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DDE is a metabolite of DDT, which was formerly used as a pesticide. DDE can inhibit androgen binding to the androgen receptor, and androgen-induced gene expression in vivo . DDE treatment in utero reduces androgen-dependent organ weights in adult rats and anogenital distance of male pups at birth; therefore, DDE acts as an anti-androgen.

17α,20β-Dihydroxy-4-pregnen-3-one (17α,20β-DP), also known as a maturation-inducing hormone (MIH), is required for the meiotic maturation of oocytes and sperm maturation in teleost fish. It is produced in the ovarian follicle from the precursor, 17α-hydroxyprogesterone, by the enzyme 20β-hydroxysteroid dehydrogenase. The 17α,20β-DP acts via its receptor, 17α,20β-DPR, on the plasma membrane of oocytes. It also has a nuclear receptor-mediated action in the testis of the Japanese eel.

Polybrominated diphenyl ethers (PBDEs) are a group of organobromine compounds, used as flame retardants, which affect thyroid hormone systems and estrogen and androgen systems. There are 209 possible congeners. PBDE congeners inhibit androgen receptor binding, and in utero treatment reduces sex hormone levels in male offspring of rats.

Some chemicals in the environment disrupt thyroid hormone (TH) systems leading to alterations in organism development, but their effect mechanisms are poorly understood. In fish, this has been limited by a lack of fundamental knowledge on thyroid gene ontogeny and tissue expression in early life stages. Here we established detailed expression profiles for a suite of genes in the hypothalamic-pituitary-thyroid (HPT) axis of zebrafish (Danio rerio) between 24-120 h post fertilisation (hpf) and quantified their responses following exposure to 3,3',5-triiodo-L-thyronine (T3) using whole mount in situ hybridisation (WISH) and qRT-PCR (using whole-body extracts). All of the selected genes in the HPT axis demonstrated dynamic transcript expression profiles across the developmental stages examined. The expression of thyroid receptor alpha (thraa) was observed in the brain, gastrointestinal tract, craniofacial tissues and pectoral fins, while thyroid receptor beta (thrb) expression occurred in the brain, otic vesicles, liver and lower jaw. The TH deiodinases (dio1, dio2 and dio3b) were expressed in the liver, pronephric ducts and brain and the patterns differed depending on life stage. Both dio1 and dio2 were also expressed in the intestinal bulb (96-120 hpf), and dio2 expression occurred also in the pituitary (48-120 hpf). Exposure of zebrafish embryo-larvae to T3 (30 and 100 μg L-1) for periods of 48, 96 or 120 hpf resulted in the up-regulation of thraa, thrb, dio3b, thyroid follicle synthesis proteins (pax8) and corticotropin-releasing hormone (crhb) and down-regulation of dio1, dio2, glucuronidation enzymes (ugt1ab) and thyroid stimulating hormone (tshb) (assessed via qRT-PCR) and responses differed across life stage and tissues. T3 induced thraa expression in the pineal gland, pectoral fins, brain, somites, gastrointestinal tract, craniofacial tissues, liver and pronephric ducts. T3 enhanced thrb expression in the brain, jaw cartilage and intestine, while thrb expression was suppressed in the liver. T3 exposure suppressed the transcript levels of dio1 and dio2 in the liver, brain, gastrointestinal tract and craniofacial tissues, while dio2 signalling was also suppressed in the pituitary gland. Dio3b expression was induced by T3 exposure in the jaw cartilage, pectoral fins and brain. The involvement of THs in the development of numerous body tissues and the responsiveness of these tissues to T3 in zebrafish highlights their potential vulnerability to exposure to environmental thyroid-disrupting chemicals.

The medaka extended one-generation test (MEOGRT) was developed as a multigenerational toxicity test for chemicals, particularly endocrine-disrupting chemicals. Briefly, 3 generations of Japanese medaka (Oryzias latipes) are exposed to a chemical over a 20-wk period: 3 wk in the parental generation (F0), 15 wk in the first generation (F1), and 2 wk in the second generation (F2). The present study reports the first MEOGRT results concerning branched isomer mixtures of 4-nonylphenol (NP). Adult F0 medaka exposed to NP at 5 actual concentrations (1.27, 2.95, 9.81, 27.8, 89.4 µg/L) were unaffected in terms of reproduction, although vitellogenin in the male liver was increased dose-dependently at concentration of 2.95 µg/L and higher. In F1, in contrast, total egg (fecundity), fertile egg, and fertility decreased as NP increased; lowest-observed-effect concentrations (LOECs) for total egg, fertile egg, and fertility were 1.27, 1.27, 27.8 µg/L, respectively. In F1, but not in F0, secondary sex characteristics (i.e., anal fin papillae in males) were suppressed at 27.8 µg/L NP. Vitellogenin induction in adult male fish was slightly weaker in F1 than it was in F0, however. Gonadal sex abnormality and sex reversal occurred at 27.8 and 89.4 µg/L NP in F1 subadults. At 89.4 µg/L NP, all genotypic F1 males in breeding pairs had female phenotype, and some even demonstrated spawning. Concentrations of NP lower than 89.4 µg/L did not affect F2 survival or hatching. The highest detected NP level in environmental freshwater in Japan was approximately a half of the LOEC (1.27 µg/L for F1 fecundity); in other countries, however, environmental concentrations above the LOEC are reported, suggesting that NP may be affecting fish populations. Environ Toxicol Chem 2017;36:3254-3266. © 2017 SETAC.

Many concerns have been expressed regarding the possible adverse effects of thyroid hormone-disrupting chemicals in the environment. The disruption of thyroid hormones in the neonatal period may lead to permanent effects on thyroid hormone homeostasis as well as related developmental disorders, as thyroid hormones are essential for regulating the growth and differentiation of many tissues. To understand the long-term alteration in gene expressions by neonatal administration of thyroid hormone-like chemicals in general, we identified genes whose expression was altered in the liver, an important component of the thyroid hormone axis, by neonatal exposure to triiodothyronine (T3). T3 was administered to male F344 rats on postnatal days 1, 3, and 5 (week 0). At 8 weeks of age, cDNA microarray analysis was used to identify hepatic genes whose expression was altered by neonatal exposure to T3. Among the up-regulated genes that were identified, the expression of Olr59, Ethe1, and Slc10a2 increased specifically in rats neonatally exposed to T3. Interestingly, altered hepatic expression of these genes indeed increased when a hydroxylated polybrominated diphenyl ether (PBDE), OH-BDE42, which is capable of binding to the TR, was given neonatally. Our data demonstrated that neonatal exposure to thyroid hormones could affect the long-term expression of the genes, which could be useful markers for neonatal effects by thyroid hormone-disrupting chemicals. Copyright © 2017 John Wiley & Sons, Ltd.

