Abstract

Wildlife species have been recognized as sentinels of environmental health for decades. In fact, ecological data on various wildlife populations provided the impetus for banning some organochlorine pesticides over the last few decades. Alligators are important sentinels of ecosystem health in the wetlands of the southeastern United States. Over the last 15 years, a series of studies have demonstrated that environmental exposure to a complex mixture of contaminants from agricultural and municipal activities alters the development and functioning of alligators' reproductive and endocrine systems. Further studies of basic developmental and reproductive endocrinology in alligators and exposure studies performed under controlled laboratory conditions support the role of contaminants as causal agents of abnormalities in gonadal steroidogenesis and in reproductive tract development. These studies offer potential insight into environmentally induced defects reported in other wildlife and human populations exposed to a wide array of endocrine-disruptive contaminants.

Over the past few decades, the field of toxicology expanded from a focus on lethality and carcinogenicity to include alterations in development and reproduction resulting from low-dose exposure to anthropogenic contaminants. As early as 1962, Rachel Carson brought reproductive impairment in wildlife exposed to pesticides to the public's attention in Silent Spring (Carson 1962); however, a multidisciplinary approach to toxicology—popularized by the publication of Our Stolen Future (Colborn et al. 1996)—did not become widespread until the past 10 to 15 years. During this time, a host of man-made chemicals, ranging from organochlorine (OC) pesticides to phthalates and surfactants to polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (used as flame retardants in various industrial applications) have been implicated in developmental and reproductive abnormalities in numerous species.

The development and functioning of the reproductive system is largely dependent on hormonal signaling in a dose-dependent fashion. Interruption of hormonal signaling can lead to the improper development of reproductive organs and tissues or to the mistiming of reproductive processes. Many xenobiotics that have shown adverse effects at low doses are known to interfere with the normal functioning of the endocrine system, and therefore have been designated collectively as endocrine-disrupting chemicals (EDCs). Our intent is to provide a brief overview of the mechanisms of endocrine disruption characterized through controlled laboratory experiments. We will then present a case study from the alligators of Lake Apopka, Florida, a wildlife population that has experienced low reproductive success in association with exposure to EDCs.

Mechanisms of endocrine disruption

EDCs can mimic or antagonize endogenous hormones, alter synthesis or degradation, and affect hormone transport in the bloodstream (figure 1). Recent evidence also suggests that exposure to EDCs can lead to changes in transcriptional activity and gene expression brought about by epigenetic effects. Many aspects of the endocrine system are highly conserved across vertebrate evolution, and thus the mechanisms through which exogenous chemicals affect hormonal signaling in one species often hold true for others. That said, organismal response to EDCs does vary among individuals and species. The use of in vitro models, in which differences as subtle as single amino-acid substitutions in receptors can be assessed, is elucidating some sources of individual and interspecies variation (see, for example, Katsu et al. 2008).

Figure 1.

Schematic representation of the hypothalamo-pituitary-gonad/thyroid axis. Circulating hormones interact with receptors in target tissues throughout the body, altering gene expression profiles. Plasma steroid and thyroid hormone concentrations are also affected by plasma-binding proteins, synthesized by the liver, as well as hepatic biotransformation and clearance. It is worth noting that the alligator's endocrine system is nearly identical to that of the human and other mammals, making it an effective sentinel of potential environmental and human health concerns.

Figure 1.

Schematic representation of the hypothalamo-pituitary-gonad/thyroid axis. Circulating hormones interact with receptors in target tissues throughout the body, altering gene expression profiles. Plasma steroid and thyroid hormone concentrations are also affected by plasma-binding proteins, synthesized by the liver, as well as hepatic biotransformation and clearance. It is worth noting that the alligator's endocrine system is nearly identical to that of the human and other mammals, making it an effective sentinel of potential environmental and human health concerns.

