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Male and female reproductive tracts develop from anterior intermediate mesoderm with similar differentiation processes. The anterior intermediate mesoderm develops into the mesonephros, and the Wolffian duct initiates by epithelialization in the mesonephros. The Müllerian duct invaginates from the coelomic epithelium of the cranial mesonephros for ductal formation and is then regionalized into proximal to caudal female reproductive tracts. In this study, we focused on the epithelialization of the Wolffian duct, initiation of the Müllerian duct, and the regionalization step of the Müllerian ducts as a continuous process. By using intermediate mesodermal cells from mouse pluripotent stem cells, we identified that inhibition of SMAD2/3 signaling might be involved in the differentiation into mesenchymal cells, after which mesonephric cells might be then epithelialized during differentiation of the Wolffian duct. Aggregation of coelomic epithelial cells might be related to initiation of the Müllerian duct. Transcriptomic analysis predicted that consensus sequences of SMAD3/4 were enriched among highly expressed genes in the proximal Müllerian duct. SMAD2/3 signaling to regulate differentiation of the Wolffian duct was continuously activated in the proximal Müllerian duct and was involved in proximal and oviductal regionalization. Therefore, SMAD2/3 signaling may be finely tuned to regulate differentiation from initiation to regionalization steps., (© 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Contaminants of emerging concern (CECs; e.g., pharmaceuticals, flame retardants, pesticides, and industrial chemicals) are omnipresent throughout tributaries to the Great Lakes. Furthermore, CECs are often present at concentrations that are potentially hazardous to aquatic species. Since 2010, we characterized the presence of CECs at 309 sites within 47 Great Lakes tributaries and characterized responses of fathead minnow (Pimephales promelas) exposed to river water at a subset of 26 sites within four tributaries. Our work resulted in three independent lines of evidence related to the potential hazards of CEC exposure to fish. First, vulnerability (where vulnerability refers to likelihood) of surface waters to CEC presence was predicted using select watershed characteristics. Second, hazard to fish (where hazard means the potential for adverse biological responses) was predicted using screening values for a subset of CECs. Third, biological responses of fathead minnow exposed to river water in streamside exposures were measured. We assessed the congruence of these three lines of evidence for identifying sites with elevated hazards to CEC exposure. Predicted vulnerability and hazards agreed at 66% of all sites. Where the two indices did not agree, vulnerability often underestimated predicted hazard. When compared with measured biological responses from streamside exposures, predicted hazards agreed for 42% of samples. Furthermore, when predicted hazards for specific effect categories were compared with similar measured biomarkers, 26% and 46% of samples agreed for reproductive and physiological effect categories, respectively. Overall, vulnerability and hazard predictions tended to overestimate the measured biological responses, providing a protective estimate of the potential hazards of CEC exposure to fish. When used together, these three approaches can help resource managers prioritize management activities in minimizing hazards of CEC exposure and can be used by researchers to prioritize studies focused on understanding the hazards of CEC exposure to fish. Integr Environ Assess Manag 2022;18:1246-1259. © 2021 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC). This article has been contributed to by US Government employees and their work is in the public domain in the USA., (© 2021 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC). This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Orthologs of human glucocorticoid receptor (GR) and human mineralocorticoid receptor (MR) first appear in cartilaginous fishes. Subsequently, the MR and GR diverged to respond to different steroids: the MR to aldosterone and the GR to cortisol and corticosterone. We report that cortisol, corticosterone and aldosterone activate full-length elephant shark GR, and progesterone, which activates elephant shark MR, does not activate elephant shark GR. However, progesterone inhibits steroid binding to elephant shark GR, but not to human GR. Together, this indicates partial functional divergence of elephant shark GR from the MR. Deletion of the N-terminal domain (NTD) from elephant shark GR (truncated GR) reduced the response to corticosteroids, while truncated and full-length elephant shark MR had similar responses to corticosteroids. Swapping of NTDs of elephant shark GR and MR yielded an elephant shark MR chimera with full-length GR-like increased activation by corticosteroids and progesterone compared to full-length elephant shark MR. Elephant shark MR NTD fused to GR DBD + LBD had similar activation as full-length MR, indicating that the MR NTD lacked GR-like NTD activity. We propose that NTD activation of human GR evolved early in GR divergence from the MR., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Due to the mounting evidence that phthalates, specifically di-2-ethylhexyl phthalate and dibutyl phthalate, produce adverse endocrine effects in humans and wildlife, the use of other chemicals as replacements has increased. One of the most commonly encountered phthalate replacements is di(isononyl)cyclohexane-1,2-dicarboxylate (DINCH). Currently, little is known about the prevalence of human exposure, bioactivity, and endocrine disrupting potential of DINCH. We sampled urine from 100 pregnant women during the second trimester of pregnancy living in Charleston, SC between 2011 and 2014 and measured the following DINCH metabolites by LC-MS/MS: cyclohexane-1,2-dicarboxylic acid-mono(hydroxy-isononyl) ester (OH-MINCH), cyclohexane-1,2-dicarboxylic acid-mono(oxo-isononyl) ester (oxo-MINCH), and cyclohexane-1,2-dicarboxylic acid-monocarboxy isooctyl ester (cx-MINCH). These metabolites were also tested on human estrogen receptor alpha and progesterone receptor beta transactivation assays in vitro. OH-MINCH was detected in 98% of urine samples. The specific gravity-adjusted median (interquartile range) OH-MINCH concentration was 0.20 (0.25) ng/mL, and concentrations were significantly higher in African American women compared to Caucasian women (p = 0.01). DINCH metabolite concentrations were consistent between years, and they did not exhibit estrogenic or progestogenic activity in vitro. Human exposure to these emerging compounds should continue to be monitored, especially in vulnerable populations, to ensure the replacement of phthalates by DINCH is not a case of regrettable substitution., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

