sequenced in the rock shell T. clavigera (Nishikawa et al. 2004), the freshwater bloodfluke planorb Biomphalaria glabrata (Bouton et al. 2005), the dogwhelk N. lapillus (Castro et al. 2007), and the mud snail I. obsoleta (Sternberg et al. 2008b). Further experimental characterization indicated that the ligandbinding domain of the RXR of T. clavigera and N. lapillus binds the putative RXR ligand 9-cis retinoic acid with high

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affinity in vitro (Castro et al. 2007; Nishikawa et al. 2004). In addition, the B. glabrata RXR transactivated transcription of a reporter gene equipped with an RXR response element from the Apo-A1 promotor in the presence of 9-cis retinoic acid (Bouton et al. 2005). Finally, injections of 9-cis retinoic acid induced imposex in T. clavigera (Nishikawa et al. 2004) and N. lapillus (Castro et al. 2007). Interestingly, (Oehlmann et al. 2007) were unable to induce imposex in N. lapillus with 9-cis retinoic acid. However, this discrepancy in results may have been due to the mode of retinoid delivery employed by the latter investigators (Castro et al. 2007). These collective observations suggest that RXR signaling may contribute to the regulation of normal male reproductive tract recrudescence in gastropods. If so, then the RXR should be expressed in concert with this phenomenon. Sternberg et al. (Sternberg et al. 2008b) quantified seasonal levels of RXR mRNA in I. obsoleta and found that the expression of RXR is elevated in concert with recrudescence in both sexes even though males begin to recrudesce three months in advance of females. This result suggested that RXR-mediated signaling may stimulate sex-specific recrudescence during critical temporal windows.

Effects of TBT on endocrine parameters of gastropods Several endocrine-based hypotheses have been proposed for how TBT causes imposex. However, the precise mechanism remains elusive. Nevertheless, exploration of the various purported mechanisms of TBT action has resulted in significant advances in the understanding of the endocrine system and reproductive physiology of gastropods. Hypotheses for the mechanism of TBT-induced imposex are critically reviewed below. Elevated testosterone hypothesis The most widely touted mechanism for TBT-induced imposex was formulated as a result of the observation that imposex-affected females consistently exhibit elevated free testosterone titres compared to non-imposexed females (Bettin et al. 1996; Gooding et al. 2003; Santos et al. 2005; Spooner et al. 1991). The hypothesis proposes that TBT increases free testosterone levels, and this upsurge in free testosterone initiates a biochemical cascade with the ultimate consequence being the imposition of male sex characteristics onto female neogastropods. However, in at least one laboratory experiment, imposex was evident prior to the detection of a statistically significant increase in testosterone titers (Bettin et al. 1996). Empirical support for this hypothesis can be found in studies where exogenously administered testosterone increased the incidence of imposex in female dogwhelks, N. lapillus (Bettin et al. 1996; Spooner et al.


1991) and N. reticulatus (Bettin et al. 1996), mud snails (I. obsoleta) (Oberdorster and McClellan-Green 2000), and ramshorn snails (M. cornuarietis) (Tillmann et al. 2001). Furthermore, exposure to the androgen receptor antagonist cyproterone acetate (CPA) alone reduced accessory sex organs and penis length in immature male M. cornuarietis and adult male N. lapillus and N. reticulatus; and CPA attenuated TBT’s ability to induce imposex in these three species (Tillmann et al. 2001). For this hypothesis to be accepted: (1) the means by which TBT increases testosterone must be established; and (2) the underlying assumption that testosterone functions in processes related to the development of the reproductive tract in neogastropods must be verified.