Sex determination of Daphnia pulex is decided by environmental conditions. We established a suitable experimental system for this study using D. pulex WTN6 strain, in which the sex of the offspring can be controlled by photoperiod. Long-day conditions induced females and short-day conditions induced males. Using this system, we previously found that methy farnesoate (MF), which is a putative innate juvenile hormone molecule in daphnids, is necessary for male sex determination and that protein kinase C (PKC) is a candidate factor of male sex determiner. In this study, we demonstrated that a PKC inhibitor [bisindolylmaleimide IV (BIM)] application strongly suppressed male offspring induction in the short-day condition. Moreover, co-treatment of BIM with MF revealed that PKC signaling acts upstream of MF signaling for male sex determination. This is the first experimental evidence that PKC is involved in the male sex determination process associated with methyl farnesoate signaling in daphnid species., Summary: This study is the first experimental evidence that PKC is involved in the male sex determination process associated with methyl farnesoate signaling in daphnid species.

Estrogens regulate many physiological responses in vertebrates by binding to the estrogen receptor (ER), a ligand-activated transcription factor. To understand the evolution of vertebrate ERs and to investigate how estrogen acts in a jawless vertebrate, we used degenerate primer sets and PCR to isolate DNA fragments encoding two distinct ER subtypes, Esr1a and Esr1b from the Japanese lamprey, Lethenteron japonicum. Phylogenetic analysis indicates that these two ERs are the result of lineage-specific gene duplication within the jawless fishes, different from the previous duplication event of Esr1 (ERα) and Esr2 (ERβ) within the jawed vertebrates. Reporter gene assays show that lamprey Esr1a displays both constitutive and estrogen-dependent activation of gene transcription. Domain swapping experiments indicate that constitutive activity resides in the A/B domain of lamprey Esr1a. Unexpectedly, lamprey Esr1b does not bind estradiol and is not stimulated by other estrogens, androgens or corticosteroids. A 3D model of lamprey Esr1b suggests that although estradiol fits into the steroid binding site, some stabilizing contacts between the ligand and side chains that are found in human Esr1 and Esr2 are missing in lamprey Esr1b.

The freshwater zooplankton Daphnia magna has been extensively employed in chemical toxicity tests such as OECD Test Guidelines 202 and 211. Previously, it has been demonstrated that the treatment of juvenile hormones (JHs) or their analogues to female daphnids can induce male offspring production. Based on this finding, a rapid screening method for detection of chemicals with JH-activity was recently developed using adult D. magna. This screening system determines whether a chemical has JH-activity by investigating the male offspring inducibility. Although this is an efficient high-throughput short-term screening system, much remains to be discovered about JH-responsive pathways in the ovary, and whether different JH-activators act via the same mechanism. JH-responsive genes in the ovary including developing oocytes are still largely undescribed. Here, we conducted comparative microarray analyses using ovaries from Daphnia magna treated with fenoxycarb (Fx; artificial JH agonist) or methyl farnesoate (MF; a putative innate JH in daphnids) to elucidate responses to JH agonists in the ovary, including developing oocytes, at a JH-sensitive period for male sex determination. We demonstrate that induction of hemoglobin genes is a well-conserved response to JH even in the ovary, and a potential adverse effect of JH agonist is suppression of vitellogenin gene expression, that might cause reduction of offspring number. This is the first report demonstrating different transcriptomics profiles from MF and an artificial JH agonist in D. magna ovary, improving understanding the tissue-specific mode-of-action of JH. Copyright © 2016 John Wiley & Sons, Ltd.

The Larval Amphibian Growth and Development Assay (LAGDA) is a globally harmonized chemical testing guideline developed by the U.S. Environmental Protection Agency in collaboration with Japan's Ministry of Environment to support risk assessment. The assay is employed as a higher tiered approach to evaluate effects of chronic chemical exposure throughout multiple life stages in a model amphibian species, Xenopus laevis. To evaluate the utility of the initial LAGDA design, the assay was performed using a mixed mode of action endocrine disrupting chemical, benzophenone-2 (BP-2). X. laevis embryos were exposed in flow-through conditions to 0, 1.5, 3.0 or 6.0 mg l-1 BP-2 until 2 months post-metamorphosis. Overt toxicity was evident throughout the exposure period in the 6.0 mg l-1 treatment due to elevated mortality rates and observed liver and kidney pathologies. Concentration-dependent increases in severity of thyroid follicular cell hypertrophy and hyperplasia occurred in larval tadpoles indicating BP-2-induced impacts on the thyroid axis. Additionally, gonads were impacted in all treatments with some genetic males showing both testis and ovary tissues (1.5 mg l-1 ) and 100% of the genetic males in the 3.0 and 6.0 mg l-1 treatments experiencing complete male-to-female sex reversal. Concentration-dependent vitellogenin induction occurred in both genders with associated accumulations of protein in the livers, kidneys and gonads, which was likely vitellogenin and other estrogen-responsive yolk proteins. This is the first study that demonstrates the endocrine effects of this mixed mode of action chemical in an amphibian species and demonstrates the utility of the LAGDA design for supporting chemical risk assessment. Copyright © 2016 John Wiley & Sons, Ltd.