All crocodilians exhibit temperature-dependent sex determination without sex chromosomes. This temperature dependency can be overridden by exposure to estrogen via estrogen receptor 1. Thus, the sex ratio of crocodilian species is vulnerable to estrogenic xenobiotics. Multiple investigations of the mechanism and effects of xenobiotics in crocodilian species have been conducted since the early 1990s. This review focuses on the impact of xenobiotics on sex determination rather than gonadal functions in crocodilians. The thermosensitive and estrogen-sensitive periods that commit the bipotential gonad to develop as an ovary end by stages 24.5 and 25.3, respectively. In contrast, it is ambiguous when the estrogen-sensitive stage begins for ovarian development, although the thermosensitive period for ovarian development initiates around developmental stage 15 at an extreme female-producing temperature of 30°C. To accurately assess the effect of xenoestrogens on sex ratio in crocodilians, it is critical to collect eggs before the sex-determining period and to incubate them under precisely controlled temperatures. A well-studied system of xenobiotic effects on crocodilians is Lake Apopka (FL, USA), an EPA superfund clean-up site heavily contaminated with Dieldrin, Endrin, and p,p'-DDE. The sum of estimated estrogenicity of xenobiotics measured in Lake Apopka was insufficient to activate the estrogen receptor 1 of Alligator mississippiensis, which is an essential receptor to induce ovarian development. Although juvenile A. mississippiensis showed gonadal alterations in sex hormone production and histology, the environmentally relevant concentration of xenobiotics in Lake Apopka was unlikely to alter the sex ratio of A. mississippiensis. Experimental exposure to xenobiotics such as 17α-ethynylestradiol, p,p'-dichlorodiphenyldichloroethylene, and 2,3,7,8-tetrachlorodibenzodioxin at environmentally relevant concentrations in ovo induced more female offspring in A. mississippiensis as compared with the control group. Bisphenol-A, atrazine, 2,4-dichlorophenoxyacetic acid, endosulfan, and Corexit did not alter the sex ratio of A. mississippiensis or Caiman latirostris under the tested conditions. Egg-incubation temperature has pronounced effects on estrogen sensitivity in crocodilian sex determination. Therefore, crocodilians are vulnerable to xenobiotic contamination and climate change in the wild. It is vital to further investigate the detailed mechanism and effects of environmental xenobiotics in crocodilian sex determination to mitigate their effect on sex ratio and conserve this ancient lineage., (© 2021 S. Karger AG, Basel.)

Many reptiles, including the American alligator, exhibit temperature-dependent sex determination (TSD), whose thermo-sensitive period for the female alligator begins at stages-15 and ends at stage-24. Estrogen signaling plays a central role in TSD, which can be overridden by an estrogen-exposure during the thermo-sensitive period. As some environmental contaminants are estrogenic, there is growing concern about their effects on the sex ratio and reproductive health of TSD-species. It is crucial to identify the timing of gonadal commitment to either ovary or testis for a better understanding of TSD and estrogen-signals. In the current study, eggs were exposed to 5 µg/g egg of 17β-estradiol (E 2 ) or vehicle ethanol alone at three developmental stages-22, 24, and 26 at a male-promoting temperature, which produced 81% testis in all controls. E 2 -exposure at stages-22 and 24 induced more ovaries than the control group, whereas the exposure at stage-26 did not induce the same outcome. These results indicated that there is a critical commitment in the testicular development between the developmental stage 24 (100% ovary in E 2 Exposure) and 26 (39% ovary with E 2 ). Based on these results, we estimated a pivotal stage as stage-25.28. Thus, a gonadal commitment to testis could be later than a known temperature-sensitive period for promoting male in TSD., (Copyright © 2020 Elsevier Inc. All rights reserved.)

To monitor physiological and toxicological conditions in an endangered species, noninvasive to minimally invasive sampling methods are needed. We analyzed peripheral blood cells to determine if we could monitor some physiological responses of the American alligator following capture stress. Juvenile American alligators were restrained for 16 h to examine the stress response in plasma and blood cells. Plasma corticosterone concentrations were increased by restraint as were plasma concentrations of aspartate aminotransferase (AST), creatine kinase (CK), uric acid, and glucose; a sexually dimorphic response was seen in AST and CK concentrations. The lapse time of restraint was associated with altered messenger RNA (mRNA) levels of the glucocorticoid receptor (GCR) in red blood cells and JUN proto-oncogene in both white and red blood cells. A two-way cluster analysis revealed that two major clusters of factors were associated with the responses seen: (a) mRNA levels of GCR and heat-shock proteins in both blood cells were associated with plasma corticosterone concentration, whereas (b) androgen receptors and JUN mRNA levels in both blood cells were associated with cloacal temperature and body composition. Blood cells appear to be an excellent source to examine the cellular stress response to steroid hormone signals in mRNA levels. We propose that this approach, using blood cells, could provide essential insights into the molecular responses associated with stress in reptiles as well as many other nontraditional model species, including endangered species., (© 2019 Wiley Periodicals, Inc.)

During the Deepwater Horizon oil spill, vast quantities of a chemical dispersant Corexit 9500 were applied in remediation efforts. In addition to the acute toxicity, it is essential to evaluate Corexit further with a broader scope of long-term sublethal endocrine endpoints. The American alligator (Alligator mississippiensis) is an excellent organism for such an endeavor. It exhibits temperature-dependent sex determination, in which egg incubation temperatures during a thermosensitive period (TSP) in embryonic development determine the sex of embryos. Estrogen signals play a critical role in this process. For example, a single exposure to exogenous estrogen during the TSP overrides the effects of temperature and leads to skewed sex ratios. At a concentration of 100 ppm, Corexit significantly induced transcriptional activity of both alligator nuclear estrogen receptors 1 and 2 in vitro in reporter gene assays. To investigate the estrogenic effects of Corexit on gonadal development, alligator eggs were exposed to Corexit at environmentally relevant concentrations (0.25, 2.5 and 25 ppm) before the TSP in ovo. Exposure to Corexit at 0.25 and 25 ppm significantly delayed hatching and growth. Corexit exposure at any treatment level did not affect sex ratios or testicular mRNA abundance as measured at 1-week post-hatching, suggesting that the combination of Corexit components did not synergize enough to induce ovarian development in ovo. These results point to a need for further investigations on individual and combined components of Corexit to understand better their long-term effects on the development and reproductive health of alligators and other coastal aquatic wildlife., (© 2019 John Wiley & Sons, Ltd.)