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17b-estradiol in TBT-exposed female dogwhelks (N. lapillus) were unchanged (Bettin et al. 1996; Spooner et al. 1991) or elevated (Santos et al. 2005) when compared to control females. In addition, no differences in aromatase activity were found between imposexed and control populations of purple dye murex (Bolinus brandaris), and measured aromatase activity in this specie represented less than 1.0% of the total phase I metabolism of testosterone (Morcillo and Porte 1999). Similarly, testosterone appears to be minimally transformed to estradiol in I. obsoleta (Gooding, unpublished data). Collectively, these observations suggest that the overall contribution of aromatase activity to testosterone metabolism may be negligible in these organisms, and therefore its inhibition would not result in a significant increase in testosterone levels.

How does TBT elevate testosterone? The means by which TBT elevates free testosterone may involve the inhibition of enzymes that convert the steroid to various metabolites. Cytochrome P450 aromatase (Bettin et al. 1996; Spooner et al. 1991), sulfotransferase (Ronis and Mason 1996), and acyl coenzyme A:steroid acyltransferase (Gooding et al. 2003; Janer et al. 2005c) are three classes of enzymes that have been considered as targets for inhibition by TBT. Cytochrome P450 aromatase After being the first to document elevated testosterone titers in TBT-exposed imposex-affected female dogwhelks (N. lapillus), Spooner et al. (Spooner et al. 1991) speculated that TBT might inhibit the cytochrome P450-dependent aromatase which converts androgens to estrogens. Indeed, TBT has been shown to be an in vitro inhibitor of cytochrome P450 aromatase activity in humans (Cooke 2002; Heidrich et al. 2001) and periwinkles (L. littorea, Ronis and Mason 1996). Furthermore, in a comparison of common whelk (Buccinum undatum) females collected from areas of low and high shipping density in the North Sea, cytochrome P450 aromatase activity was significantly higher in normal females from the low shipping density area than in imposex-affected individuals from the high shipping density area (Santos et al. 2002). Bettin et al. (1996) pursued the possibility that TBT inhibits aromatase by exposing dogwhelks (N. lapillus and H. reticulata) to the steroidal and nonsteroidal aromatase inhibitors, SH 489 and flavone, respectively. Both pharmacological compounds induced imposex in the tested species. Furthermore, Santos et al. (2005) reported that the P450 aromatase inhibitor formestane was capable of inducing imposex in dogwhelks (N. lapillus). An elevation of androgen titers as a result of aromatase inhibition should be accompanied by a stoichiometric reduction of estrogen titers; yet concentrations of

Sulfotransferase Ronis and Mason (1996) reported that periwinkles (L. littorea) metabolize testosterone almost completely to water-soluble sulfur conjugates. In contrast, testosterone-injected, TBT-exposed periwinkles retained more unmetabolized testosterone and its phase I metabolites (Ronis and Mason 1996). Therefore, they concluded that TBT inhibits the metabolism of testosterone to more polar, and thus excretable, metabolites and in doing so decreases the ability of L. littorea to eliminate androgens. However, as described previously, periwinkles are not susceptible to the phenomenon of imposex but rather the phenotypically distinct condition of intersex, and the investigators did not demonstrate that TBT directly inhibits sulfotransferase activity. In contrast, Gooding and LeBlanc (2001) showed that the metabolism of testosterone to polar sulfate conjugates is negligible in the imposex-susceptible mud snail (I. obsoleta). Rather, these organisms conjugate testosterone primarily to non-polar fatty acid esters. Furthermore, the ramshorn snail (M. cornuarietis), an imposex-susceptible mesogastropod (Janer et al. 2006a), had low testosterone sulfotransferase activity (0.05–0.18 pmol/min/mg) when compared to fatty acid conjugating activity (102 pmol/min/mg) (Janer et al. 2005a). This implies that sulfotransferases do not contribute significantly to the metabolism of testosterone in species that are susceptible to TBT-induced imposex. Acyl coenzyme A:testosterone acyltransferase As described previously, Gooding and LeBlanc (2001, 2004) found the fatty acid esterification of testosterone to be the major regulatory process by which mud snails (I. obsoleta) maintain free testosterone homeostasis. The microsomal enzyme ATAT biotransforms free testosterone to testosterone-fatty acid esters that are retained by the organism. Additional laboratory and field studies by these investigators showed that exposure to TBT not only elevates free testosterone in I. obsoleta, but also decreases the percentage of total