EDCs are commonly classified in accordance with the receptor-signaling pathway they are shown to disrupt, as opposed to chemical structure. For instance, the pesticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (o,p'-DDT) and the plastic monomer bisphenol A (BPA) are often referred to as environmental estrogens, and in vitro assays have shown that both chemicals activate estrogen receptor (ER)–dependent pathways (Andersen et al. 1999). It is important to note, however, that in vitro experiments do not always predict in vivo effects, as the timing and route of exposure can also affect the outcome. A case in point: The production of vitellogenin, or yolk protein, in rainbow trout is stimulated through the activation of ER in the liver; a single injection of DDT fails to induce vitellogenin production in rainbow trout (Andersen et al. 1999), whereas a similar dose distributed over three injections does induce it (Donohoe and Curtis 1996). Endocrine disruption can also occur by inhibiting, rather than activating, receptor-dependent pathways. The pesticide metabolite p,p'-DDE (1,1-dichloro-2,2-bis[p-chlorophenyl]ethylene) and a metabolite of the fungicide vinclozolin act as antiandrogens by competitively binding the androgen receptor (AR) without activating AR-dependent pathways (Gray et al. 2001). Classifying EDCs by the signaling pathway they disrupt has also been applied to complex mixtures in the environment such as sewage effluent. In such cases, the physiological effects evident in animals environmentally exposed to the effluent are similar to those seen in animals experimentally exposed to estrogenic or antiandrogenic positive controls (Filby et al. 2007). Some environmental contaminants have been shown to inhibit specific enzymes involved in hormone synthesis. The initial and rate-limiting step of de novo steroidogenesis is the transfer of cholesterol from the outer to the inner mitochondrial membrane by the steroidogenic acute regulatory protein (StAR; Stocco and Clark 1996). Cholesterol is then converted to pregnenolone by the cytochrome P450 (CYP) enzyme CYP11A, and pregnenolone converted to other steroid hormones by a series of hydroxysteroid dehydrogenases and CYP enzymes (figure 2). The insecticides lindane (organochlorine) and dimethoate (organophosphate) inhibit steroidogenesis by disrupting the transcription of StAR messenger RNA (mRNA; Walsh and Stocco 2000, Walsh et al. 2000a), whereas posttranscriptional disruption of StAR was observed following treatment of MA-10 (mouse Leydig tumor) cells with the antifungal drugs econazole and miconazole (Walsh et al. 2000b). Similarly, chronic exposure to PCBs inhibited CYP11A activity in the testes of bulls, contributing to low serum testosterone concentrations (Machala et al. 1998).

Figure 2.

A representative model of vertebrate steroidogenesis, including the initial steps that occur in the mitochondria. A gonadotropin, such as luteinizing hormone (LH) is delivered via the blood and binds to a specific membrane receptor (LH-R) that is associated with a guinine nucleotide-binding protein–adenyl cyclase (AC) complex. This complex generates a second messenger, cAMP, that activates a kinase cascade and enzyme activity associated with steroidogenesis. Initial stages of steroidogenesis occur in the mitochondria following NR5A1-induced production of steroid acute regulatory (StAR) protein. StAR is essential for the transport of cholesterol across the mitochondial membrane. Cholesterol is then modified by enzyme action both within the mitochondria and in the smooth endoplasmic reticulum of the cytoplasm to generate steroid hormones that are released into the blood. Enzymes: P450scc, P450 side chain cleavage; 3ßHSD, 3ß hydroxysteroid dehydrogenase; 17ßHSD, 17ß hydroxysteroid dehydrogenase; P450arom, P450 aromatase. Abbreviation: ATP, adenosine-5'-triphosphate.

Figure 2.

A representative model of vertebrate steroidogenesis, including the initial steps that occur in the mitochondria. A gonadotropin, such as luteinizing hormone (LH) is delivered via the blood and binds to a specific membrane receptor (LH-R) that is associated with a guinine nucleotide-binding protein–adenyl cyclase (AC) complex. This complex generates a second messenger, cAMP, that activates a kinase cascade and enzyme activity associated with steroidogenesis. Initial stages of steroidogenesis occur in the mitochondria following NR5A1-induced production of steroid acute regulatory (StAR) protein. StAR is essential for the transport of cholesterol across the mitochondial membrane. Cholesterol is then modified by enzyme action both within the mitochondria and in the smooth endoplasmic reticulum of the cytoplasm to generate steroid hormones that are released into the blood. Enzymes: P450scc, P450 side chain cleavage; 3ßHSD, 3ß hydroxysteroid dehydrogenase; 17ßHSD, 17ß hydroxysteroid dehydrogenase; P450arom, P450 aromatase. Abbreviation: ATP, adenosine-5'-triphosphate.