Environmental estrogens from anthropogenic activities are ubiquitous in aquatic ecosystems. Ambient temperature in these systems also fluctuates in daily, seasonal, and long-term rhythms. While both factors have been studied extensively, their interaction on aquatic life is critical to understand. The objective of this study was, therefore, to examine how behavior and gene expression are impacted by estrogenic exposure across a range of environmental temperatures. Larval fathead minnows (Pimephales promelas) were exposed to estrone (E 1 ) at two concentrations (nominal 625 and 1250 ng/L) or to an ethanol solvent control, at one of four temperatures (15, 18, 21 and 24 °C) from fertilization to 21 days post-hatch. Exposed larvae were assessed for alterations in predator evasion performance and mRNA abundances of two genes for calcium channel receptors found in muscles - dihydropyridine receptor (dhpr) and ryanodine receptor 1, and the gonadal genes anti-Müllerian hormone, cytochrome P450 gonadal aromatase (cyp19a), doublesex and mab-3 related transcription factor 1 (dmrt1) and estrogen receptor 1 (esr1). Larval escape angle, escape latency, as well as systemic esr1 and cyp19a mRNA abundances were altered by an interaction between E 1 concentration and temperature. E 1 -exposed larval exhibited reduced escape performance across all tested temperatures, whereas decreased systemic dhpr mRNA abundance was observed only at 18 °C. E 1 -exposure reduced systemic mRNA abundances of amh, cyp19a, dhpr, and ryr1, while temperature significantly reduced systemic cyp19a and dhpr mRNA abundances. E 1 -exposure and temperature significant enhanced systemic mRNA abundances of esr1 and cyp19a, respectively. These complex results illustrate the importance of considering how abiotic factors may moderate the effects of contaminant exposure during the sensitive larval developmental stage, as temperature modulates effects of estrogenic exposure on animal performance and mRNA abundances., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The mineralocorticoid receptor (MR) is a nuclear receptor and part of a large and diverse family of transcription factors that also includes receptors for glucocorticoids, progesterone, androgens, and estrogens. The corticosteroid aldosterone is the physiological activator of the MR in humans and other terrestrial vertebrates; however, its activator is not known in cartilaginous fish, the oldest group of extant jawed vertebrates. Here, we analyzed the ability of corticosteroids and progesterone to activate the full-length MR from the elephant shark ( Callorhinchus milii ). On the basis of their measured activities, aldosterone, cortisol, 11-deoxycorticosterone, corticosterone, 11-deoxcortisol, progesterone, and 19-norprogesterone are potential physiological mineralocorticoids. However, aldosterone, the physiological mineralocorticoid in humans and other terrestrial vertebrates, is not found in cartilaginous or ray-finned fish. Although progesterone activates MRs in ray-finned fish, progesterone does not activate MRs in humans, amphibians, or alligator, suggesting that during the transition to terrestrial vertebrates, progesterone lost the ability to activate the MR. Both elephant shark MR and human MR are expressed in the brain, heart, ovary, testis, and other nonepithelial tissues, suggesting that MR expression in diverse tissues evolved in the common ancestor of jawed vertebrates. Our data suggest that 19-norprogesterone- and progesterone-activated MR may have unappreciated functions in reproductive physiology., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Agricultural and urban contaminants are an environmental concern because runoff may contaminate aquatic ecosystems, resulting in stress for exposed fish. The objective of the present controlled, field-based study was to assess the impacts of high-intensity agriculture and urban land use on multiple life stages of the fathead minnow (Pimephales promelas), using the Maumee River (Toledo, OH, USA) as a case study. Laboratory cultured adult and larval fathead minnows were exposed for 21 d, and embryos were exposed until hatching to site-specific water along the lower reach of the Maumee River. Adult minnows were analyzed for reproduction and alterations to hematologic characteristics (vitellogenin, glucose, estradiol, 11-ketotestosterone). Water and fish tissue samples were analyzed for a suite of multiresidue pesticides, hormones, and pharmaceuticals. Contaminants were detected in every water and tissue sample, with 6 pesticides and 8 pharmaceuticals detected in at least 82% of water samples and at least half of tissue samples. Effects differed by exposed life stage and year of exposure. Fecundity was the most sensitive endpoint measured and was altered by water from multiple sites in both years. Physiological parameters associated with fecundity, such as plasma vitellogenin and steroid hormone concentrations, were seldom impacted. Larval fathead minnows appeared to be unaffected. Embryonic morphological development was delayed in embryos exposed to site waters collected in 2016 but not in 2017. A distinction between agricultural and urban influences in the Maumee River was not realized due to the great overlap in contaminant presence and biological effects. Differences in precipitation patterns between study years likely contributed to the observed biological differences and highlight the need for environmental exposure studies to assess the environmental risk of contaminants. Environ Toxicol Chem 2019;00:1-17. © 2019 SETAC., (© 2019 SETAC.)

Background: Phthalates are used extensively in commercial and personal care products and maternal exposure is ubiquitous. Phthalates are anti-androgenic, but the potential effects of phthalates on male penile development have not been assessed in utero., Objective: The study aims to investigate the association between early pregnancy phthalate exposure and fetal penile development, overall and by race., Methods: Prospective cohort study of women with singleton pregnancies presenting for prenatal ultrasound between 18 and 22 weeks' gestation. Maternal urine samples were assayed for eight phthalate monoester metabolites. We used maternal phthalate levels at 18 to 22 weeks' gestation as predictors of fetal size using multiple linear regression models, adjusted for fetal gestational age, maternal age, race, smoking, and education. We incorporated a phthalate by race interaction into a second set of regression models., Results: We detected statistically significant race interactions for continuous phthalates with penile width. Race interactions were also suggested for penile length and volume using tertiles of phthalates with point estimates generally positive for whites and negative for African Americans., Conclusion: Penile development is significantly influenced by race, and the impact of maternal phthalates on penile measurements also varies by race. Maternal phthalate exposure can adversely affect in utero penile growth and development, especially among African Americans., (© 2018 John Wiley & Sons, Ltd.)

Sex steroid hormones including estrogens and androgens play fundamental roles in regulating reproductive activities and they act through estrogen and androgen receptors (ESR and AR). These steroid receptors have evolved from a common ancestor in association with several gene duplications. In most vertebrates, this has resulted in two ESR subtypes (ESR1 and ESR2) and one AR, whereas in teleost fish there are at least three ESRs (ESR1, ESR2a and ESR2b) and two ARs (ARα and ARβ) due to a lineage-specific whole genome duplication. Functional distinctions have been suggested among these receptors, but to date their roles have only been characterized in a limited number of species. Sexual differentiation and the development of reproductive organs are indispensable for all animal species and in vertebrates these events depend on the action of sex steroid hormones. Here we review the recent progress in understanding of the functions of the ESRs and ARs in the development and expression of sexually dimorphic characteristics associated with steroid hormone signaling in vertebrates, with representative fish, amphibians, reptiles, birds and mammals., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Deepwater Horizon spilled over 200 million gallons of oil into the waters of the Gulf of Mexico in 2010. In an effort to contain the spill, chemical dispersants were applied to minimize the amount of oil reaching coastal shorelines. However, the biological impacts of chemically-dispersed oil are not well characterized, and there is a particular lack of knowledge concerning sublethal long-term effects of exposure. This study examined potential estrogenic effects of CWAF, Corexit 9500-enhanced water-accommodated fraction of oil, by examining its effect on estrogen receptors and sex determination in the American alligator, Alligator mississippiensis. The alligator exhibits temperature-dependent sex determination which is modulated by estrogen signals, and exposure to 17β-estradiol (E 2 ) and estrogenic compounds in ovo during the thermosensitive period of embryonic development can induce ovarian development at a male-producing temperature (MPT). CWAF induced transactivation up to 50% of the maximum induction by E 2 via alligator estrogen receptors in vitro. To determine potential endocrine-disrupting effects of exposure directly on the gonad, gonad-adrenal-mesonephric (GAM) organ complexes were isolated from embryos one day prior to the thermosensitive period and exposed to E 2 , CWAF, or medium alone in vitro for 8-16 days at MPT. Both CWAF and E 2 exposure induced a significant increase in female ratios. CWAF exposure suppressed GAM mRNA abundances of anti-Müllerian hormone (AMH), sex determining region Y-box 9, and aromatase, whereas E 2 exposure suppressed AMH and increased Forkhead box protein L2 mRNA abundances in GAM. These results indicate that the observed endocrine-disrupting effects of CWAF are not solely estrogenically mediated, and further investigations are required., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Contaminants of emerging concern (CECs) are ubiquitous in aquatic environments with well-established endocrine-disrupting effects. A data matrix of 559 water samples was queried to identify two commonly occurring CECs mixtures in Great Lakes tributaries. One mixture consisted of eight agricultural CECs (AG), while another contained 11 urban CECs (UB). The known estrogenic compounds bisphenol A, estrone and nonylphenol were present in both mixtures. According to the EPA Tox21 in ToxCast database, AG and UB mixture at an environmentally relevant concentration were estimated to account for 6.5% and 3.4% estrogenicity of the model endocrine disruptor estradiol-17β, respectively. Two isoforms of the estrogen receptor (Esr1 and -2, former Erα and Erβ) cloned from fathead minnow, bluegill sunfish, American alligator and human, responded differently to AG and UB mixtures. Human and bluegill Esr1 were the most sensitive to AG and UB mixtures, respectively. Fathead minnow Esr1 and Esr2b were the least sensitive to 10× AG and UB in estrogen dose equivalents, respectively. Even at environmentally documented concentrations, UB significantly activated bluegill Esr1. Moreover, 100× concentrated UB hyperstimulated fathead minnow Esr1 beyond the maximum induction of estradiol-17β. These results indicate that efficacious receptors and species differ in their response to CEC mixtures. Furthermore, estrogenicity may be present in some CECs not previously considered estrogenic, or, alternatively, estrogenicity of a mixture may be enhanced through chemical interactions. Our study highlights the need for further studies of CECs utilizing a variety of receptors cloned from diverse species., (Copyright © 2017 John Wiley & Sons, Ltd.)