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testosterone found as a testosterone-fatty acid esters (Gooding et al. 2003). Collectively, these observations suggested that TBT suppresses the ability of these organisms to produce or retain testosterone-fatty acid esters (Gooding et al. 2003). Still, ATAT activity in microsomes isolated from TBT-exposed mud snails was not altered regardless of imposex condition implying that TBT does not reduce ATAT protein levels provided that ATAT activity is directly correlated with ATAT protein expression (Gooding et al. 2003). Rather, TBT competitively inhibited ATAT at toxicologically-relevant in vivo concentrations, i.e., concentrations of TBT in imposex-affected snails collected from TBT-contaminated areas (Sternberg and LeBlanc 2006). Thus, TBT most likely elevates free testosterone in neogastropods by inhibiting their major regulatory process for maintaining free testosterone homeostasis—the fatty acid esterification of testosterone.

Does testosterone regulate reproductive tract recrudescence? Sternberg et al. (2008a) used a weight-of-evidence approach to investigate putative roles for androgens in reproductive recrudescence of molluscs, i.e., the annual development of the reproductive tract using I. obsoleta as a model species. The endpoint of recrudescence was selected because imposex is essentially an aberration in the development of the reproductive tract in female neogastropods. The study targeted the specific hormone— testosterone—and its associated receptor. Analyses of the relationships among testosterone, sex, and reproductive status in the model species, in conjunction with the analyses of similar studies performed with other molluscan species, provided no significant evidence for a role of testosterone in reproductive recrudescence (Sternberg et al. 2008a). Consistent with phylogenic analyses as discussed previously, these investigators found no evidence for a nuclear androgen receptor in this species (Sternberg et al. 2008a). Collectively, the lack of sexspecific differences in testosterone levels through reproductive development and the absence of an androgen receptor tipped the weight-of-evidence against a role for this hormone in the regulation of molluscan recrudescence (Sternberg et al. 2008a). Thus, although elevated free testosterone in TBTexposed neogastropods can be explained by the inhibition of ATAT by TBT, there is a lack of support for testosterone as a functional hormone in processes related to reproductive recrudescence in these organisms. Consequently, accepting the hypothesis regarding the elevation of testosterone by TBT as the mechanism by which TBT induces imposex in neogastropods is problematic.

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Neurotoxicant hypothesis Another hypothesis for the mechanism of TBT-induced imposex was developed as a result of experiments by Fe´ral and Le Gall (1982) followed by those of Oberdorster and McClellan-Green (2000, 2002). The former demonstrated that excised ganglia from female sting winkles (Ocenebra erinacea) were activated by exposure to TBT. The activated ganglia induced the development of a penis in the excised penis-forming area tissues of slipper limpets (Crepidula fornicata). The latter showed that APGWamide, a putative penis morphogenic factor (PMF), significantly induced imposex in I. obsoleta. Consequently, a hypothesis implicating TBT as a neurotoxicant was proposed: TBT causes the aberrant secretion of neurohormones that contribute to male sexual differentiation (e.g., PMF) in gastropods. However, in a more recent study, APGWamide failed to promote imposex in B. brandaris (Santos et al. 2006), and a causal relationship among TBT exposure, abnormal APGWamide release, and imposex has yet to be established. RXR agonist hypothesis Several studies (Grun et al. 2006; Kanayama et al. 2005; Nishikawa et al. 2004) have shown that some organotins, including TBT, are high-affinity ligands of human RXRs. In addition, Kanayama et al. (2005) used a reporter-gene assay to demonstrate that TBT stimulates the transactivation of gene expression via RXRa. Collectively, these findings provided the foundation for investigations into whether TBT could act via a signaling pathway involving RXR to induce imposex (Castro et al. 2007; Nishikawa et al. 2004). As discussed previously, a RXR has been identified in several molluscan species (Bouton et al. 2005; Castro et al. 2007; Nishikawa et al. 2004; Sternberg et al. 2008b). This molluscan RXR has been shown to bind the putative RXR ligand 9-cis retinoic acid with high affinity (Castro et al. 2007; Nishikawa et al. 2004) as well as transduce RXR-mediated gene transcription in a reporter gene assay (Bouton et al. 2005). Most importantly, injections of 9-cis retinoic acid stimulated the development of imposex in female rock shells (T. clavigera, Nishikawa et al. 2004) and dogwhelks (N. lapillus) (Castro et al. 2007), and TBT inhibited the binding of 9-cis retinoic acid to the RXR of the rock shell (T. clavigera, Nishikawa et al. 2004; Nishikawa 2006). The involvement of retinoid signaling via RXR in the aberrant development of male sex characteristics is strengthened by a study suggesting that RXR-mediated signaling may be an important regulator of sex-specific recrudescence in these organisms, with the timing of RXR expression being critical to sex-specific differentiation (Sternberg et al. 2008b). More specifically,


