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The thyroid gland is sensitive to steroid hormone signaling, and many thyroid disrupting contaminants also disrupt steroid hormone homeostasis, presenting the possibility that thyroid disruption may occur through altered steroid hormone signaling. To examine this possibility, we studied short-term and persistent impacts of embryonic sex steroid exposure on thyroid physiology in the American alligator. Alligators from a lake contaminated with endocrine disrupting contaminants (Lake Apopka, FL, USA) have been shown to display characteristics of thyroid and steroid hormone disruption. Previous studies suggest these alterations arise during development and raise the possibility that exposure to maternally deposited contaminants might underlie persistent organizational changes in both thyroidal and reproductive function. Thus, this population provides a system to investigate contaminant-mediated organizational thyroid disruption in an environmentally-relevant context. We assess the developmental expression of genetic pathways involved in thyroid hormone biosynthesis and find that expression of these genes increases prior to hatching. Further, we show that nuclear steroid hormone receptors are also expressed during this period, indicating the developing thyroid is potentially responsive to steroid hormone signaling. We then explore functional roles of steroid signaling during development on subsequent thyroid function in juvenile alligators. We exposed alligator eggs collected from both Lake Apopka and a reference site to 17β-estradiol and a non-aromatizable androgen during embryonic development, and investigated effects of exposure on hatchling morphometrics and thyroidal gene expression profiles at 5 months of age. Steroid hormone treatment did not impact the timing of hatching or hatchling size. Furthermore, treatment with steroid hormones did not result in detectable impacts on thyroid transcriptional programs, suggesting that precocious or excess estrogen and androgen exposure does not influence immediate or long-term thyroidal physiology.

Animal manures, used as a nitrogen source for crop production, are often associated with negative impacts on nutrient levels in surface water. The concentrations of estrogens in streams from these manures also are of concern due to potential endocrine disruption in aquatic species. Streams associated with livestock operations were sampled by discrete samples (n = 38) or by time-integrated polar organic chemical integrative samplers (POCIS, n = 19). Samples were analyzed for estrogens by gas chromatography-tandem mass spectrometry (GC-MS(2)) and estrogenic activity was assessed by three bioassays: Yeast Estrogen Screen (YES), T47D-KBluc Assay, MCF-7 Estrogenicity Screen (E-Screen). Samples were collected from 19 streams within small (≈ 1-30 km(2)) watersheds in 12 U.S. states representing a range of hydrogeologic conditions, dominated by: dairy (3), grazing beef (3), feedlot cattle (1); swine (5); poultry (3); and 4 areas where no livestock were raised or manure was applied. Water samples were consistently below the United Kingdom proposed Lowest Observable Effect Concentration for 17β-estradiol in fish (10 ng/L) in all watersheds, regardless of land use. Estrogenic activity was often higher in samples during runoff conditions following a period of manure application. Estrone was the most commonly detected estrogen (13 of 38 water samples, mean 1.9, maximum 8.3 ng/L). Because of the T47D-KBluc assay's sensitivity towards estrone (1.4 times 17β-estradiol) it was the most sensitive method for detecting estrogens, followed by the E-Screen, GC-MS(2), and YES. POCIS resulted in more frequent detections of estrogens than discrete water samples across all sites, even when applying the less-sensitive YES bioassay to the POCIS extracts.