The Larval Amphibian Growth and Development Assay (LAGDA) is a globally harmonized test guideline developed by the U.S. Environmental Protection Agency in collaboration with Japan's Ministry of the Environment. The LAGDA was designed to evaluate apical effects of chronic chemical exposure on growth, thyroid-mediated amphibian metamorphosis and reproductive development. During the validation phase, two well-characterized endocrine-disrupting chemicals were tested to evaluate the performance of the initial assay design: xenoestrogen 4-tert-octylphenol (tOP) and xenoandrogen 17β-trenbolone (TB). Xenopus laevis embryos were exposed, in flow-through conditions, to tOP (nominal concentrations: 0.0, 6.25, 12.5, 25 and 50 µg l-1 ) or TB (nominal concentrations: 0.0, 12.5, 25, 50 and 100 ng l-1 ) until 8 weeks post-metamorphosis, at which time growth measurements were taken, and histopathology assessments were made of the gonads, reproductive ducts, liver and kidneys. There were no effects on growth in either study and no signs of overt toxicity, sex reversal or gonad dysgenesis. Exposure to tOP caused a treatment-related decrease in circulating thyroxine and an increase in thyroid follicular cell hypertrophy and hyperplasia (25 and 50 µg l-1 ) during metamorphosis. Mullerian duct development was affected after exposure to both chemicals; tOP exposure caused dose-dependent maturation of oviducts in both male and female frogs, whereas TB exposure caused accelerated Mullerian duct regression in males and complete regression in >50% of the females in the 100 ng l-1 treatment. Based on these results, the LAGDA performed adequately to evaluate apical effects of chronic exposure to two endocrine-active compounds and is the first standardized amphibian multiple life stage toxicity test to date. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Estrogen-mediated actions in female reproductive organs are tightly regulated, mainly through estrogen receptor 1 (ESR1). The mouse vaginal epithelium cyclically exhibits cell proliferation and differentiation in response to estrogen and provides a unique model for analyzing the homeostasis of stratified squamous epithelia. To address the role of ESR1-mediated tissue events during homeostasis, we analyzed mice with a vaginal epithelium-specific knockout of Esr1 driven by keratin 5-Cre (K5-Esr1KO). We show here that loss of epithelial ESR1 in the vagina resulted in aberrant epithelial cell proliferation in the suprabasal cell layers and led to failure of keratinized differentiation. Gene expression analysis showed that several known estrogen target genes, including erbB growth factor ligands, were not induced by estrogen in the K5-Esr1KO mouse vagina. Organ culture experiments revealed that the addition of erbB growth factor ligands, such as amphiregulin, could activate keratinized differentiation in the absence of epithelial ESR1. Thus, epithelial ESR1 integrates estrogen and growth factor signaling to mediate regulation of cell proliferation in squamous differentiation, and our results provide new insights into estrogen-mediated homeostasis in female reproductive organs.

Androgens are essential for the development, reproduction, and health throughout the life span of vertebrates, particularly during the initiation and maintenance of male sexual characteristics. Androgen signaling is mediated by the androgen receptor (AR), a member of the steroid nuclear receptor superfamily. Mounting evidence suggests that environmental factors, such as exogenous hormones or contaminants that mimic hormones, can disrupt endocrine signaling and function. The American alligator (Alligator mississippiensis), a unique model for ecological research in that it exhibits environment-dependent sex determination, is oviparous and long lived. Alligators from a contaminated environment exhibit low reproductive success and morphological disorders of the testis and phallus in neonates and juveniles, both associated with androgen signaling; thus, the alterations are hypothesized to be related to disrupted androgen signaling. However, this line of research has been limited because of a lack of information on the alligator AR gene. Here, we isolated A mississippiensis AR homologs (AmAR) and evaluated receptor-hormone/chemical interactions using a transactivation assay. We showed that AmAR responded to all natural androgens and their effects were inhibited by cotreatment with antiandrogens, such as flutamide, p,p'-dichlorodiphenyldichloroethylene, and vinclozolin. Intriguingly, we found a spliced form of the AR from alligator cDNA, which lacks seven amino acids within the ligand-binding domain that shows no response to androgens. Finally, we have initial data on a possible dominant-negative function of the spliced form of the AR against androgen-induced AmAR.

Exposure to endocrine disrupting chemicals (EDCs) can elicit adverse effects on development, sexual differentiation, and reproduction in fish. Teleost species exhibit at least three subtypes of estrogen receptor (ESR), ESR1, ESR2a, and ESR2b; thus, estrogenic signaling pathways are complex. We applied in vitro reporter gene assays for ESRs in five fish species to investigate the ESR subtype-specificity for better understanding the signaling pathway of estrogenic EDCs. Responses to bisphenol A, 4-nonylphenol, and o,p'-DDT varied among ESR subtypes, and the response pattern of ESRs was basically common among the different fish species. Using a computational in silico docking model and through assays quantifying transactivation of the LBD (using GAL-LBD fusion proteins and chimera proteins for the ESR2s), we found that the LBD of the different ESR subtypes generally plays a key role in conferring responsiveness of the ESR subtypes to EDCs. These results also indicate that responses of ESR2s to EDCs cannot necessarily be predicted from the LBD sequence alone, and an additional region is required for full transactivation of these receptors. Our data thus provide advancing understanding on receptor functioning for both basic and applied research.

Estrogen receptor α (ERα) plays a pivotal role in the mouse uterine and vaginal epithelial cell proliferation stimulated by estrogen, whereas ERβ inhibits cell proliferation. ERβ mRNA is expressed in neonatal uteri and vaginae; however, its functions in neonatal tissues have not been ascertained. In this study, we investigated the ontogenic mRNA expression and localization of ERβ, and its roles in cell proliferation in neonatal uteri and vaginae of ERβ knockout (βERKO) mice. ERβ mRNA and protein were abundant in the uterine and vaginal epithelia of 2-day-old mice and decreased with age. In uterine and vaginal epithelia of 2-day-old βERKO mice, cell proliferation was greater than that in wild-type animals and in uterine epithelia of 90- and 365-day-old βERKO mice. In addition, p27 protein, known as a cyclin-dependent kinase inhibitor, was decreased in the uteri of 90- and 365-day-old βERKO mice. Inhibition of neonatal ERs by ICI 182780 (an ER antagonist) treatment stimulated cell proliferation and decreased p27 protein in the uterine luminal epithelium of 90-day-old mice but not in the vaginal epithelium. These results suggest that neonatal ERβ is important in the persistent inhibition of epithelial cell proliferation with accumulation of p27 protein in the mouse uterus. Thus, suppression of ERβ function in the uterine epithelium during the neonatal period may be responsible for a risk for proliferative disease in adults.