early retinoid signaling via RXR may stimulate male recrudescence, whereas later signaling may stimulate female recrudescence (Sternberg et al. 2008b). If so, then TBT may induce the development of male sex characteristics in females (imposex) by initiating RXR signaling prematurely in females during the temporal window of normal male recrudescence. This supposition was supported by the observation that TBT stimulated the development of male sex characteristics in females only when exposure occurred during the temporal window of male recrudescence (Gooding 2002). Collectively, these results provide promise that TBT may cause imposex by disrupting retinoid signaling in neogastropods.

Dumpton syndrome The emergence of a mutation in some dogwhelk (N. lapillus) populations may ultimately provide additional insight into the mechanism of imposex. Populations of dogwhelks have emerged in TBT-contaminated regions of Brittany and north-west Spain in which development of the male reproductive tract is compromised in both males and imposex-affected females (Barreiro et al. 1999; Gibbs 1993; Huet et al. 1996b, Huet et al. 2008). While females are typically sterilized by TBT in these locals, reproduction is preserved among the mutant females due to the suppression of imposex. This condition, known as Dumpton Syndrome, appears to be the consequence of a recessive mutation that conforms to principals of Mendelian inheritance (Gibbs 2005) and is selected for in TBT-contaminated regions. Putative molecular targets of TBT that result in imposex (e.g., APGWamide, testosterone, RXR) should be evaluated in these organisms for changes in accumulation or function. The detection of an aberrant target in these organisms would provide strong supportive evidence for the role of the target in imposex development.

Summary and conclusions Reproductive tract differentiation and development in gastropods is mediated by various environmental cues that permit reproduction to occur at the most favorable time. Photoperiod appears to function as the master regulator of reproductive tract recrudescence during the season that is energetically most favorable for reproduction and survival of offspring (Fig. 3). Temperature and nutritional status appear to serve as regulators of the precise timing of egg laying (Fig. 3). These secondary environmental cues may control year-to-year variations in environmental conditions that could compromise successful reproduction.