Hepatic degradation, transport, and clearance of endogenous hormones are also susceptible to alterations following exposure to xenobiotics (Gunderson et al. 2001). Oxido-reductase enzymes from the CYP2 and CYP3 families, conjugating enzymes such as glutathione S-transferases and sulfotranserases, and membrane-transport proteins such as organic anion transport protein are all involved in the breakdown and elimination of steroids and lipophilic xenobiotic compounds. Transcriptional regulation of these enzymes and transport proteins is largely under the control of the orphan nuclear receptors, the steroid and xenobiotic receptor, and the constitutive androstane receptor (Maglich et al. 2002). Each of these receptors is characterized by its broad ligand specificity relative to related nuclear receptors, and each has been shown prone to activation by numerous xenobiotics (Kretschmer and Baldwin 2005, Milnes et al. 2008a). Ligand-dependent activation of these receptors by xenobiotics typically results in increased metabolism of the compound, but it also increases the biotransformation and elimination of endogenous signaling molecules, potentially disrupting steroid homeostasis (Zhai et al. 2007). Additionally, some man-made compounds have been shown to interact with plasma proteins that transport specific hormones and protect them from hepatic metabolism. PCBs have been implicated in the disruption of the hypothalamus-pituitary-thyroid axis (Desaulniers et al. 1999). Cheek and colleagues (1999) found that hydroxylated PCBs (OH-PCBs) and the thyroid hormone thyroxine (T4) have the same affinity for transthyretin, a serum thyroid-hormone transport protein. By competitively binding transthyretin, OH-PCBs could increase the bio-availability of T4 and increase its susceptibility to hepatic degradation.

Finally, transcriptional and epigenetic effects, in which gene expression or DNA is modified without altering nucleotide sequence, has been associated with exposure to xenobiotics (reviewed in Tabb and Blumberg 2006, Edwards and Myers 2007). For example, nuclear hormone receptors, including steroid receptors, are typically degraded through a ubiquitin-proteosome pathway. Masuyama and Hiramatsu (2004) found that BPA inhibits ubiquitination and degradation of estrogen receptor ß (ERß), potentially increasing the transcription of ERß target genes. Another mechanism for epigenetic effects of EDCs involves the sex-specific methylation patterns of DNA, which can be passed along through the germ cell line. Reduced spermatogenic capacity has been observed in association with altered DNA methylation patterns in rats following in utero exposure to vinclozolin and methoxychlor. This phenotype was perpetuated in male offspring for four generations after exposure (Anway et al. 2005).

Alligators as a model species for ecosystem health

As a long-lived top predator and a species of aesthetic and commercial importance in the southeastern United States, the alligator serves as a sentinel of wetland ecosystem health. The alligator is an oviparous species that produces a single clutch of eggs during a synchronous reproductive cycle. This allows for the coordinated collection of a large number of eggs enabling adequate sample sizes for ecotoxicological monitoring and experimental designs. Alligators also exhibit temperature-dependent sex determination (TSD)—that is, the egg incubation temperature during a thermosensitive period of embryonic development determines the sex of the offspring (Lang and Andrews 1994). Females are typically produced at temperatures below 31 degrees Celsius (°C), males are produced at or above 33°C, and a mixed ratio of males and females are produced at intermediate temperatures (Lang and Andrews 1994). Although the molecular mechanisms of sex determination have not been worked out in detail for TSD species, experimental exposure to natural and synthetic steroids and some EDCs has been shown to override the effects of temperature on sex determination in alligators (Crain et al. 1997, Milnes et al. 2005a). Thus, alligators are susceptible to experimental manipulations of incubation temperature and hormonal milieu to facilitate the investigation of temperature and sex-specific developmental patterns and indices. Although their large size and time to sexual maturity (10 or more years) makes transgenerational studies problematic, alligators are an excellent model for investigating the effects of chronic exposure to xenobiotic chemicals on reproductive success and embryonic development.

Lake Apopka, Florida: A case study in endocrine disruption

The initial indication of reproductive impairment in alligators from Lake Apopka, Florida, was the lack of recruitment of juveniles into the population. Following nearly 40 years of pesticide and nutrient runoff from adjacent agricultural operations, Lake Apopka experienced a pesticide spill in 1980 from the Tower Chemical Company site. The spill, which resulted from the overflow of holding ponds during heavy rainfall, consisted primarily of dicofol and derivatives of DDT (USEPA 1994). Unfortunately, data from before the spill are unavailable, but rates for alligator egg viability and juvenile population density declined significantly in the years after the spill (Woodward et al. 1993). Compared with yolk samples taken from alligator eggs at Lake Griffin, Florida, an area of intermediate anthropogenic influence (Heinz et al. 1991), samples taken at Lake Apopka in 1985 had higher concentrations of several OC pesticides and metabolites, including toxaphene, dieldrin, p,p'-DDE, p,p'-dichlorodiphenyl dichloroethane, p,p'-DDT, cis-chlordane, and trans-nonachlor. In the following decade, egg viability rates rose but still remained below those at two reference sites, Lake Woodruff National Wildlife Refuge (NWR) and Orange Lake (Masson 1995, Rice et al. 1999). More recent studies from eggs collected between 2000 and 2002 show that concentrations of OC pesticide residues in Lake Apopka eggs still have not appreciably decreased, and Lake Apopka egg viability and posthatching survival remain low relative to Orange Lake and Lake Woodruff NWR (Rauschenberger et al. 2007, Milnes et al. 2008b).