Androgen receptors (ARs) mediate the physiological effects of androgens in vertebrates. In fishes, AR-mediated pathways can be modulated by aquatic contaminants, resulting in the masculinisation of female fish or diminished secondary sex characteristics in males. The Murray-Darling rainbowfish (Melanotaenia fluviatilis) is a small-bodied freshwater teleost used in Australia as a test species for environmental toxicology research. We determined concentration-response profiles for selected agonists and antagonists of rainbowfish ARα and ARβ using transient transactivation assays. For both ARα and ARβ, the order of potency of natural agonists was 11-ketotestosterone (11-KT)>5α-dihydrotestosterone>testosterone>androstenedione. Methyltestosterone was a highly potent agonist of both receptors relative to 11-KT. The relative potency of the veterinary growth-promoting androgen, 17β-trenbolone, varied by more than a factor of 5 between ARα and ARβ. The non-steroidal anti-androgen bicalutamide exhibited high inhibitory potency relative to the structurally related model anti-androgen, flutamide. The inhibitory potency of the agricultural fungicide, vinclozolin, was approximately 1.7-fold relative to flutamide for ARα, but over 20-fold in the case of ARβ. Fluorescent protein tagging of ARs showed that the rainbowfish ARα subtype is constitutively localised to the nucleus, while ARβ is cytoplasmic in the absence of ligand, an observation which agrees with the reported subcellular localisation of AR subtypes from other teleost species. Collectively, these data suggest that M. fluviatilis ARα and ARβ respond differently to environmental AR modulators and that in vivo sensitivity to contaminants may depend on the tissue distribution of the AR subtypes at the time of exposure.

There have been many concerns about the possible adverse effects of thyroid hormone-disrupting chemicals in the environment. Because thyroid hormones are essential for regulating the growth and differentiation of many tissues, disruption of thyroid hormones during the neonatal period of an organism might lead to permanent effects on that organism. We postulated that there are target genes that are sensitive to thyroid hormones particularly during the neonatal period and that would thus be susceptible to thyroid hormone-disrupting chemicals. Global gene expression analysis was used to identify these genes in the liver of rat neonates. The changes in hepatic gene expression were examined 24 h after administering 1.0, 10, and 100 ng/g body weight (bw) triiodothyronine (T3) to male rats on postnatal day 3. Thirteen upregulated and four downregulated genes were identified in the neonatal liver. Among these, Pdp2 and Slc25a25 were found to be upregulated and more sensitive to T3 than the others, whereas Cyp7b1 and Hdc were found to be downregulated even at the lowest dose of 1.0 ng/g bw T3. Interestingly, when the responses of gene expression to T3 were examined in adult rats (8-week old), one-third of them did not respond to T3. The environmental chemicals with thyroid hormone-like activity, hydroxylated polybrominated diphenyl ethers, were then administered to neonatal rats to examine the effects on expression of the identified genes. The results showed that these chemicals were indeed capable of changing the expression of Slc25a25 and Hdc. Our results demonstrated a series of hepatic T3-responsive genes that are more sensitive to hormones during the neonatal period than during adulthood. These genes might be the potential targets of thyroid hormone-disrupting chemicals in newborns.

Testis-ova differentiation in sexually mature male medaka (Oryzias latipes) is easily induced by estrogenic chemicals, indicating that spermatogonia persist in sexual bipotentiality, even in mature testes in medaka. By contrast, the effects of estrogen on testicular somatic cells associated with testis-ova differentiation in medaka remain unclear. In this study, we focused on the dynamics of sex-related genes (Gsdf, Dmrt1, and Foxl2) expressed in Sertoli cells in the mature testes of adult medaka during estrogen-induced testis-ova differentiation. When mature male medaka were exposed to estradiol benzoate (EB; 800ng/L), testis-ova first appeared after EB treatment for 14days (observed as the first oocytes of the leptotene-zygotene stage). However, the testis remained structurally unchanged, even after EB treatment for 28days. Although Foxl2 is a female-specific sex gene, EB treatment for 7days induced Foxl2/FOXL2 expression in all Sertoli cell-enclosed spermatogonia before testis-ova first appeared; however, Foxl2 was not detected in somatic cells in control testes. Conversely, Sertoli-cell-specific Gsdf mRNA expression levels significantly decreased after EB treatment for 14days, and no changes were observed in DMRT1 localization following EB treatment, whereas Dmrt1 mRNA levels increased significantly. Furthermore, after EB exposure, FOXl2 and DMRT1 were co-localized in Sertoli cells during testis-ova differentiation, although FOXL2 localization was undetectable in Sertoli-cell-enclosed apoptotic testis-ova, whereas DMRT1 remained localized in Sertoli cells. These results indicated for the first time that based on the expression of female-specific sex genes, feminization of Sertoli cells precedes testis-ova differentiation induced by estrogen in mature testes in medaka; however, complete feminization of Sertoli cells was not induced in this study. Additionally, it is suggested strongly that Foxl2 and Gsdf expression constitute potential molecular markers for evaluating the effects of estrogenic chemicals on testicular somatic cells associated with estrogen-induced testis-ova differentiation in mature male medaka.

Various receptor bioassays, including estrogens, androgens and thyroid hormones, have been developed and applied successfully for assessing hormone function in a wide range of animal species, including fish. In fish, corticosteroids play a pivotal role in physiology as they do in mammals, but far less is known about the corticosteroid receptor system in fish compared with in mammals. Here we established a transient transactivation assay using the Japanese medaka, Oryzias latipes, glucocorticoid receptors (olGRs) and mineralocorticoid receptor to analyse their functional properties in a fish. We found that olGR2 was highly responsive to glucocorticoids, similar to the human GR, whereas the olGR1 subtype was minimally responsive. Thus, olGR2 most likely mediates glucocorticoid signaling in medaka. We further tested crosstalk between GRs and other steroid hormones, and found that progestins could activate or inactivate olGR2-mediating transcription, depending on the presence or absence of cortisol. The transactivation assays developed for medaka GRs provide tools to gain useful insights into corticosteroid signaling in fish and for in vitro screening of environmental substances activating GRs.