The American alligator, Alligator mississippiensis , like all crocodilians, has temperature-dependent sex determination, in which the sex of an embryo is determined by the incubation temperature of the egg during a critical period of development. The lack of genetic differences between male and female alligators leaves open the question of how the genes responsible for sex determination and differentiation are regulated. Insight into this question comes from the fact that exposing an embryo incubated at male-producing temperature to estrogen causes it to develop ovaries. Because estrogen response elements are known to regulate genes over long distances, a contiguous genome assembly is crucial for predicting and understanding their impact. We present an improved assembly of the American alligator genome, scaffolded with in vitro proximity ligation (Chicago) data. We use this assembly to scaffold two other crocodilian genomes based on synteny. We perform RNA sequencing of tissues from American alligator embryos to find genes that are differentially expressed between embryos incubated at male- versus female-producing temperature. Finally, we use the improved contiguity of our assembly along with the current model of CTCF-mediated chromatin looping to predict regions of the genome likely to contain estrogen-responsive genes. We find that these regions are significantly enriched for genes with female-biased expression in developing gonads after the critical period during which sex is determined by incubation temperature. We thus conclude that estrogen signaling is a major driver of female-biased gene expression in the post-temperature sensitive period gonads., (© 2017 Rice et al.; Published by Cold Spring Harbor Laboratory Press.)