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All of the environmental cues are likely converted to neuro-endocrine signals that either function directly in the regulation of reproductive maturation or serve as penultimate hormones that regulate the ultimate reproductive hormones. A variety of hormones whose action is mediated by nuclear hormone receptors (retinoids, steroids) have been suggested to function as the ultimate hormones. However, the role of these hormones in gastropod reproductive development and function remains equivocal. TBT has been shown to interfere with normal reproductive differentiation causing the development of male sex characteristics in females—a phenomenon known as imposex. TBT also is known to elevate testosterone levels in gastropods. The elevated testosterone levels have long been presumed to be responsible for the development of imposex. TBT elevates free testosterone levels in gastropods through the inhibition of the fatty acid esterification of testosterone. However, we view this effect as inconsequential with regards to the development of imposex, since exposure to very high concentrations of testosterone is required to stimulate imposex and no evidence exists to suggest that the androgen signaling pathway exists in gastropods. Indeed, phylogenetic analysis has revealed that androgen signaling is a product of vertebrate evolution. Alternatively, the inhibition of fatty acid esterification by TBT could result in the elevation of free levels of the relevant hormone that controls the development of the male reproductive tract. To date, the strongest candidate for the ultimate regulator of male reproductive tract development is 9-cis retinoic acid or some other ligand of the retinoid X-receptor (RXR). Retinoids are known to be subject to fatty acid esterification (Ross 1982) and the inhibition of the fatty acid esterification of retinoids by TBT could elevate levels of the relevant hormone. Furthermore, TBT also has been shown to be a high-affinity activator of RXR. Thus, two mechanisms exist by which TBT could stimulate aberrant RXR signaling resulting in imposex (Fig. 4). Elevated free testosterone levels could serve as a biomarker of TBT action without causal involvement in the process of imposex (Fig. 4). Experiments performed with pharmacological agents or environmental chemicals should be viewed with caution when attempting to ascribe a mechanism by which these chemicals interfere with recrudescence. For example, exposure to the vertebrate androgen receptor antagonist cyproterone acetate has been shown to inhibit TBTinduced imposex in snails (Tillmann et al. 2001). These observations suggest that the action of TBT is mediated via an androgen receptor. However, cyproterone acetate is not a specific antagonist of the androgen receptor as it is also capable of binding the glucocorticoid receptor, the progesterone receptor, and the pregnane X-receptor (Honer et al. 2003; Lehmann et al. 1998). At sufficiently high

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Fig. 3 Proposed coordinate action of environmental cues on reproductive maturation in gastropods

B

A

C

Fig. 4 Proposed mechanism for TBT-induced imposex. TBT activates the retinoid X receptor (RXR) signaling pathway to initiate the transcription of genes necessary for male reproductive system development (a) directly, by binding to and activating RXR; or (b) indirectly, by inhibiting acyl coenzyme A:acyltransferase (AXAT), resulting in an increase in endogenous retinoid (and testosterone) levels. RXR is then activated by the endogenous free retinoid. RXR

stimulates gene transcription through interaction with RXR response elements (RXRRE). Similarly, exogenously-administered testosterone (c) competitively inhibits retinoid esterification resulting in elevated free, endogenous retinoid levels that are capable of activating the RXR signaling pathway, leading to male reproductive organ development

dosages, cyproterone acetate might also inhibit other nuclear receptors such as RXR. Furthermore, cyproterone acetate is capable of inhibiting the action of enzymes that are involved in hormonal biosynthetic pathways (Ayub and Levell 1987; Pham-Huu-Trung et al. 1984). Therefore, this compound may elicit multiple effects on hormone signaling pathways beyond its recognized ability to inhibit androgen signaling.

Taken together, studies of environmental signaling of reproductive tract recrudescence (Fig. 1) along with indices of neuro-endocrine regulators of reproductive tract development (Table 1) provide for the inference of a model of the environmental-endocrine control of reproductive development in gastropods (Fig. 5). We propose that the environmental-endocrine regulation of reproductive tract recrudescence and reproduction in gastropods is primarily