Comparatively, little data are available detailing the geographic variation that exists in the reproductive endocrinology of adult alligators, especially those living in barrier islands. The Merritt Island National Wildlife Refuge (MI) is a unique barrier island environment and home to the Kennedy Space Center (FL, USA). Seasonal patterns of sex steroids were assessed in adult female American alligators from MI monthly from 2008 to 2009, with additional samples collected at more random intervals in 2006, 2007, and 2010. Plasma 17β-estradiol and vitellogenin concentrations peaked in April, coincident with courtship and mating, and showed patterns similar to those observed in adult female alligators in other regions. Plasma concentrations of progesterone, however, showed patterns distinctly different than those reported for alligator populations in other regions and remained relatively constant throughout the year. Plasma DHEA peaked in July around the time of oviposition, decreased in August, and then remained constant for the remaining months, except for a moderate increase in October. Circulating concentrations of DHEA have not been previously assessed in a female crocodilian, and plasma concentrations coincident with reproductive activity suggest a reproductive and/or behavioral role. Interestingly, plasma testosterone concentrations peaked in May of 2008, as has been shown in female alligator populations in other regions, but showed no peak in 2009, demonstrating dramatic variability from year to year. Surveys showed 2009 to be particularly depauperate of alligator nests in MI, and it is possible that testosterone could serve as a strong indicator of breeding success.

The endocrine responses in male Murray rainbowfish (Melanotaenia fluviatilis) were evaluated after exposures to biologically active concentrations of the nonsteroidal pharmaceutical, flutamide. Fish were exposed to nominal concentrations of 125 µg/L, 250 µg/L, 500 µg/L, and 1000 µg/L of flutamide for 7 d, after which plasma vitellogenin concentration; brain aromatase activity; and hepatic expression of the genes for vitellogenin, choriogenin, and androgen and estrogen receptors were assessed. Qualitative assessment of the testes of the fish exposed to flutamide exhibited hindrance in the transformation of spermatogonia to spermatozoa and increased testicular anomalies, such as multinucleated and pyknotic cells and interstitial fibrosis. An increase in the hepatosomatic index with respect to the controls was noted after treating the fish with flutamide at all concentrations. Vitellogenin was induced in plasma in the 1000 µg/L flutamide group. The activity of aromatase in the brain declined significantly after exposures to flutamide at all concentrations. Males exposed to 1000 µg/L of flutamide showed a downregulation in the genes encoding androgen receptors α and β. The expression of the gene for the estrogen receptor α was induced and of vitellogenin was downregulated after treatment with 250 µg/L to 1000 µg/L of flutamide. The results suggest that 7-d exposures to 125 µg/L to 1000 µg/L flutamide can impair the reproductive endocrine system in male Murray rainbowfish at multiple levels by an antiandrogenic mode of action.

The most potent chemicals potentially causing adverse effects on fish species are estrogens in human waste.Sewage is a source of these estrogens and it is difficult to reduce. In particular, although the bioactivity of estrone is estimated to be about half of that of estradiol, multiple studies report that more than 100 ng l(–1) of estrone can be detected in urban rivers, including discharges from sewage treatment works; approximately two times as high as estradiol. Few studies have been conducted to investigate the long-term effects of estrone on wildlife; therefore, we conducted fish multigeneration test using Japanese medaka (Oryzias latipes). Medaka were exposed to estrone for 27 weeks across three generations in environmentally relevant concentrations, being 5.74, 11.4, 24.0, 47.1 and 91.4 ng l(–1). No effects on reproduction were observed in the first generation; however, a decline in egg production and fertility was observed in the second generation exposed to 91.4 ng l(–1) estrone, which is lower than some known environmental concentrations in urban environments. Furthermore, histopathological abnormalities were observed in the third generation exposed to both 47.1 and 91.4 ng l(–1), suggesting that estrone possibly exerts severe effects on the third or later generations. However, appearances of testis–ova were observed in the second and third generation they were not consistent with actual effects on reproduction, notwithstanding the testis-ovais regarded as the key evidence for endocrine disruption. Accordingly, we consider that qualitative measurement of abnormalities using histopathological observations is required for appropriate evaluation of endocrine disruption.

Androgens play key roles in the morphological specification of male type sex attractive and reproductive organs, whereas little is known about the developmental mechanisms of such secondary sex characters. Medaka offers a clue about sexual differentiation. They show a prominent masculine sexual character for appendage development, the formation of papillary processes in the anal fin, which has been induced in females by exogenous androgen exposure. This current study shows that the development of papillary processes is promoted by androgen-dependent augmentation of bone morphogenic protein 7 (Bmp7) and lymphoid enhancer-binding factor-1 (Lef1). Androgen receptor (AR) subtypes, ARα and ARβ, are expressed in the distal region of outgrowing bone nodules of developing papillary processes. Development of papillary processes concomitant with the induction of Bmp7 and Lef1 in the distal bone nodules by exposure to methyltestosterone was significantly suppressed by an antiandrogen, flutamide, in female medaka. When Bmp signaling was inhibited in methyltestosterone-exposed females by its inhibitor, dorsomorphin, Lef1 expression was suppressed accompanied by reduced proliferation in the distal bone nodules and retarded bone deposition. These observations indicate that androgen-dependent expressions of Bmp7 and Lef1 are required for the bone nodule outgrowth leading to the formation of these secondary sex characteristics in medaka. The formation of androgen-induced papillary processes may provide insights into the mechanisms regulating the specification of sexual features in vertebrates.

Juvenile hormone (JH) and JH agonists have been shown to induce male offspring production in various daphnids, including Daphnia magna using OECD TG211. The critical period (about 1h) for JH action on ova in the parent's ovary to induce male offspring is existing at 7-8h later from ovulation. Therefore, we considered that adult D. magna could be used to produce a short-term screening method for detecting JH analogs. Using this method, we successfully demonstrated male offspring induction in the second broods after exposure to JH or JH agonists. After investigating the exposure time, the number of repetitions and the exposure concentration, we established a short-term, in vivo screening method for detecting JH analogs using adult D. magna. We examined positive and negative control chemicals using a previously developed method and verified the validity of our new testing method. Copyright © 2014 John Wiley & Sons, Ltd.