In 2010, approximately 2.1 million gallons of chemical dispersants, mainly Corexit 9500, were applied in the Gulf of Mexico to prevent the oil slick from reaching shorelines and to accelerate biodegradation of oil during the Deepwater Horizon oil spill. Recent studies have revealed toxic effects of Corexit 9500 on marine microzooplankton that play important roles in food chains in marine ecosystems. However, there is still little known about the toxic effects of Corexit 9500 on freshwater zooplankton, even though oil spills do occur in freshwater and chemical dispersants may be used in response to these spills. The cladoceran crustacean, water flea Daphnia magna, is a well-established model species for various toxicological tests, including detection of juvenile hormone-like activity in test compounds. In this study, we conducted laboratory experiments to investigate the acute and chronic toxicity of Corexit 9500 using D. magna. The acute toxicity test was conducted according to OECD TG202 and the 48 h EC 50 was 1.31 ppm (CIs 0.99-1.64 ppm). The reproductive chronic toxicity test was performed following OECD TG211 ANNEX 7 and 21 days LOEC and NOEC values were 4.0 and 2.0 ppm, respectively. These results indicate that Corexit 9500 has toxic effects on daphnids, particularly during the neonatal developmental stage, which is consistent with marine zooplankton results, whereas juvenile hormone-like activity was not identified. Therefore, our findings of the adverse effects of Corexit 9500 on daphnids suggest that application of this type of chemical dispersant may have catastrophic impacts on freshwater ecosystems by disrupting the key food chain network. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, binds to a variety of chemical compounds including various environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin. This receptor regulates expression of target genes through dimerization with the AHR nuclear translocator (ARNT). Since AHR-ARNT signaling pathways differ among species, characterization of AHR and ARNT is important to assess the effects of environmental contamination and for understanding the molecular mechanism underlying the intrinsic function. In this study, we isolated the cDNAs encoding three types of AHR and two types of ARNT from a reptile, the American alligator (Alligator mississippiensis). In vitro reporter gene assays showed that all complexes of alligator AHR-ARNT were able to activate ligand-dependent transcription on a xenobiotic response element. We found that AHR-ARNT complexes had higher sensitivities to a ligand than AHR-ARNT2 complexes. Alligator AHR1B showed the highest sensitivity in transcriptional activation induced by indigo when compared with AHR1A and AHR2. Taken together, our data revealed that all three alligator AHRs and two ARNTs were functional in the AHR signaling pathway with ligand-dependent and isoform-specific transactivations in vitro., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Dr. Louis J. Guillette Jr. thought of himself as a reproductive biologist. However, his interest in reproductive biology transcended organ systems, life history stages, species, and environmental contexts. His integrative and collaborative nature led to diverse and fascinating research projects conducted all over the world. He doesn't leave us with a single legacy. Instead, he entrusts us with several. The purpose of this review is to highlight those legacies, in both breadth and diversity, and to illustrate Dr. Guillette's grand contributions to the field of reproductive biology. He has challenged the field to reconsider how we think about our data, championed development of novel and innovative techniques to measure endocrine function, helped define the field of endocrine disruption, and lead projects to characterize new endocrine disrupting chemicals. He significantly influenced our understanding of evolution, and took bold and important steps to translate all that he has learned into advances in human reproductive health. We hope that after reading this manuscript our audience will appreciate and continue Dr. Guillette's practice of open-minded and passionate collaboration to understand the basic mechanisms driving reproductive physiology and to ultimately apply those findings to protect and improve wildlife and human health., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Steroid hormones are a key regulator of reproductive biology in vertebrates, and are largely regulated via nuclear receptor families. Estrogen signaling is regulated by two estrogen receptor (ER) subtypes alpha and beta in the nucleus. In order to understand the role of estrogen in vertebrates, these ER from various species have been isolated and were functionally analyzed using luciferase reporter gene assays. Interestingly, species difference in estrogen sensitivity has been noted in the past, and it was reported that snake ER displayed highest estrogen sensitivity. Here, we isolated additional ER from three lizards: chameleon (Bradypodion pumilum), skink (Plestiodon finitimus), and gecko (Gekko japonicus). We have performed functional characterization of these ERs using reporter gene assay system, and found high estrogen sensitivity in all three species. Furthermore, comparison with results from other tetrapod ER revealed a seemingly uniform gradual pattern of ligand sensitivity evolution. In silico 3D homology modeling of the ligand-binding domain revealed structural variation at three sites, helix 2, and juncture between helices 8 and 9, and caudal region of helix 10/11. Docking simulations indicated that predicted ligand-receptor interaction also correlated with the reporter assay results, and overall squamates displayed highest stabilized interactions. The assay system and homology modeling system provides tool for in-depth comparative analysis of estrogen function, and provides insight toward the evolution of ER among vertebrates., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The molecular signaling processes involved the differentiation of the Müllerian duct (MD) into the female reproductive tract, or oviduct, in non-mammalian vertebrates are not well understood. Studies in mammals and birds indicate that steroid hormones play a role in this process, as the embryonic MD has been shown to be vulnerable to exogenous estrogens and progestins and environmental endocrine disrupting contaminants. In a previous study, developmental treatment with an estrogen receptor α (ERα) agonist, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), induced significant enlargement of the MD in alligator embryos incubated at a male-producing temperature, which was not observed in embryos treated with an estrogen receptor β (ERβ) agonist, 7-bromo-2-(4-hydroxyphenyl)-1,3-benzoxazol-5-ol (WAY 200070), or with 17β-estradiol (E 2 ). In order to understand the role of estrogen signaling in female alligator oviduct development, we incubated eggs at a female-producing temperature and treated them with E 2 and these ER selective agonists, PPT and WAY 200070, just prior to the thermosensitive window of sex determination. At stage 27, one stage prior to hatching, PPT induced significant enlargement of the MD with precocious development of secretory glands and connective tissue differentiation similar to characteristics of mature adult oviduct. PPT treatment in ovo increased mRNA expression of ERβ, progesterone receptor, androgen receptor and insulin-like growth factor 1 in MD at stage 27, while expression of ERα was decreased. Neither WAY 200070 nor E 2 treatment induced these effects seen in PPT-treated MD. The results of this study provide insight into the critical factors for healthy reproductive system formation in this sentinel species, although further investigation is needed to determine whether the observed phenomena are directly due to selective stimulation of ERα or related to some other aspect of PPT treatment., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We investigated the evolution of the response of human, chicken, alligator and frog glucocorticoid receptors (GRs) to dexamethasone, cortisol, cortisone, corticosterone, 11-deoxycorticosterone, 11-deoxycortisol and aldosterone. We find significant differences among these vertebrates in the transcriptional activation of their full length GRs by these steroids, indicating that there were changes in the specificity of the GR for steroids during the evolution of terrestrial vertebrates. To begin to study the role of interactions between different domains on the GR in steroid sensitivity and specificity for terrestrial GRs, we investigated transcriptional activation of truncated GRs containing their hinge domain and ligand binding domain (LBD) fused to a GAL4 DNA binding domain (GAL4-DBD). Compared to corresponding full length GRs, transcriptional activation of GAL4-DBD_GR-hinge/LBD constructs required higher steroid concentrations and displayed altered steroid specificity, indicating that interactions between the hinge/LBD and other domains are important in glucocorticoid activation of these terrestrial GRs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: The American alligator (Alligator mississippiensis) displays temperature-dependent sex determination (TSD), in which incubation temperature during embryonic development determines the sexual fate of the individual. However, the molecular mechanisms governing this process remain a mystery, including the influence of initial environmental temperature on the comprehensive gonadal gene expression patterns occurring during TSD., Results: Our characterization of transcriptomes during alligator TSD allowed us to identify novel candidate genes involved in TSD initiation. High-throughput RNA sequencing (RNA-seq) was performed on gonads collected from A. mississippiensis embryos incubated at both a male and a female producing temperature (33.5 °C and 30 °C, respectively) in a time series during sexual development. RNA-seq yielded 375.2 million paired-end reads, which were mapped and assembled, and used to characterize differential gene expression. Changes in the transcriptome occurring as a function of both development and sexual differentiation were extensively profiled. Forty-one differentially expressed genes were detected in response to incubation at male producing temperature, and included genes such as Wnt signaling factor WNT11, histone demethylase KDM6B, and transcription factor C/EBPA. Furthermore, comparative analysis of development- and sex-dependent differential gene expression revealed 230 candidate genes involved in alligator sex determination and differentiation, and early details of the suspected male-fate commitment were profiled. We also discovered sexually dimorphic expression of uncharacterized ncRNAs and other novel elements, such as unique expression patterns of HEMGN and ARX. Twenty-five of the differentially expressed genes identified in our analysis were putative transcriptional regulators, among which were MYBL2, MYCL, and HOXC10, in addition to conventional sex differentiation genes such as SOX9, and FOXL2. Inferred gene regulatory network was constructed, and the gene-gene and temperature-gene interactions were predicted., Conclusions: Gonadal global gene expression kinetics during sex determination has been extensively profiled for the first time in a TSD species. These findings provide insights into the genetic framework underlying TSD, and expand our current understanding of the developmental fate pathways during vertebrate sex determination.

Temperature-dependent sex determination (TSD), commonly found among reptiles, is a sex determination mode in which the incubation temperature during a critical temperature sensitive period (TSP) determines sexual fate of the individual rather than the individual's genotypic background. In the American alligator (Alligator mississippiensis), eggs incubated during the TSP at 33 °C (male producing temperature: MPT) yields male offspring, whereas incubation temperatures below 30 °C (female producing temperature: FPT) lead to female offspring. However, many of the details of the underlying molecular mechanism remains elusive, and the molecular link between environmental temperature and sex determination pathway is yet to be elucidated. Here we show the alligator TRPV4 ortholog (AmTRPV4) to be activated at temperatures proximate to the TSD-related temperature in alligators, and using pharmacological exposure, we show that AmTRPV4 channel activity affects gene expression patterns associated with male differentiation. This is the first experimental demonstration of a link between a well-described thermo-sensory mechanism, TRPV4 channel, and its potential role in regulation of TSD in vertebrates, shedding unique new light on the elusive TSD molecular mechanism.

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.