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controlled by photoperiodic signals that simulate the nocturnal secretion of 5-methoxyindoles. These hormones allow the gastropod to discern seasons that are conducive to reproduction. The 5-methoxyindoles regulate the occupancy of the RXR gene promoter region by repressor proteins (e.g., SMRT). The vertebrate RXR gene is known to be under the regulatory control of such repressor proteins (Cohen et al. 2000). The RXR gene is thus largely silenced during periods that are not conducive to reproduction. At the appropriate season, the repressor proteins dissociate from the RXR gene which renders the organism permissive for recrudescence and reproduction to occur. A second environmental cue, temperature or nutrition, may stimulate the neuroendocrine cascade responsible for the synthesis of RXR ligand, which, in association with RXR, then initiates recrudescence. Temperature or nutritional cues also stimulate various activities and processes associated with egg laying to precisely control the timing of the release of eggs into the environment (Table 2). According to this model, recrudescence of both male and female reproductive tracts is ultimately regulated by retinoid signaling via RXR. Sex-specific recrudescence may be a function of the timing of the retinoid signal (Sternberg et al. 2008b). Precedence exists for the timing of retinoid signaling resulting in sex-specific development. Germ cells in the rodent ovary undergo meiosis and

Fig. 5 Proposed environmental-endocrine regulation of reproductive tract recrudescence and reproduction in gastropods. a Photoperiod information is translated into a biological signal by the nocturnal secretion of 5-methoxyindoles which serve to decipher seasons. b Repressor proteins (e.g., SMRT) occupy the promoter region of the RXR gene during periods of reproductive senescence and dissociate from the RXR gene to allow recrudescence (c) and reproduction (d). e A second environmental cue, temperature or nutrition, stimulates the neuroendocrine cascade responsible for the synthesis of RXR ligand, which, in association with RXR, initiates recrudescence. f Temperature or nutritional cues also stimulate various activities and processes associated with egg laying to precisely control the timing of the release of eggs into the environment

17

Table 2 Proposed seasonal regulation of reproductive tract recrudescence via RXR transcriptional repression and RXR ligand levels Reproductive stages Senescence

Recrudescence

Reproduction

Male

Female

-

?

?

?

Male

-

?

-

?

Female

-

-

?

?

RXR Ligand

Reproductive stages are depicted when RXR is inactive (-), perhaps due to transcriptional repression of the RXR gene, or active (?), perhaps due to loss of the repressor. Differences in RXR levels among reproductive stages are based upon the work of Sternberg et al. (2008b). Sex-specific RXR ligand levels are depicted as low (-) or high (?). Receptor levels may be regulated by photoperiod; while, ligand levels may be regulated by a secondary environmental cue such as temperature or nutrition

progress into oocytes prenatally whereas germ cells in the testis undergo meiosis and develop into spermatocytes postnatally. Retinoic acid is the key determinant for the timing of germ cell meiosis by stimulating the expression of Stra8 (Stimulated by retinoic acid gene 8), a protein that is necessary for the commencement of meiotic division. Elevated retinoic acid levels in the prenatal ovary induce

F A

E

B

C

D

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Stra8 to initiate germ cell meiosis (Koubova et al. 2006). In contrast, prenatal testes expressed high levels of CYP26B1, an enzyme that metabolizes retinoid acid to an inactive form, and thus prevents retinoid signaling from stimulating Stra8 expression (Bowles et al. 2006; Koubova et al. 2006). CYP26B1 is suppressed in postnatal testes, allowing for the accumulation of retinoic acid, the induction of Stra8, and the progression of germ cells into spermatocytes (Bowles et al. 2006; Koubova et al. 2006). Therefore, retinoid signaling determines the ultimate sex-specific fate of rodent germ cells by controlling the timing of meiosis. Retinoic acid signaling holds promise as being the target through which TBT causes imposex. However, too little is currently known of the role of retinoids in molluscan reproductive tract recrudescence. Additional study is required to definitively establish whether retinoids and the RXR are responsible for the initiation or progression of male reproductive tract development. Mechanistic studies are warranted to identify the molecular events that lead to recrudescence. Finally, the ability of TBT to either masquerade as the hormone or elevate levels of the relevant hormone, perhaps by inhibiting its fatty acid esterification, need to be established. Only then can the TBT/imposex enigma be resolved. Acknowledgments 0234676.

This work was supported by NSF grant IBN

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