Exposure to EDCs does not appear to be an immediate threat to the existence of alligators in Lake Apopka or elsewhere; however, this population of alligators offers the opportunity to study and better understand the various modes of action through which environmental exposure to EDCs affects development and reproduction in a long-lived species, enabling improved population monitoring and more-informed policy decisions. It is difficult to compare contaminant concentrations across species and tissues, and although yolk concentrations of OC pesticides in Lake Apopka are one to two orders of magnitude above concentrations reported in fish across the southeastern United States (Hinck et al. 2008), this situation is not unique. For instance, the concentrations of p,p'-DDE and toxaphene in the visceral fat and liver of fresh-and saltwater crocodiles (Crocodylus johnstoni and Crocodylus porosus) from the Ord River, Western Australia, are among the highest ever reported in wildlife, even though it has been nearly 30 years since pesticides were applied to this drainage (Yoshikane et al. 2006). Although variation in size and reproductive status among the Ord River sampling sites prevented statistical comparisons of phenotypic effects reported in Lake Apopka alligators, the study nonetheless illustrates the bioaccumulative nature and long-lasting legacy of these chemicals in top predators.

That the reduced egg viability reported in Lake Apopka alligators could be caused by exposure to OC pesticides rather than industrial chemicals or metals is supported by analyses of contaminant concentrations in various alligator tissues. Whereas yolk and serum OC concentrations in samples from Lake Apopka were elevated in comparison with samples from Lake Woodruff NWR and Orange Lake (Heinz et al. 1991, Guillette et al. 1999a), no differences in PCB concentrations were found among these lakes (Guillette et al. 1999a). Metal and metalloid concentrations in all three lakes (Burger et al. 2000) were well below suggested toxic levels and levels previously reported in alligators from the Everglades and Georgia (Heaton-Jones et al. 1997).

Other proposed hypotheses to explain the low reproductive success reported in Lake Apopka alligators include nutritional and genetic differences. One as yet unstudied but plausible explanation is that nutritional differences resulting from several decades of nutrient and contaminant loading may affect the composition of prey species. Previous research has shown dietary differences and altered egg yolk lipid composition to be associated with reduced egg viability in captive alligators (Noble et al. 1993). If the prey species composition has been drastically altered on Lake Apopka, it is possible that essential nutrients are lacking in the diet, thus leading to deficiencies in specific fatty acids or nutrients incorporated into the egg yolk during vitellogenesis.

Genetic differences resulting from contaminant-induced selection or natural variation seem an unlikely cause of low reproductive success on Lake Apopka at this point. The maximum life span of American alligators is not known, but conservative estimates place the number well beyond 30 years. No mass-mortality events have been reported for adult alligators at Lake Apopka, suggesting that a portion of the current breeding population hatched before the Tower Chemical Company spill. Given alligators' long life span and time to maturation, and the population's connectivity with other lakes in the same drainage basin, it seems unlikely that exposure to contaminants has significantly altered allele frequency in Lake Apopka's alligators relative to those in nearby reference sites. Indeed, microsatellite DNA analysis revealed insignificant variation among alligator populations (including Lake Apopka and Lake Woodruff NWR) across Florida and southern Georgia (Davis et al. 2002).

Field studies.

For the purposes of this review, neonates are defined as alligators one month of age or younger; hatchlings are more than one month but less than or equal to one year of age; and juveniles are older than one year but less than 90 centimeters (cm) long from snout to vent, the approximate minimum size at sexual maturity (Wilkinson and Rhodes 1997). All fieldwork described in this review from our laboratory was permitted by the Florida Fish and Wildlife Conservation Commission, and the use of experimental animals was performed under the guidelines specified by the Institutional Animal Care and Use Committee at the University of Florida.

Because natural systems are infinitely complex and contain numerous biotic and abiotic characters that can potentially influence development, true replication of the scenario at Lake Apopka cannot be achieved. Rather, our laboratory and others have used Lake Apopka as a case study of a chronically exposed alligator population that has experienced poor reproductive success. In many of the comparative studies, Lake Woodruff NWR was used as a reference population, as it has been subjected to fewer anthropogenic influences and exhibits consistently high reproductive success relative to the population at Lake Apopka. It is important to note, however, that physiological differences described in alligators from Lake Apopka relative to those from Lake Woodruff NWR are merely associative, not causative, and complementary experimental studies are required to characterize the specific effects of EDCs.