Summary There is growing concern of exposure of fish, wildlife and humans to water sources contaminated with oestrogens and the potential impact on reproductive health. Environmental oestrogens can come from various sources including concentrated animal feedlot operations (CAFO), municipal waste, agricultural and industrial effluents. US EPA’s drinking water contaminant candidate list 3 (CCL3) includes several oestrogenic compounds. Although these contaminants are currently not subject to any proposed or promulgated national primary drinking water regulations, they are known or anticipated to occur in public water systems and may require future regulation under the Safe Drinking Water Act. Using an in vitro transcriptional activation assay, this study evaluated oestrogens from CCL3 both individually and as a seven oestrogen mixture (fixed ray design) over a broad range of concentrations, including environmentally relevant concentrations. Log EC50 and Hillslope values for individual oestrogens were as follows: estrone, −11.92, 1.283; estradiol-17α, −9.61, 1.486; estradiol-17β, 11.77, 1.494; estriol, −11.14, 1.074; ethinyl estradiol-17α, −12.63, 1.562; Mestranol, −11.08, 0.809 and Equilin, −11.48, 0.946. In addition, mixtures that mirrored the primary oestrogens found in swine, poultry and dairy CAFO effluent (fixed-ratio ray design), and a ternary mixture (4 × 4 × 4 factorial design) of oestrogens found in hormone replacement therapy and/or oral contraceptives were tested. Mixtures were evaluated for additivity using both the concentration addition (CA) model and oestrogen equivalence (EEQ) model. For each of the mixture studies, a broad range of concentrations were tested, both above and below environmentally relevant concentrations. Results show that the observed data did not vary consistently from either the CA or EEQ predictions for any mixture. Therefore, either the CA or EEQ model should be useful predictors for modelling oestrogen mixtures.

The expression of estrogen receptors, ESR1 (ERα) and ESR2 (ERβ), and androgen receptors (AR) in the thyroid gland has been reported in few vertebrate species other than a few mammals. This study reports the presence of sex steroid hormone receptors and thyroid receptors (ERα, ERβ, AR, TRα, and TRβ) in the thyroid gland of the American alligator at several life stages. It provides a semiquantification and distribution of ERα in the thyroid follicle cells using an immunohistochemical approach as well as reports quantitative differences in mRNA expression of ERα, ERβ, TRα, TRβ, and AR in the same tissue using quantitative real time-PCR (Q-PCR) with primers designed specifically for alligators. The thyroid tissue of the American alligator expresses ERα, ERβ, and AR at all of the life stages examined here although no statistically significant differences were observed between male and female in thyroid mRNA expression for any of the genes analyzed. No sexual dimorphism was observed in ERα immunostaining. No statistical analysis across life stages were performed due to confounding factor of season.

Exposure to xenoestrogens occurs against a backdrop to physiological levels of endogenous estrogens. Endogenous estrogen levels vary from low levels in early childhood to high levels during pregnancy and in young women. However, few studies have addressed how xenoestrogens interact with endogenous estrogens. The current study was designed to characterize the individual dose-response curves of estradiol-17beta (E(2)), bisphenol A (BPA), tetrabromo-bisphenol A (TBBPA), and bisphenol AF (BPAF, 4,4'-hexafluoroisopropylidene diphenol) on estrogen-dependent luciferase expression in T47D-KBluc cells and to determine how binary (8 x 8 factorial) and ternary (4 x 4 x 4 factorial) mixtures of an endogenous estrogen (E(2)) interact with BPA and/or BPAF. Log EC(50) and hillslope values with SEs, respectively, for individual compounds were as follows: E(2), -12.10M +/- 0.06071, 0.7702 +/- 0.1739; BPA, -6.679M +/- 0.08505, 1.194 +/- 0.2137; and BPAF, -7.648M +/- 0.05527, 1.273 +/- 0.1739. TBBPA was not evaluated in mixture studies because of its minimally estrogenic response at 3 x10(-5)M and elicited cytotoxicity at higher concentrations. Both the binary mixtures of E(2) with BPA and BPAF and the ternary mixture of E(2), BPA, and BPAF behaved in an additive manner. For binary mixtures, as E(2) concentration increased, higher concentrations of BPA and BPAF were necessary to induce a significant increase in the estrogenic response. Understanding the behavior of mixture interactions of xenoestrogens, like BPA and BPAF, with endogenous estrogens will allow a better assessment of the potential risk associated with exposure to these chemicals, individually or as mixtures.