The Mullerian duct develops into the oviduct, uterus, and vagina, all of which are quite distinct in their morphology and function. The epithelial fate of these female reproductive organs in developing mice is determined by factors secreted from the stroma; however, how stromal differentiation occurs in the female reproductive organs derived from the Mullerian duct is still unclear. In the present study, roles of retinoic acid (RA) signaling in developing female reproductive tracts were investigated. Retinol dehydrogenase 10 (RDH10) and aldehyde dehydrogenase family 1 subfamily A2 (ALDH1A2) mRNAs and proteins and transactivation activity of endogenous RA were found in the stroma of proximal Mullerian ducts and gradually decreased from the proximal to caudal regions in fetal mice. In organ-cultured Mullerian ducts, retinaldehyde or RA treatment induced uterine epithelial differentiation, defined as a layer of columnar epithelial cells negative for oviductal and vaginal epithelial markers. In contrast, inhibition of RA receptor (RAR) signaling induced vaginal epithelial differentiation, characterized as vaginal epithelial marker genes–positive stratified epithelium. Grafting experiments of the organ-cultured Mullerian duct revealed irreversible epithelial fate determination. Although RAR did not directly bind to the homeobox A10 (Hoxa10) promoter region, RA–RAR signaling stimulated Hoxa10 expression. Thus, RA–RAR signaling in the Mullerian duct determines the fate of stroma to form the future uterus and vagina.

Daphnia magna has been used extensively to evaluate organism- and population-level responses to pollutants in acute toxicity and reproductive toxicity tests. We have previously reported that exposure to juvenile hormone (JH) agonists results in a reduction of reproductive function and production of male offspring in a cyclic parthenogenesis, D. magna. Recent advances in molecular techniques have provided tools to understand better the responses to pollutants in aquatic organisms, including D. magna. DNA microarray was used to evaluate gene expression profiles of neonatal daphnids exposed to JH agonists: methoprene (125, 250 and 500 ppb), fenoxycarb (0.5, 1 and 2 ppb) and epofenonane (50, 100 and 200 ppb). Exposure to these JH analogs resulted in chemical-specific patterns of gene expression. The heat map analyses based on hierarchical clustering revealed a similar pattern between treatments with a high dose of methoprene and with epofenonane. In contrast, treatment with low to middle doses of methoprene resulted in similar profiles to fenoxycarb treatments. Hemoglobin and JH epoxide hydrolase genes were clustered as JH-responsive genes. These data suggest that fenoxycarb has high activity as a JH agonist, methoprene shows high toxicity and epofenonane works through a different mechanism compared with other JH analogs, agreeing with data of previously reported toxicity tests. In conclusion, D. magna DNA microarray is useful for the classification of JH analogs and identification of JH-responsive genes. Copyright © 2013 John Wiley & Sons, Ltd.

In the ovary of neonatally DES-treated mice, lipid droplets accumulation was observed in the hypertrophied interstitial tissues. Our previous results demonstrated that the impaired steroidogenesis in the ovary of neonatally DES-treated mice was caused by altered gonadotropins levels, and resulted in the hypertrophy of ovarian interstitial cells. We speculated that lipid droplets in the ovary mainly consisted of cholesterol. This study was aimed to examine the effects of neonatal DES on cholesterol homeostasis in the ovary. The serum and ovarian total cholesterol concentrations in 3-month-old neonatally DES-treated mice were significantly higher than those in the neonatally oil-treated mice, but triglyceride concentrations were not altered. In the ovary of neonatally DES-treated mice, expression of Hmgcr, a rate-limiting enzyme in de novo cholesterol biosynthesis, was reduced but expression of Ldlr and Scarb1, involved in cholesterol uptake, was not changed. These results suggest that cholesterol uptake is not altered in the ovary of 3-month-old neonatally DES-treated mice. However, the expression of Acat1, the microsomal acyl coenzyme A cholesterol acyltransferase which is involved in cholesterol esterification and storing was increased compared with that in the ovary of neonatally oil-treated mice. Since ovarian steroidogenesis in neonatally DES-treated mice was impaired, synthesized and/or obtained cholesterol from the blood may not be used sufficiently. Thus, in the ovary of neonatally DES-treated mice, cholesterol is esterified by ACAT1 and stored in the interstitial cells.

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

Under favorable conditions, the micro-crustacean Daphnia pulex produces female offspring by parthenogenesis, whereas under unfavorable conditions, they produce male offspring to induce sexual reproduction (environmental sex determination: ESD). We recently established a suitable system for ESD studies using D. pulex WTN6 strain, in which the sex of the offspring can be regulated by alterations in day-length; long-day and short-day conditions can induce female and male offspring, respectively. Taking advantage of this system, we have already demonstrated that methyl farnesoate (MF) synthesis is necessary for male offspring production and identified ionotropic glutamate receptors as an upstream regulator of MF signaling. Despite these findings, the molecular mechanisms associated with MF signaling have not yet been well elucidated. In this study, we analyzed the whole metabolic profiles of mother daphnids reared under long-day (female-producing) and short-day (male-producing) conditions and discovered that pantothenate (vitamin B5), a known precursor to coenzyme A, was significantly accumulated in response to the short-day condition. To confirm the innate role of pantothenate in D. pulex, this metabolite was administered to mother daphnids resulting in a significantly increased proportion of male offspring producing mothers. This study provides novel insights of the metabolic mechanisms of the ESD system in D. pulex.

Vinclozolin is a dicarboximide fungicide and exhibits anti-androgenic effects by inhibiting androgen binding to the androgen receptor. Vinclozolin treatment reduces androgen-dependent organ weights in adult rats and anogenital distance of male pups at birth by in utero exposure.

Androgens are critical steroid hormones that regulate male sexual development and differentiation, including the formation of the reproductive system and maintenance of its function. Androgens are also essential in muscle development and psychosexual behavior. Testosterone and dihydrotestosterone act as the predominant androgens in mammals. Androgens mediate their effects through the androgen receptor (AR), a member of the nuclear receptor superfamily. Liganded AR is translocated from cytoplasm to the nucleus and regulates the transcription of androgen-responsive genes. Mutations in the AR result in diseases such as androgen insensitivity syndrome, prostate cancer, Kennedy's disease, and infertility.

18-Hydroxycorticosterone (18-OHB), which has a weak electrolyte action, is a derivative of corticosterone. Corticosterone is converted into 18-OHB by two steps, 18-hydroxylation and 18-oxidation. These two steps are catalyzed by CYP11B2. There is no difference between the sexes in the concentration of this 18-OHT; however, ACTH stimulation increases the plasma concentration of 18-OHT, while dexamethasone decreases this steroid. Very little is known about the physiological function of 18-OHT. In adrenalectomized rats, a physiological dose of 18-OHT has a positive effect on the brush border Na+/K+ exchanger. In sodium-replete patients with low-renin essential hypertension, 18-OHT seems to be a better marker than aldosterone.