The initial indicator that exposure to EDCs could be causing reproductive problems in alligators at Lake Apopka—aside from the relatively low percentage of eggs hatching there—was the observation of morphological abnormalities in the gonads of six-month-old hatchlings. Multioocytic ovarian follicles and multinuclear oocytes (figure 3), which resembled the gonads of female mice exposed in utero to the synthetic estrogen diethylstilbestrol, were observed in the ovaries of captive-raised alligator hatchlings from Lake Apopka (Guillette et al. 1994). Recent studies have reported observations of multioocytic follicles in caiman (Caiman latirostris) or mice following embryonic or neonatal experimental exposure to estradiol, BPA, or phytoestrogens, such as genistein (Jefferson et al. 2006, Stoker et al. 2008). Further morphological evidence of chemicals interfering with endocrine signaling was evident when the size of the phallus, an androgen-dependent tissue, was compared among alligators from different lakes: alligators from Lake Apopka show reduced phallus size when compared with similar-sized animals from less-polluted lakes (reviewed in Guillette et al. 2000). Other morphological differences noted between juvenile alligators from Lake Apopka and Lake Woodruff NWR include thymus and spleen histological structure (Rooney et al. 2003) and long-bone mineral density and composition (Lind et al. 2004)

Figure 3.

Photomicrographs of normal and multioocytic ovarian follicles (a, b) and a multinuclear oocyte (c) from juvenile American alligators. Multioocytic (polyovular) follicles have been reported from females alligators from Lake Apopka, Florida, as well as from female caiman treated with various estrogenic contaminants. Abbreviations: o, oocyte; mof, multioocytic follicle; mno, multinuclear oocyte. Photomicrographs: Brandon C. Moore, University of Florida.

Figure 3.

Photomicrographs of normal and multioocytic ovarian follicles (a, b) and a multinuclear oocyte (c) from juvenile American alligators. Multioocytic (polyovular) follicles have been reported from females alligators from Lake Apopka, Florida, as well as from female caiman treated with various estrogenic contaminants. Abbreviations: o, oocyte; mof, multioocytic follicle; mno, multinuclear oocyte. Photomicrographs: Brandon C. Moore, University of Florida.

Just as hormonally active xenobiotics can affect development, alterations in endogenous hormone concentrations can have similar deleterious effects. The thyroid and steroid hormones of alligators from Lake Apopka and from reference sites such as Lake Woodruff NWR have been compared multiple times; the results of these comparisons vary, depending on many factors such as age or size and time of year. Juvenile alligators display seasonal variation in plasma sex steroids (Rooney et al. 2004), with the degree of the response depending on body size; alligators above a size threshold of approximately 38 cm from snout to vent begin to show pronounced seasonal variation in estradiol-17ß(E2) and testosterone, suggesting a peripubescent period. Although seasonal patterns among lakes are somewhat variable, some trends are evident. Juvenile males living in Lake Apopka have lower concentrations of circulating testosterone than males living in Lake Woodruff NWR (figure 4; Crain et al. 1998, Guillette et al. 1999a). Egg-hatch rates that are higher than Lake Apopka's and lower than Lake Woodruff NWR's have been reported at other Florida lakes that have experienced agricultural or municipal influences that are intermediate to those of Lake Apopka and Lake Woodruff NWR. Several of these lakes—including Lake Okeechobee (Crain et al. 1998, Gunderson et al. 2004) and Lakes Griffin and Jessup (Guillette et al. 1999b)—also show depressed plasma testosterone concentrations in juvenile males. Females from Lakes Apopka, Griffin, Okeechobee, and Orange exhibit elevated E2 compared with Lake Woodruff NWR juveniles (Guillette et al. 1999b). Seasonal variation has also been examined in thyroid hormones in juvenile alligators from several Florida lakes, and the overall pattern of variation was similar to that for sex steroids (Bermudez et al. 2005). Crain and colleagues (1998) found a negative relationship between thyroxine (T4) and tri-iodothyronine (T3) and body size in juveniles from Lake Woodruff NWR, but no such relationship existed in alligators from Lake Apopka.

Figure 4.

Plasma testosterone concentrations in size-matched juvenile male alligators from Lakes Woodruff NWR and Lake Apopka. Bars represent means ± the standard error of the mean. At all juvenile body sizes represented, males from Lake Apopka have significantly lower plasma testosterone concentrations than do those from Lake Woodruff NWR. Data are from Guillette and colleagues (1999b) and Crain and colleagues (1998).

Figure 4.

Plasma testosterone concentrations in size-matched juvenile male alligators from Lakes Woodruff NWR and Lake Apopka. Bars represent means ± the standard error of the mean. At all juvenile body sizes represented, males from Lake Apopka have significantly lower plasma testosterone concentrations than do those from Lake Woodruff NWR. Data are from Guillette and colleagues (1999b) and Crain and colleagues (1998).