Many chemicals introduced into the environment by humans adversely affect embryonic development and the functioning of the male reproductive system. It has been hypothesized that these developmental alterations are due to the endocrine-disruptive effects of various environmental contaminants. The endocrine system exhibits an organizational effect on the developing embryo. Thus, a disruption of the normal hormonal signals can permanently modify the organization and future function of the male reproductive system. A wide range of studies examining wildlife either in laboratories or in natural settings have documented alterations in the development of males. These studies have begun to provide the causal relationships between embryonic contaminant exposure and reproductive abnormalities that have been lacking in pure field studies of wild populations. An understanding of the developmental consequences of endocrine disruption in wildlife can lead to new indicators of exposure and a better understanding of the most sensitive life stages as well as the consequences of exposure during these periods.

Reduced reproductive success, altered reproductive tract development, and differences in circulating hormones have been documented in American alligators (Alligator mississippiensis) from Lake Apopka, FL, compared to less contaminated sites, such as the Lake Woodruff National Wildlife Refuge, FL. Comparative studies among alligators of varying size and age suggest that in ovo contaminant-induced alterations of endocrine function are further modified during postembryonic development and/or through environmental exposure. In the present study, we examined developmental and endocrine-related indices in neonatal (age

Circulating concentrations of thyroxine (T(4)) vary seasonally in many vertebrates. This study examined the seasonal variation in plasma concentrations of T(4) in juvenile American alligators (Alligator mississippiensis) from three populations in central Florida, USA. One site, Lake Woodruff National Wildlife Refuge, is considered a reference site whereas the other two lakes, Lake Apopka and Orange Lake, are significantly impacted by human activity. Juvenile American alligators ranging from 75-150 cm in total length were hand-captured at night from November 2000-April 2002. Plasma thyroxine concentrations were analyzed using a radioimmunoassay (RIA) previously validated for alligator plasma. Juvenile American alligators display seasonal variation in circulating T(4) concentrations. Plasma T(4) concentrations decrease from August/September to November and then begin a slow rise until April, at which point they plateau. Sex of juveniles influenced plasma concentrations of T(4) in some months but did not appear to alter the pattern in seasonal variation. The pattern we observed in plasma T(4) concentrations is not directly related to an environmental factor such as ambient temperature but is similar to that seen in plasma sex steroid concentrations during the reproductive cycle of adult alligators. Although the pattern and plasma concentration of T(4) exhibits significant variation among the three lakes studied, the pattern in seasonal variation appears similar. Comparing the seasonal pattern in plasma T(4) with plasma concentrations of sex steroids (testosterone and estradiol-17beta) or corticosterone could provide important information on the peripubescent life stage of the American alligator.

This 3-year study was designed to examine variation in plasma sex steroids, phallus size, and the standard error (S.E.) associated with these endpoints in juvenile alligators collected from 3 sites within the Kissimmee-Everglades drainage (Florida, USA) with varying concentrations of sediment organochlorine contaminants. We hypothesized that decreased plasma sex steroid concentrations and phallus size would be observed in the higher contaminant site when compared to the intermediate and lower contaminant sites. Furthermore, we hypothesized that greater S.E. associated with these endpoints would be observed for the populations from more contaminated sites. We found that differences existed with females from the higher contaminant site exhibiting lower plasma estradiol-17β (E 2 ) and testosterone (T) concentrations. Males from the higher contaminant site exhibited smaller phallus sizes than males from the intermediate and lower contaminant sites. Smaller phallus size in this case differed from that reported in Lake Apopka male alligators [Gen. Comp. Endocrinol. 116 (1999) 356] in that a significant positive relationship between body size and phallus size existed. No difference among sites was observed in plasma T for males. Lower S.E. was associated with E 2 and T concentrations in females from the higher contaminant site and in phallus size in males from the higher contaminant site. This pattern was opposite to what we had hypothesized. We concluded that variation in plasma E 2 and T concentrations, phallus size, and the S.E. associated with these endpoints exists among the 3 sites with the patterns matching the patterns of organochlorine contamination, although S.E. patterns were opposite to what was predicted.