In 1966, Idler and Truscott isolated 1α-hydroxycorticosterone (1α-OHB) from elasmobranchs. This steroid is a dominant corticosteroid produced by the interrenal tissue. The normal biosynthetic pathway of adrenocortical steroids is used for 1α-OHB production until corticosterone synthesis, and most steroidogenic enzymes required for 1α-OHB, including StAR, CYP, and HSD, have been identified. However, there are a few unidentified key enzymes that have to be examined including 1α-hydroxylase and CYP11B. 1α-OHB may act as a dual ligand in both the glucocorticoid and mineralocorticoid signaling pathways, likely in the regulation of salt and osmolyte balance and glucose balance in elasmobranchs.

Cortisol is one of the major glucocorticoids synthesized in the zona fasciculata of the adrenal cortex. Cortisol secretion is regulated by the hypothalamic hormone, CRH, and the pituitary hormone, ACTH, in the hypothalamus-pituitary-adrenal axis. Cortisol is known as a stress hormone involved in the response to physical and/or emotional stress. Cortisol also participates in various homeostatic maintenance actions: blood pressure; immune system; metabolism of protein, carbohydrate, and adipose; and anti-inflammatory action. Decrease of cortisol induces Addison’s disease, while overproduction of cortisol is related to Cushing’s syndrome.

DHEA and its sulfated form, DHEAS, are the most abundant endogenous steroids in human circulation, and are mainly produced in the adrenal cortex. The synthesis of DHEA is stimulated by ACTH secreted by the pituitary gland. DHEA is converted from pregnenolone by CYP17 (17α-hydroxylase, 17,20-lyase). DHEA can be converted to androstenedione by 3β-HSD, and subsequently to active androgens and estrogens. The decline of both circulating and brain DHEA with age is implicated in cardiovascular and neural diseases and an increased risk of carcinogenesis. High levels of DHEA and DHEAS induce precocious puberty in children.

Progesterone (P4) is an an intermediate of sex steroids produced by all steroidogenic tissues. It maintains pregnancy and the secretory condition of the uterine endometrium during the luteal phase, and inhibits release of gonadotropins. P4 can bind to progesterone receptor (PR), which is a member of the nuclear receptor superfamily of transcription factors. PR has two natural isoforms, PRA and PRB, which are transcribed from the same gene. Since PRA knockout (PRAKO) mice exhibit severely reduced ovulation rates and implantation defects, PRA is responsible for many functions in the reproductive organs including uterus. PRB is critical for mammary gland branching and ductal development in mice.

17,20β-Dihydroxy-4-pregnen-3-one (17α,20β-DP) and 17,20β,21-Trihydroxy-4-pregnen-3-one (20β-S) are identified as maturation inducing steroids in teleost fish. 20β-S is produced from 11-deoxycortisol by 20β-hydroxysteroid dehydrogenase (20β-HSD) in ovarian follicular cells under the influence of pituitary gonadotropin. It stimulates the meiotic maturation of oocytes during GVBD. Biological actions of these steroids are mediated by both membrane and nuclear receptors.

Estradiol-17β (E2) steroid hormone is produced mainly in the ovary, stimulates proliferation of the uterine endometrium, induces the LH surge for ovulation, and inhibits the pituitary hormone release via negative feedback. E2 is also produced in the testis but in low concentrations. E2 is converted from testosterone by aromatase or from estrone by 17β-hydroxysteroid dehydrogenase in the granulosa cell and luteal cell of the ovary, Leydig cells of the testis, adrenal gland, placenta, brain, and adipose tissue. The estrogen receptors (ERs) for E2 are ERα and ERβ in mammals, avians, reptilians, amphibians, and ancient fish species. ERα is considered to have a pivotal role because ERα knockout (ERα KO) female mice are sterile due to the inefficient levels of gonadotropins and lack of sensitivity to estradiol treatment, and ERα KO male mice are also sterile. ERβ KO female mice are fertile but exhibit reduced ovulation rates, while ERβ KO male mice exhibit fertility.

Aldosterone is a steroid hormone produced by the adrenal cortex in the adrenal gland to regulate sodium and potassium balance in the blood. Specifically, it regulates electrolyte and water balance by increasing the renal retention of sodium and the excretion of potassium. It is synthesized from cholesterol by aldosterone synthase in the zona glomerulosa of the adrenal gland, and its secretion is regulated via the renin–angiotensin system, potassium status, and ACTH. Aldosterone is a critical regulator of serum sodium and electrolytes via mineralocorticoid receptors.

Estrone is one of the steroid hormones produced in the ovary, testis, and placenta. Estrone is converted from androstenedione by aromatase (P450arom encoded by the CYP19 gene) or from estradiol-17β by 17β-hydroxysteroid dehydrogenase (17β-HSD) type 2. Estrone can bind to both types of estrogen receptors (ERs), ERα and ERβ; however, it exhibits weak estrogenic activity.

Corticosteroids, glucocorticoids, and mineralocorticoids are classes of chemicals that include steroid hormones naturally produced in the adrenal cortex of vertebrates. Glucocorticoids are stress hormones and influence glucose metabolism, and mineralocorticoids affect Na+ and K+ balance. The glucocorticoid actions are mediated through the glucocorticoid receptor (GR), whereas the mineralocorticoid actions are mediated through the mineralocorticoid receptor (MR). Both receptors belong to the superfamily of nuclear receptors, a diverse group of transcriptional regulators.

Bisphenol A (BPA) is a synthetic estrogen found in numerous consumer products, and its exposure to humans and wildlife is widespread. In particular, developmental animals may be more susceptible to BPA (and other endocrine disruptors) which can cause various changes in offspring, including alterations in postnatal growth, reproductive organ development and function, and behavior. Some effects of BPA are observed at extremely low concentrations (low-dose effects). Although the primary effect is considered to be estrogenic, BPA also affects the metabolic, thyroid hormone, and androgen systems.

Equilin is an equine estrogen isolated from pregnant mares. Equilin is one of the major components of estrogens for hormone replacement therapy, along with estrone and 17α-dihydroequilin. These estrogens are administered under the commercial name Premarin™, as salts of their sulfate esters. It is known that sewage treatment works effluents contain contaminants that can be estrogenic to aquatic animals. Estrogens used in hormone replacement therapy, including equilin, are implicated as being some of the causative compounds.