All crocodilians and many turtles exhibit temperature-dependent sex determination where the temperature of the incubated egg, during a thermo-sensitive period (TSP), determines the sex of the offspring. Estrogens play a critical role in sex determination in crocodilians and turtles, as it likely does in most nonmammalian vertebrates. Indeed, administration of estrogens during the TSP induces male to female sex reversal at a male-producing temperature (MPT). However, it is not clear how estrogens override the influence of temperature during sex determination in these species. Most vertebrates have 2 forms of nuclear estrogen receptor (ESR): ESR1 (ERα) and ESR2 (ERβ). However, there is no direct evidence concerning which ESR is involved in sex determination, because a specific agonist or antagonist for each ESR has not been tested in nonmammalian species. We identified specific pharmaceutical agonists for each ESR using an in vitro transactivation assay employing American alligator ESR1 and ESR2; these were 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) and 7-bromo-2-(4-hydroxyphenyl)-1,3-benzoxazol-5-ol (WAY 200070), respectively. Alligator eggs were exposed to PPT or WAY 200070 at a MPT just before the TSP, and their sex was examined at the last stage of embryonic development. Estradiol-17β and PPT, but not WAY 200070, induced sex reversal at a MPT. PPT-exposed embryos exposed to the highest dose (5.0 μg/g egg weight) exhibited enlargement and advanced differentiation of the Müllerian duct. These results indicate that ESR1 is likely the principal ESR involved in sex reversal as well as embryonic Müllerian duct survival and growth in American alligators.

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., (© 2014 Society for Reproduction and Fertility.)

Epigenetic modifications are key mediators of the interactions between the environment and an organism's genome. DNA methylation represents the best-studied epigenetic modification to date and is known to play key roles in regulating transcriptional activity and promoting chromosome stability. Our laboratory has previously demonstrated the utility of the American alligator (Alligator mississippiensis) as a sentinel species to investigate the persistent effects of environmental contaminant exposure on reproductive health. Here, we incorporate a liquid chromatography-tandem mass spectrometry method to directly measure the total (global) proportion of 5-methyl-2'-deoxycytidine (5mdC) in ovarian and whole blood DNA from alligators. Global DNA methylation in ovaries was significantly elevated in comparison with that of whole blood. However, DNA methylation appeared similar in juvenile alligators reared under controlled laboratory conditions but originating from three sites with dissimilar environmental qualities, indicating an absence of detectable site-of-origin effects on persistent levels of global 5mdC content. Analyses of tissues across individuals revealed a surprising lack of correlation between global methylation levels in blood and ovary. In addition, global DNA methylation in blood samples from juvenile alligators was elevated compared with those from adults, suggesting that age, as observed in mammals, may negatively influence global DNA methylation levels in alligators. To our knowledge, this is the first study examining global levels of DNA methylation in the American alligator and provides a reference point for future studies examining the interplay of epigenetics and environmental factors in a long-lived sentinel species.

Environmental factors are known to influence sex determination in many nonmammalian vertebrates. In all crocodilians studied thus far, temperature is the only known determinant of sex. However, the molecular mechanisms mediating the effect of temperature on sex determination are not known. Aromatase (CYP19A1) and SOX9 play critical roles in vertebrate sex determination and gonadogenesis. Here, we used a variety of techniques to investigate the potential roles of DNA methylation patterning on CYP19A1 and SOX9 expression in the American alligator, an organism that relies on temperature-dependent sex determination. Our findings reveal that developing gonads derived from embryos incubated at a male-producing temperature (MPT) show elevated CYP19A1 promoter methylation and decreased levels of gene expression relative to incubation at a female-producing temperature (FPT). The converse was observed at the SOX9 locus, with increased promoter methylation and decreased expression occurring in embryonic gonads resulting from incubation at FPT relative to that of MPT. We also examined the gonadal expression of the three primary, catalytically active DNA methyltransferase enzymes and show that they are present during critical stages of gonadal development. Together, these data strongly suggest that DNA methylation patterning is a central component in coordinating the genetic cascade responsible for sexual differentiation. In addition, these data raise the possibility that DNA methylation could act as a key mediator integrating temperature into a molecular trigger that determines sex in the alligator.

As temperature-dependent sex determination (TSD) and homozygote or heterozygote genetic sex determination (GSD) exist in multiple reptilian taxa, they represent sex determination systems that have emerged de novo. Current investigations have revealed that the genetic mechanisms used by various reptilian species are similar to those used by other vertebrates. However, the recent completion or near completion of various reptilian genome projects suggests that new studies examining related species with and without TSD could begin to provide additional insight into the evolution of TSD and GSD in vertebrate ancestors. Major questions still remain concerning germ cell migration and specification, the differentiation of gonadal accessory cells, such as the Sertoli cells and granulosa cells of the developing testis and ovary, respectively, and the mechanisms by which gene expression is regulated during TSD events. Further, reptilian sentinels and their mechanisms of gonadogenesis will likely remain important indicator species for environmental health. Thus, ongoing and new investigations need to tie molecular information to gonadal morphogenesis and function in reptiles. Such data will not only provide important information for an understanding of the evolution of these phenomena in vertebrates, but could also provide an important understanding of the health of the environment around us.

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., (Copyright © 2012 John Wiley & Sons, Ltd.)

Endocrine properties of extraembryonic membranes have traditionally been viewed as a characteristic of placental amniotes. However, our laboratory recently demonstrated that this ability extends to the extraembryonic membranes of two oviparous amniotes (chicken and alligator) indicating that endocrine extraembryonic membranes are not an innovation of placental amniotes and suggesting that this could be a shared amniote characteristic. In this study, we test our hypothesis that the chorioallantoic membrane (CAM) obtained from non-archosaurian obligate oviparous amniotes such as turtles, have the potential for steroid hormone activity. To investigate synthesis of a major placental hormone, we performed explant culture and found that the turtle CAM synthesizes progesterone in vitro in the presence of a steroid precursor. In addition, to examine whether the CAM has the ability to respond to steroid signaling, we quantified mRNA expression of the progesterone, androgen, and two estrogen receptors. Finally, to determine if steroid receptor mRNA is translated to protein, we performed immunolocalization of the progesterone receptor. Our data demonstrate that the turtle CAM exhibits steroid synthesis and has steroid hormone signaling capabilities. To that end, steroid hormone activity has now been demonstrated in the CAMs of three oviparous species that represent three independent lineages within oviparous Reptilia that have never exhibited viviparity; thus these data support our hypothesis that endocrine activity of extraembryonic membranes is a conserved trait of Amniota., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Amniotes, mammals, reptiles, and birds form common extraembryonic membranes during development to perform essential functions, such as protection, nutrient transfer, gas exchange, and waste removal. Together with the maternal uterus, extraembryonic membranes of viviparous (live-bearing) amniotes develop as an endocrine placenta that synthesizes and responds to steroid hormones critical for development. The ability of these membranes to synthesize and respond to steroid hormone signaling has traditionally been considered an innovation of placental amniotes. However, our laboratory recently demonstrated that this ability extends to the chorioallantoic membrane (CAM) of an oviparous (egg-laying) amniote, the domestic chicken, and we hypothesized that steroidogenic extraembryonic membranes could be an evolutionarily conserved characteristic of all amniotes because of similarities in basic structure, function, and shared evolutionary ancestry. In this study, we examined steroid hormone synthesis and signaling in the CAM of another oviparous amniote, the American alligator (Alligator mississippiensis). We quantified mRNA expression of a steroidogenic factor involved in the regulation of steroidogenesis (NR5A1), the key steroidogenic enzymes involved in the synthesis of progestins (HSD3B1), androgens (CYP17A1), and estrogens (CYP19A1), and the receptors involved in the signaling of progestins (PR), androgens (AR), estrogens (ESR1 and ESR2), and glucocorticoids (GR). Furthermore, we performed protein immunolocalization for PR and ESR1. Collectively, our findings indicate that the alligator CAM has the capability to regulate, synthesize, and respond to steroid hormone signaling, thus, supporting our hypothesis that the extraembryonic membranes of Amniota share a unifying characteristic, that is, the ability to synthesize and respond to steroid hormones.