Reproductive disorders have been found in pesticide-exposed alligators living in Lake Apopka, Florida (USA). These disorders have been hypothesized to be caused by exposure to endocrine- disruptive estrogen-like contaminants. The aim of this study was to expand our analysis beyond previous studies by investigating whether bone tissue, known to be affected by sex steroid hormones, is a potential target of endocrine disruptors. Long bones from 16 juvenile female alligators from Lake Apopka (pesticide-contaminated lake) and Lake Woodruff (control lake) were evaluated by peripheral quantitative computed tomography. We observed significant differences in bone composition, with female alligators from the contaminated lake having greater trabecular bone mineral density (BMD), total BMD, and trabecular mineral content compared with females from the control lake (p < 0.05). Increased trabecular and total BMD measurements suggest that juvenile female alligators from Lake Apopka were exposed to contaminants that created an internal environment more estrogenic than that normally observed. This estrogenic environment could be caused by both natural and anthropogenic compounds. Effects on BMD indicate interference with bone homeostasis. We hypothesize that contaminants present in the lake inhibit the natural and continuous resorption of bone tissue, resulting in increased bone mass. Although this is the only study performed to date examining effects of environmental estrogenic compounds on alligator bones, it supports previous laboratory-based studies in rodents. Further, this study is important in demonstrating that the alterations in morphology and physiology induced in free-ranging individuals living in environments contaminated with endocrine-active compounds are not limited to a few systems or tissues; rather, effects can be observed in many tissues affected by these hormones.

Morphological differences in spleen and thymus are closely related to functional immune differences. Hormonal regulation of the immune system has been demonstrated in reptilian splenic and thymic tissue. Spleens and thymus were obtained from juvenile alligators at two reference sites in Florida, USA: Orange Lake and a National Wildlife Refuge, Lake Woodruff, as well as from a contaminated lake, Lake Apopka. Lake Apopka has been extensively polluted with agricultural pesticides. Tissues were prepared for histological analysis to determine if previously detected endocrine abnormalities associated with contaminant exposure might also be reflected in morphological differences in splenic and thymic structures important for immunological response. Similar tissues were taken from captive-raised juvenile female alligators (3 years old) that were hatched from eggs collected on Lake Woodruff and Lake Apopka. Differences in thymic ratios (medulla/cortex) were found among alligators collected from the two lakes (P = 0.0051). Alligators from Lake Apopka had smaller thymic ratios than animals from either reference lake. Males from Lake Woodruff had significantly smaller lymphocyte sheaths in the spleen than females (P = 0.0009), indicative of a normal sexual dimorphism. Lymphocyte sheath width differed among females obtained from the three lakes, with females from Lake Apopka having the smallest sheath width and those from Orange Lake having the largest. Malpighian body area was largest in alligators from Orange Lake, intermediate in Lake Woodruff, and smallest in Lake Apopka. In contrast to that observed for wild-caught animals, no difference was found in the thymic medulla/cortex ratio of captive-raised female alligators (P = 0.378). Captive-raised female alligators from Lake Apopka and Lake Woodruff displayed lake-associated differences in lymphocyte sheath width as observed in wild animals; Lake Apopka alligators had smaller lymphocyte sheath width compared to Woodruff alligators (P = 0.0396). In contrast to wild-caught animals, area of the Malpighian bodies did not differ by lake in the captive-raised female alligators (P = 0.066). The enlarged thymic cortex suggests a change in T-lymphocyte maturation within the thymus of alligators from a contaminated environment, Lake Apopka. The results point to alterations in the histology of the thymus and spleen. Further studies are required to examine the functional significance of these observations.