Corticosterone is a steroid-based hormone of 21 carbons among adrenal corticosteroids synthesized in the adrenal cortex. Corticosterone, a major glucocorticoid in rodents, birds, reptiles, and amphibians, controls metabolism and stress response. In humans, corticosterone is mainly synthesized in the zona glomerulosa of the adrenal cortex; however, the activity of corticosterone is weak in humans. Corticosterone is important as an intermediate product for production of aldosterone from pregnenolone in the steroidogenic pathway.

Thyroid hormones are essential for the regulation of a wide range of biological processes associated with normal development and metabolism in vertebrates. For the screening of chemicals with a potential thyroid hormone and anti-thyroid hormone activities, we have established transient transactivation assay systems using thyroid hormone receptors (TRα and TRβ) from three frog species (Xenopus laevis, Silurana tropicalis and Rana rugosa), a fish (Oryzias latipes), an alligator (Alligator mississippiensis) and a human (Homo sapiens). In all species examined, similar transcriptional activities were found for triiodothyronine (T3 : 10(-11) M in TRα and 10(-10) M in TRβ) and thyroxine (T4 : 10(-9) M in TRα and 10(-8) M in TRβ). Analogs of thyroid hormone (3,5,3',-triiodothyroacetic acid and 3,3',5,5'-tetraiodothyroacetic acid) exhibited weaker activity, requiring 10-fold higher concentrations for induction of activity when compared with T3 and T4 . These results provide support for the usefulness of in vitro screening assay systems as part of an approach to test chemicals for potential thyroid hormone receptor activity. In addition, we observed that T3 -stimulated transcriptional activity of the O. latipes TRα was inhibited by 10(-5) M tetrabromobisphenol A (TBBPA). In contrast, TR antagonist activities on TRα were not encountered in other species, even with TBBPA concentrations at 10(-5) M. In vitro transactivation assay systems using TRs from various species can be used for the screening of chemicals with thyroid-receptor agonist and antagonist activities. They also can be used for studies that examine evolutionary differences among species in the potency of TR activation.

Proliferation and differentiation of cells in female reproductive organs, the oviduct, uterus and vagina, are regulated by endogenous estrogen. In utero exposure to a synthetic estrogen, diethylstilbestrol (DES), induces vaginal clear-cell adenocarcinoma in humans. In mice, perinatal exposure to DES results in abnormalities such as polyovular follicles, uterine circular muscle disorganization and persistent vaginal epithelial cell proliferation. We reported the persistent gene expression change such as interleukin-1 (IL-1) related genes, insulin-like growth factor-I (IGF-I) and its downstream signaling in the mouse vagina exposed neonatally to DES. In this study, we found persistent up-regulation of Wnt4 and persistent down-regulation of Wnt11 in the vagina of mice exposed neonatally to DES and estrogen receptor α specific ligand. Also Wnt4 expression in vagina is correlated to the stratification of epithelial cells with the superficial keratinization of vagina, but not epithelial cell stratification only.

The occurrence of oocytes in the testis (testis-ova) of several fish species is often associated with exposure of estrogenic chemicals. However, induction mechanisms of the testis-ova remain to be elucidated. To develop marker genes for detecting testis-ova in the testis, adult male medaka were exposed to nominal concentration of 100 ng L(-1) of 17α-ethinylestradiol (EE2) for 3-5 weeks, and 800 ng estradiol benzoate (EB) for 3 weeks (experiment I), and a measured concentration of 20 ng L(-1) EE2 for 1-6 weeks (experiment II). Histological analysis was performed for the testis, and microarray analyses were performed for the testis, liver and brain. Microarray analysis in the estrogen-exposed medaka liver showed vitellogenin and choriogenin as estrogen responsive genes. Testis-ova were induced in the testis after 4 weeks of exposure to 100 ng L(-1) EE2, 3 weeks of exposure to 800 ng EB, and 6 weeks of exposure to 20 ng L(-1) EE2. Microarray analysis of estrogen-exposed testes revealed up-regulation of genes related to zona pellucida (ZP) and the oocytes marker gene, 42Sp50. Using quantitative RT-PCR we confirmed that Zpc5 gene can be used as a marker for the detection of testis-ova in male medaka.

Knowledge of the regulation of molluscan reproductive systems is limited. Many studies have described how the development and maturation of molluscan gonads are regulated by oestrogen. Vertebrate-type oestrogen, 17b-oestradiol, has been detected in molluscs and its concentration in the gonads has been suggested to change in conjunction with reproductive cycles. However, it is unclear if molluscan gonadal tissues are relatively high in aromatase activities. Thais clavigera has been shown to possess a preferential expression of the oestrogen receptor gene in the ovary and ganglia, suggesting an association with gonadal maturation and expression of the oestrogen receptor gene. Therefore, it is interesting to explore the existence of vertebrate-type sex steroid hormones and aromatase activity in T. clavigera. We applied gas chromatography–mass spectrometry techniques to detect steroids in the gonadal tissues of T. clavigera and identified testosterone, androstenedione, 17b-oestradiol and oestrone. The aromatase activities converting androstenedione and testosterone into oestrone and 17boestradiol were much higher in gonads than in digestive glands. These data provide insights into the endocrinological system of neogastropods, prompting molecular evidence for the aromatase gene.

Environmental contaminant exposure can influence gonadal steroid signaling milieus; however, little research has investigated the vulnerability of non-steroidal signaling pathways in the gonads. Here we use American alligators (Alligator mississippiensis) hatched from field-collected eggs to analyze gonadal mRNA transcript levels of the activin–inhibin–follistatin gene expression network and growth differentiation factor 9. The eggs were collected from Lake Woodruff National Wildlife Refuge, a site with minimal anthropogenic influence, and Lake Apopka, a highly contaminated lake adjacent to a former EPA Superfund site. The hatchling alligators were raised for 13 months under controlled conditions, thus limiting differences to embryonic origins. Our data reveal sexually dimorphic mRNA expression in 13-month-old alligator gonads similar to patterns established in vertebrates with genetic sex determination. In addition, we observed a relationship between lake of origin and mRNA expression of activin/inhibin subunits α and βB, follistatin, and growth differentiation factor 9. Our study suggests that embryonic exposure to environmental contaminants can affect future non-steroidal signaling patterns in the gonads of a long-lived species.