Developing organisms interpret and integrate environmental signals to produce adaptive phenotypes that are prospectively suited for probable demands in later life. This plasticity can be disrupted when embryos are impacted by exogenous contaminants, such as environmental pollutants, producing potentially deleterious and long-lasting mismatches between phenotype and the future environment. We investigated the ability for in ovo environmental contaminant exposure to alter the growth trajectory and ovarian function of alligators at five months after hatching. Alligators collected as eggs from polluted Lake Apopka, FL, hatched with smaller body masses but grew faster during the first five months after hatching, as compared to reference-site alligators. Further, ovaries from Lake Apopka alligators displayed lower basal expression levels of inhibin beta A mRNA as well as decreased responsiveness of aromatase and follistatin mRNA expression levels to treatment with follicle stimulating hormone. We posit that these differences predispose these animals to increased risks of disease and reproductive dysfunction at adulthood., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Oviducts respond to hormonal cues from ovaries with tissue proliferation and differentiation in preparation of transporting and fostering gametes. These responses produce oviducal microenvironments conducive to reproductive success. Here, we investigated changes in circulating plasma sex steroid hormones concentrations and ovarian and oviducal mRNA expression to an in vivo gonadotropin (FSH) challenge in sexually immature, five-month-old alligators. Further, we investigated differences in these observed responses between alligators hatched from eggs collected at a heavily-polluted (Lake Apopka, FL) and minimally-polluted (Lake Woodruff, FL) site. In oviducts, we measured mRNA expression of estrogen, progesterone, and androgen receptors and also beta A and B subunits which homo- or heterodimerize to produce the transforming growth factor activin. In comparison, minimal inhibin alpha subunit mRNA expression suggests that these oviducts produce a primarily activin-dominated signaling milieu. Ovaries responded to a five-day FSH challenge with increased expression of steroidogenic enzyme mRNA which was concomitant with increased circulating sex steroid hormone concentrations. Oviducts in the FSH-challenged Lake Woodruff alligators increased mRNA expression of progesterone and androgen receptors, proliferating cell nuclear antigen, and the activin signaling antagonist follistatin. In contrast, Lake Apopka alligators displayed a diminished increase in ovarian CYP19A1 aromatase expression and no increase in oviducal AR expression, as compared to those observed in Lake Woodruff alligators. These results demonstrate that five-month-old female alligators display an endocrine-responsive ovarian-oviducal axis and environmental pollution exposure may alter these physiological responses., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Anti-Müllerian hormone (AMH) plays an important role in male sex differentiation in vertebrates. AMH produced by Sertoli cells of the fetal testis induces regression of the Müllerian duct in mammalian species. In alligators, sexual differentiation is controlled by the temperature during egg incubation, termed temperature-dependent sex determination (TSD). The TSD mechanism inducing sex differentiation is thought to be unique and different from that of genetic sex determination as no gene such as the SRY of mammals has been identified. However, many of the genes associated with gonadal differentiation in mammals also are expressed in the developing gonads of species exhibiting TSD. To clarify the molecular mechanisms associated with gonad formation during the temperature-sensitive period (TSP), we have cloned the full length AMH gene in the alligator, and quantitatively compared mRNA expression patterns in the gonad-adrenal-mesonephros (GAM) complex isolated from alligator embryos incubated at male and female producing temperatures. The deduced amino acid sequence of the alligator AMH cDNA showed high identity (59-53%) to avian AMH genes. AMH mRNA expression was high in the GAM of male alligator embryos at stage 24 (immediately after sex determination) and hatchlings, but suppressed in the GAM of estrogen-exposed hatchlings incubated at the male-producing temperature. In the alligator AMH proximal promoter, a number of transcriptional factors (for SF-1. GATA, WT-1 and SOX9) binding elements were also identified and they exhibit a conserved pattern seen in other species. SOX9 up-regulates transcriptional activity through the amAMH promoter region. These results suggested that AMH and SOX9 play important roles in TSD of the American alligator., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

In many vertebrates, steroid hormones are essential for ovarian differentiation during a critical developmental stage as well as promoting the growth and differentiation of the adult female reproductive system. Although studies have been extensively conducted in mammals and a few fish, amphibians, and bird species, the molecular mechanisms of sex steroid hormone (estrogens) action have been poorly examined in reptiles. Here, we evaluate hormone receptor and ligand interactions in two species of snake, the Okinawa habu (Protobothrops flavoviridis, Viperidae) and the Japanese four-striped rat snake (Elaphe quadrivirgata, Colubridae) after the isolation of cDNAs encoding estrogen receptor α (ESR1) and estrogen receptor β (ESR2). Using a transient transfection assay with mammalian cells, the transcriptional activity of reptilian (Okinawa habu, Japanese four-striped rat snake, American alligator, and Florida red-belly freshwater turtle) ESR1 and ESR2 was examined. All ESR proteins displayed estrogen-dependent activation of transcription via an estrogen-response element-containing promoter; however, the responsiveness to various estrogens was different. Further, we determined the chromosomal locations of the snake steroid hormone receptor genes. ESR1 and ESR2 genes were localized to the short and long arms of chromosome 1, respectively, whereas androgen receptor was localized to a pair of microchromosomes in the two snake species examined. These data provide basic tools that allow future studies examining receptor-ligand interactions and steroid endocrinology in snakes and also expands our knowledge of sex steroid hormone receptor evolution.