We examined variation in plasma thyroxine (T 4 ) in juvenile American alligators (Alligator mississippiensis) collected from three sites within the Kissimmee River drainage hasin (FL, USA). Based on historical sediment data, Moonshine Bay served as the low contaminant exposure site, Water Conservation Area 3A served as an intermediate contaminant exposure site, and Belle Glade served as the high contaminate exposure site. In May 1999, alligators (n= 22) from Water Conservation Area 3A exhibited higher T 4 concentrations than animals from both Belle Glade (n= 22; p=0.0003) and Moonshine Bay (n = 33; p = 0.001). In May 2000, alligators (n = 29) Water Conservation Area 3A again exhibited higher T, concentrations than those from Belle Glade (n = 49; p = 0.02) but not those from Moonshine Bay (n = 40). No sexual dimorphism was observed among mean T 4 concentrations within any of the sites during either year (p > 0.05). Animals within all sites exhibited higher T 4 concentrations in May 2000 when compared to May 1999. When variance was examined, animals from Water Conservation Area 3A exhibited higher variance in plasma T 4 concentrations than those from either Moonshine Bay or Belle Glade. We concluded that mean plasma T 4 concentrations did not match the sediment contaminant mixture data presently available to us, whereas variance seems to he a more reliable indicator of contaminant exposure.

Reproductive and developmental abnormalities have been reported in the American alligator (Alligator mississippiensis) population from Lake Apopka, FL, that is chronically exposed to a complex mixture of environmental contaminants. To begin to understand the molecular mechanisms that could lead to the observed abnormalities of the reproductive and endocrine system, we quantified concentrations of the steroid hormones testosterone (T) and estradiol-17β (E2) and expression of steroid hormone receptors and genes relating to steroidogenesis in gonadal tissue from juvenile alligators from three lakes in Florida using enzyme immunoassay and quantitative real-time polymerase chain reaction. Alterations of ESR2 (estrogen receptor β) and SF1 (steroidogenic factor 1) mRNA expression in male gonadal tissue, without an observed difference in plasma concentrations of T, from the different lakes, begin to provide insight into potential mechanisms underlying the alterations of the reproductive system previously observed. Likewise, alterations in P450 aromatase and DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita critical region on the X chromosome, gene 1) mRNA expression, with elevated plasma E2 concentrations in females, provide leads to the potential mechanisms modifying folliculogenesis and ovarian development. The investigation of these genes also helps clarify normal endocrine and reproductive system function in the American alligator.

Inhibitor of growth 2 (ING2) belongs to a family of tumor suppressors that are important regulators of a wide range of cellular processes including proliferation, apoptosis, and DNA repair. ING family members are found in yeast, plants, invertebrates and many vertebrate species. However, to date, ING has not been characterized in reptiles. Herein we describe the isolation of expressed ING2 sequence in the American alligator, Alligator mississippiensis, and compare this sequence with that isolated in the chicken. We identify features that are unique to these two representatives of the Archosaurs including conservation of specific amino acid residues and the absence of an adenylate residue in the 5' end of the nucleotide sequence relative to frogs and mammals. The latter feature results in an alteration of the coding potential leading to distinctive N-termini. Injection of juvenile alligators with thyroid stimulating hormone (TSH), which increases endogenous thyroid hormones, results in the modulation of ING2 transcript levels. Quantitative real time polymerase chain reaction analyses revealed a reduction in the steady-state levels of ING2 mRNA in the phallus/cliterophallus, lung, and liver by 48 h after TSH injection. ING2 expression in the thyroid gland, gonad, and heart was unaffected by TSH treatment. These data indicate that control of ING2 expression by the thyroid axis may be conserved among species and is tissue-dependent.