Because of the bioaccumulating and lipophilic nature of many of the contaminants ubiquitous to Lake Apopka, individuals experience two major stages of contaminant exposure. First, the embryo is exposed to any contaminants the mother transfers to the embryonic environment during the vitellogenic stage of oocyte maturation. Embryonic exposure to hormonally active compounds can have unique consequences as a result of the profusion of cell proliferation and differentiation in the developing embryo (Bern 1992). Second, offspring experience environmental exposure throughout their growth and reproductive maturation. With this in mind, it is important to distinguish abnormalities present in Lake Apopka's alligators as a result of embryonic exposure to EDCs from alterations caused by environmental exposure. We have attempted to do this by collecting eggs from Lake Apopka and Lake Woodruff NWR and raising the hatchlings in a controlled environment, thus limiting the cause of developmental differences to embryonic or genetic origins.

When we compared neonates collected as eggs from Lake Apopka and from Lake Woodruff NWR, we found that males and females from Lake Apopka had higher plasma testosterone concentration (Milnes et al. 2005b). Interestingly, although plasma testosterone concentrations were higher, the phallus tip length and cuff diameter were smaller in males from Lake Apopka (figure 5). In contrast, the plasma testosterone concentration was lower in six-month-old males (Guillette et al. 1994) and nine-month-old females (Crain et al. 1997) from Lake Apopka than it was in animals of the same age from Lake Woodruff NWR. The results from the six- and nine-month-old hatchlings are more consistent with our observations of field-caught juveniles. Current studies in our laboratory are examining the phenomena of embryonic and posthatching sexual differentiation and ontogeny of sexual maturation of the gonads and external genitalia. Recent results suggest that significant organizational changes within the steroidogenic tissues of the immature gonad take place during the first weeks to three months of life (Moore et al. 2008).

Figure 5.

Phallus cuff diameter and tip length of male neonatal alligators from Lake Apopka (AP) and Lake Woodruff (WO). Bars represent least-square means adjusted for snout-to-vent length ± the standard error of the mean, and asterisks denote significant differences between lakes within an incubation temperature. One asterisk indicates p ≤ 0.05; two asterisks indicate p ≤ 0.005. The data are taken from Milnes and colleagues (2005b).

Figure 5.

Phallus cuff diameter and tip length of male neonatal alligators from Lake Apopka (AP) and Lake Woodruff (WO). Bars represent least-square means adjusted for snout-to-vent length ± the standard error of the mean, and asterisks denote significant differences between lakes within an incubation temperature. One asterisk indicates p ≤ 0.05; two asterisks indicate p ≤ 0.005. The data are taken from Milnes and colleagues (2005b).

Experimental exposure studies.

As previously stated, the observed differences between alligators from Lake Apopka and Lake Woodruff NWR are associative, and controlled experimental approaches must be applied to begin to elucidate proximate causes for these. Invoking Occam's razor (the principle of parsimony), we conducted experiments exposing alligators from Lake Woodruff NWR to individual contaminants relevant to Lake Apopka to reveal specific contaminant-induced effects. Many of these experiments used chemicals dissolved in ethanol and applied topically to the eggshell. The major criticism of this technique is that the amount of chemical delivered into the egg is variable, and typically only a small percentage of what was applied, thus invalidating dose-response relationships (Muller el al. 2007). On the other hand, if proper controls are employed and results replicated, such as ovarian development at male-producing temperatures following treatment with natural or synthetic estrogens, phenotypic and genetic responses can still be attributed to the treatment even if the dose is underestimated. Two of the OC contaminants found in the highest concentration in either serum or egg yolk samples from Lake Apopka are p,p'-DDE and toxaphene. Although p,p'-DDE has been shown to have a weak affinity for the alligator ER (Vonier et al. 1996), previous studies on mammals indicate that it inhibits androgen-dependent pathways (Gray et al. 2001). Embyronic exposure to p,p'-DDE did not influence sex determination at temperatures that typically produce all males, but did cause a higher than expected percentage of females at a temperature that normally produces both sexes (figure 6; Milnes et al. 2005a); in ovo exposure to toxaphene had no effect on sex determination, but did result in elevated plasma testosterone in the exposed neonates (Milnes et al. 2004). Interestingly, gonadal synthesis of testosterone was not affected, suggesting differences in hepatic degradation as a possible source of differences in circulating steroid concentrations. Intravenous exposure of juvenile alligators to toxaphene resulted in the up-regulation of CYP3A77 mRNA transcripts, an enzyme involved in biotransformation of xenobiotics and endogenous steroids (Gunderson et al. 2006).