Thyroid hormones (THs) play key regulatory roles in growth, development and metabolism in vertebrates. Modulation of the cellular hormonal response is largely through the activity of two nuclear TH receptors, TRalpha and TRbeta, which act as transcription factors and alter gene expression programs. Little information is available regarding their structure and regulation in reptiles. We have cloned the expressed sequences encoding these two receptors in the American alligator, Alligator mississippiensis. The encoded putative proteins share a high degree of amino acid sequence conservation with other vertebrates, however, both alligator TRs contain putative N-terminal truncations. This phenomenon is shared with the chicken for TRbeta, but not for TRalpha, making this the first demonstration of this type of TRalpha isoform. We measured the steady-state levels of TR transcripts in heart, lung, liver, thyroid, cliterophallus/phallus, and gonad of juvenile alligators 24 and 48 h after injection with thyroid stimulating hormone (TSH). TRalpha transcript levels were increased in the heart, decreased in the lung and cliterophallus/phallus, and unaffected in the liver, thyroid, and gonad. TRbeta transcript levels were increased in the heart, lung, and gonad whereas estrogen receptor alpha transcript levels were elevated by TSH treatment only in the gonad. Modulation of these transcripts in the gonad is consistent with TH playing an important role in this tissue's function since seasonal TH fluctuations coincide with reproductive events. These data demonstrate that alligator tissues are differentially responsive to TSH by regulation of TR expression and provide an important comparative framework among vertebrates.

Steroid hormones perform many essential roles in vertebrates during embryonic development, reproduction, growth, water balance, and responses to stress. The estrogens are essential for normal reproductive activity in female and male vertebrates and appear to have direct actions during sex determination in some vertebrates. To begin to understand the molecular mechanisms of estrogen action in alligators, we have isolated cDNAs encoding the estrogen receptors (ER) from the ovary. Degenerate PCR primers specific to ER were designed and used to amplify alligator ovary RNA. Two different DNA fragments (ERalpha and ERbeta) were obtained and the full-length alligator ERalpha cDNA was obtained using 5' and 3' RACE. The inferred amino acid sequence of alligator ERalpha (aERalpha) was very similar to the chicken ERalpha (91% identity), although phylogenetic analyses suggested profound differences in the rate of sequence evolution for vertebrate ER sequences. We also isolated partial DNA fragments encoding ERbeta and the progesterone receptor (PR) of the alligator, both of which show strong sequence similarities to avian ERbeta and PR. We examined the expression levels of these three steroid receptors (ERalpha, ERbeta, and PR) in the ovary of juvenile alligators and observed detectable levels of all three receptors. Quantitative RT-PCR showed that gonadal ERalpha transcript levels in juvenile alligators decreased after E2 treatment whereas ERbeta and PR transcripts were not changed. These results provide tools that will allow future studies examining the regulation and ontogenic expression of steroid receptors in alligators and expand our knowledge of vertebrate steroid receptor evolution.

We examined variation in plasma thyroxine (T4) in juvenile American alligators (Alligator mississippiensis) collected from three sites within the Kissimmee River drainage basin (FL, USA). Based on historical sediment data, Moonshine Bay served as the low contaminant exposure site, Water Conservation Area 3A served as an intermediate contaminant exposure site, and Belle Glade served as the high contaminate exposure site. In May 1999, alligators (n = 22) from Water Conservation Area 3A exhibited higher T4 concentrations than animals from both Belle Glade (n = 22; p = 0.0003) and Moonshine Bay (n = 33; p = 0.001). In May 2000, alligators (n = 29) Water Conservation Area 3A again exhibited higher T4 concentrations than those from Belle Glade (n = 49; p = 0.02) but not those from Moonshine Bay (n = 40). No sexual dimorphism was observed among mean T4 concentrations within any of the sites during either year (p0.05). Animals within all sites exhibited higher T4 concentrations in May 2000 when compared to May 1999. When variance was examined, animals from Water Conservation Area 3A exhibited higher variance in plasma T4 concentrations than those from either Moonshine Bay or Belle Glade. We concluded that mean plasma T4 concentrations did not match the sediment contaminant mixture data presently available to us, whereas variance seems to be a more reliable indicator of contaminant exposure.