Figure 6.

Sex ratios of alligators exposed in ovo to p,p'-DDE or estradiol-17ß and incubated at 32°C or 33.5°C. Treatments are reported in parts per billion wet egg mass. Asterisks denote a significant difference between treatment and the control and vehicle groups. The data are taken from Milnes and colleagues (2005a).

Figure 6.

Sex ratios of alligators exposed in ovo to p,p'-DDE or estradiol-17ß and incubated at 32°C or 33.5°C. Treatments are reported in parts per billion wet egg mass. Asterisks denote a significant difference between treatment and the control and vehicle groups. The data are taken from Milnes and colleagues (2005a).

In a recent study by Rauschenberger and colleagues (2007), captive alligators were exposed to an OC pesticide mixture in their diet to achieve environmentally relevant concentrations of contaminants in the egg yolk. The percentage of eggs within a clutch that hatched was lower in the treatment group than in controls, the result of a dramatic increase in non-banded eggs that were either infertile or experienced very early embryonic mortality. In the same study, differences in clutch viability among contaminated and reference populations (including Lake Apopka and Orange Lake) in the wild were accounted for by early and late embryo mortality, confirming an earlier study (Masson 1995). The stress of captivity, however, may have been a confounding factor in the more recent study. Only four of the six control females and three of the seven treated females produced clutches over the three years of the study (Rauschenberger et al. 2007), demonstrating the difficulty of captive breeding and transgenerational studies in this species.

Mechanistic studies and future directions.

We have begun to investigate the mechanistic actions of EDCs in alligators at the cellular and molecular levels. By quantifying gene expression related to endocrine function, we are beginning to understand the genetic attributes underlying the physiological basis of the abnormalities identified in Lake Apopka's alligators. Moreover, molecular cloning and the development of in vitro receptor binding and gene expression assays are providing a means of conducting predictive experiments that minimize the use of experimental animals (Kohno et al. 2008a). We hope that these experiments, in concert with existing comparative and in vivo studies, will reveal specific receptor-mediated pathways susceptible to endocrine disruption and reduce the use of experimental animals.

In a recent study, we observed lower expression of mRNA for steroidogenic factor-1 (NR5A1) and StAR in one-year old male alligators from Lake Apopka relative to those from Lake Woodruff NWR (Milnes et al. 2008b). A reduction in the expression of NR5A1 would be expected to reduce the synthesis of cholesterol by decreasing the transcription of steroidogenic enzymes such as CYP11A, and lower expression of StAR could reduce the availability of cholesterol to steroidogenic enzymes. These results provide a potential mechanism for previous studies that found that hatchling and juvenile males from Lake Apopka had lower plasma testosterone concentrations than did those from Lake Woodruff NWR.

In addition to examining steroidogenesis at the molecular level through an examination of the enzymes involved in this process, we have also examined steroid and thyroid receptor expression in various target tissues. In vertebrates, the steroid and thyroid receptors belong to a superfamily of nuclear transcription factors that include all other steroid hormone receptors (such as progestogens, androgens, gluco-corticoids, mineralocorticoids), the vitamin D receptor, and the retinoic acid receptor (Blumberg and Evans 1998). The ancestral condition for the jawed vertebrates (Gnathostomata) appears to have been the presence of two forms of ER, corresponding to ERα and ERß; two forms of thyroid receptor; one AR; and one progestin receptor (Thornton 2001). Two forms of ER have been found in mammals, fish, birds, reptiles, and amphibians. Our group cloned and sequenced ERα and ERß from the American alligator (Alligator mississippiensis; Katsu et al. 2004). The alligator steroid-receptor sequences can be divided into five domains on the basis of their sequence homology to other steroid hormone receptors. We have found that the hormone (ligand)- and DNA-binding domains of the alligator are highly conserved with birds and mammals, with the DNA-binding domain of the alligator ERa having 100% sequence similarity with the same region in the chicken, mouse, and human ERα (figure 7). Likewise, the amino acid sequence of the ligand-binding domain is also highly conserved among birds, mammals, and crocodilians such as the alligator (Katsu et al. 2004). These data suggest that environmental contaminants that act through the alligator ER are likely to act through the human ER as well, which is yet another reason to use wildlife as sentinels of environmental health: they could also forewarn us of dangers to our own health.

Figure 7.

Comparison of human ESR1 (ERα) protein with estrogen receptors of representative vertebrate species. The functional A/B to F domains are schematically represented with percentage similarities of sequence presented. Note the high sequence similarity (values at or close to 100%) between human, mouse, chicken, and alligator for the DNA-binding (DBD) and ligand-binding (LBD) domains.

Figure 7.

Comparison of human ESR1 (ERα) protein with estrogen receptors of representative vertebrate species. The functional A/B to F domains are schematically represented with percentage similarities of sequence presented. Note the high sequence similarity (values at or close to 100%) between human, mouse, chicken, and alligator for the DNA-binding (DBD) and ligand-binding (LBD) domains.

We have recently observed alterations in ERß, NR5A1, and StAR mRNA expression in testicular tissue from juvenile alligators from Lake Apopka (Kohno et al. 2008b). Likewise, alterations of aromatase and DAX1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1) mRNA expression in ovarian tissue could provide further insight into the mechanisms leading to modified folliculogenesis and ovarian development in females (Kohno et al. 2008b). Current studies are extending this work to examine the interaction between steroid hormones and their receptors and thyroid hormone action, via thyroid hormone nuclear receptors and thyroid hormone–dependent gene expression and ovarian development and functioning (Helbing et al. 2006). Given the conserved nature of the molecular mechanisms underlying the biology of reproduction among vertebrates, these studies have the potential to expand our understanding of the environmental and genetic interactions that lead to birth defects involving the reproductive system in alligators and other species.

Conclusions

Sublethal exposure to EDCs can have far-reaching consequences for both wildlife and human populations. In addition to its own intrinsic value, each wildlife population is a critical part of an ecosystem that depends upon interspecies relationships to maintain the processes that provide humans with necessary commodities such as clean air, water, soil, and food. Chemical exposure that reduces reproductive success within a population can upset the balance of processes that maintain a healthy ecosystem. Although direct extrapolation of the effects of EDCs on wildlife species to humans cannot be made, many of the molecular, cellular, and physiological processes regulated by the endocrine system are conserved throughout vertebrate evolution, and a weight-of-evidence approach should be used in guiding future policy decisions. The data presented here reveal alterations in development and endocrine function in alligators, a long-lived species that is chronically exposed to a complex mixture of environmental contaminants in Lake Apopka. The captive-raised alligator studies begin to separate the alterations of embryonic origin from those of environmental origin, and the laboratory-based studies identify mechanisms through which some of the chemicals can act.

The success of any population, human or wildlife, ultimately depends on survival and successful reproduction, not survival alone. Alligators have adapted their reproductive strategy to a type III survivorship curve, meaning they produce a large number of offspring with a low probability of survival. As individuals attain sexual maturity, the mortality rate decreases. Alligator populations in central Florida can withstand a loss of up to 50% of the estimated annual recruitment without a detectable decrease in population density, as evidenced by the monitored harvesting of eggs and hatchlings for commercial purposes on selected lakes (Rice et al. 1999). However, maintaining these populations depends on the production of viable offspring over succesive generations. The Tower Chemical Company spill on Lake Apopka occur red in 1980. From 1983 to 1986, egg viability was at an all-time low (approximately 20%) and the juvenile population declined from 1981 to 1987 (Woodward et al. 1993). This time period could indicate the acute toxicological effects of juvenile and adult contaminant exposure. Since the initial decline, egg viability and the number of juvenile alligators have gradually increased, and the current level of embryonic and posthatching mortality on Lake Apopka does not appear immediately detrimental to the population; however, nothing is yet known about the fecundity of the newly recruited individuals. From an evolutionary perspective, the cost of altered reproductive success in a species that takes 10 to 15 years to attain sexual maturity is difficult to predict. If the currently surviving offspring are selected for traits that confer resistance to contaminants, how do these traits contribute to or detract from reproductive success? We could be observing the gradual recovery of Lake Apopka's alligators in terms of population size and, simultaneously, a reduction in heritable variation and in the ability to cope with future challenges in a changing environment. As more chemicals that disrupt development are identified, the charge for the current and future generations is to evaluate the consequences of altered development on reproductive fitness. This will undoubtedly be particularly challenging to do for long-lived species such as alligators and humans.

Acknowledgments

Research described from the laboratory of L. J. G. was supported by grants from the University of Florida Opportunity Fund, NICHHD 1 R21 HD047885-01, and NIEHS R21 ES014053-01A1. We are especially grateful to Allan Woodward and the Florida Fish and Wildlife Conservation Commission for their continued support of this research. We thank Satomi Kohno for preparing the estrogen-receptor species comparison figure.

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Author notes

Matthew R. Milnes (e-mail: mmilnes@sandiegozoo.org) is a scientist at the Zoological Society of San Diego, Center for Conservation and Research for Endangered Species, in California.
Louis J. Guillette Jr. (e-mail: ljg@zoo.ufl.edu) is a professor in the Department of Zoology at the University of Florida, Gainesville.