Sex-steroid hormones are essential for normal reproductive activity in both sexes in all vertebrates. Estrogens are required for ovarian differentiation during a critical developmental stage and promote the growth and differentiation of the female reproductive system following puberty. Recent studies have shown that environmental estrogens influence the developing reproductive system as well as gametogenesis, especially in males. To understand the molecular mechanisms of estrogen actions and to evaluate estrogen receptor-ligand interactions in Elasmobranchii, we cloned a single estrogen receptor (ESR) from two shark species, the cloudy catshark (Scyliorhinus torazame) and whale shark (Rhincodon typus) and used an ERE-luciferase reporter assay system to characterize the interaction of these receptors with steroidal and other environmental estrogens. In the transient transfection ERE-luciferase reporter assay system, both shark ESR proteins displayed estrogen-dependent activation of transcription, and shark ESRs were more sensitive to 17beta-estradiol compared with other natural and synthetic estrogens. Further, the environmental chemicals, bisphenol A, nonylphenol, octylphenol and DDT could activate both shark ESRs. The assay system provides a tool for future studies examining the receptor-ligand interactions and estrogen disrupting mechanisms in Elasmobranchii., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Activins and estrogens participate in regulating the breakdown of ovarian germ cell nests and follicle assembly in mammals. In 1994, our group reported elevated frequencies of abnormal, multioocytic ovarian follicles in 6 month old, environmental contaminant-exposed female alligators after gonadotropin challenge. Here, we investigated if maternal contribution of endocrine disrupting contaminants to the egg subsequently alters estrogen/inhibin/activin signaling in hatchling female offspring, putatively predisposing an increased frequency of multioocytic follicle formation. We quantified basal and exogenous gonadotropin-stimulated concentrations of circulating plasma steroid hormones and ovarian activin signaling factor mRNA abundance in hatchling alligators from the same contaminated (Lake Apopka) and reference (Lake Woodruff) Florida lakes, as examined in 1994. Basal circulating plasma estradiol and testosterone concentrations were greater in alligators from the contaminated environment, whereas activin/inhibin betaA subunit and follistatin mRNA abundances were lower than values measured in ovaries from reference lake animals. Challenged, contaminant-exposed animals showed a more robust increase in plasma estradiol concentration following an acute follicle stimulating hormone (FSH) challenge compared with reference site alligators. Aromatase and follistatin mRNA levels increased in response to an extended FSH challenge in the reference site animals, but not in the contaminant-exposed animals. In hatchling alligators, ovarian follicles have not yet formed; therefore, these endocrine differences are likely to affect subsequent ovarian development, including ovarian follicle assembly., (2010 Wiley-Liss, Inc.)

Transforming growth factor-beta (TGFbeta) homologues form a diverse superfamily that arose early in animal evolution and control cellular function through membrane-spanning, conserved serine-threonine kinases (RII and RI receptors). Activin and inhibin are related dimers within the TGFbeta superfamily that share a common beta-subunit. The evolution of the inhibin alpha-subunit created the only antagonist within the TGFbeta superfamily and the only member known to act as an endocrine hormone. This hormone introduced a new level of complexity and control to vertebrate reproductive function. The novel functions of the inhibin alpha-subunit appear to reflect specific insertion-deletion changes within the inhibin beta-subunit that occurred during evolution. Using phylogenomic analysis, we correlated specific insertions with the acquisition of distinct functions that underlie the phenotypic complexity of vertebrate reproductive processes. This phylogenomic approach presents a new way of understanding the structure-function relationships between inhibin, activin, and the larger TGFbeta superfamily.

Gonadal steroid hormone receptors play a vital role in transforming ligand signals into gene expression. We have shown previously that gonads from wild-caught juvenile alligators express greater levels of estrogen receptor 1 (ESR1) than estrogen receptor 2 (ESR2). Furthermore, sexually dimorphic ESR2 mRNA expression (female > male) observed in animals from the reference site (Lake Woodruff, FL, USA) was lost in alligators from the contaminated Lake Apopka (FL, USA). We postulated that environmental contaminant exposure could influence gonadal steroid hormone receptor expression. Here, we address questions regarding gonadal estrogen and androgen receptor (AR) mRNA expression in 1-yr-old, laboratory-raised alligators. What are relative expression levels within gonads? Do these levels vary between sexes or incubation temperatures? Can contaminant exposure change these levels? We observed a similar pattern of expression (ESR1 > AR > ESR2) in ovary and testis. However, both incubation temperature and environment modulated expression. Males incubated at 33.5 degrees C expressed greater AR levels than females incubated at 30 degrees C; dimorphic expression was not observed in animals incubated at 32 degrees C. Compared to Lake Woodruff alligators, Lake Apopka animals of both sexes showed lesser ESR2 mRNA expression levels. Employing cluster analyses, we integrated these receptor expression patterns with those of steroidogenic factors. Elevated ESR2 and CYP19A1 expressions were diagnostic of alligator ovary, whereas elevated HSD3B1, CYP11A1, and CYP17A1 expressions were indicative of testis. In contrast, AR, ESR1, and NR5A1 showed variable expressions that were not entirely associated with sex. These findings demonstrate that the mRNA expression of receptors required for steroid hormone signaling are modified by exposure to environmental factors, including temperature and contaminants.

Estrogens are necessary for ovarian differentiation during a critical developmental stage in many vertebrates, and they promote the growth and differentiation of the adult female reproductive system. To understand the evolution of vertebrate estrogen receptors (ESRs) and to evaluate estrogen receptor-ligand interactions in phylogenetically ancient fish, we used PCR techniques to isolate the cDNA encoding ESRs from lungfish, sturgeon, and gar. Sequence analyses indicate that these fishes have two ESRs, ESR1 (ERalpha) and ESR2 (ERbeta), as previously reported for other vertebrate species, but a second type of ESR2 (ERbeta2) was not found as has been reported in a number of teleost fishes. Phylogenetic analysis of the ESR sequences indicated that the lungfish ESRs are classified to the tetrapod ESR group, not with the teleost fish ESRs as are the ESRs from gar and sturgeon. Using transient transfection assays of mammalian cells, ESR proteins from these three ancient fishes displayed estrogen-dependent activation of transcription from an estrogen-responsive-element containing promoter. We also examined the estrogenic potential of o,p'-dichloro-diphenyl-trichloroethane (o,p'-DDT) and p,p'-DDT as well as one of its common metabolites, p,p'-dichloro-diphenyl-ethylene (p,p'-DDE) on the ESRs from these fishes. Lungfish ESR1 was less sensitive to DDT/DDE than the ESR1 from the other two fishes. The response of lungfish ESR1 to these pesticides is similar to the pattern obtained from salamander ESR1. These data provide a basic tool allowing future studies examining the receptor-ligand interactions and endocrine-disrupting mechanisms in three species of phylogenetically ancient fish and also expands our knowledge of ESR evolution.

Environmental estrogens have the potential to considerably affect the reproduction and development of aquatic vertebrates by interfering with the endocrine system. In addition to the potential risk of environmental estrogens, increasing water temperatures as a result of global warming have become a serious problem in many rivers and streams. To assess the degree of estrogenic exposure, the analysis of the estrogen-dependent protein vitellogenin (Vtg) is a frequently used biomarker in field studies. Little, however, is known regarding the potential interaction between ambient water temperature and the Vtg production induced by waterborne environmental estrogens. In order to test the influence of temperature on Vtg synthesis, we exposed juvenile brown trout to an environmentally relevant concentration of ethinylestradiol (EE(2)) and held them either at low or high temperatures (12 and 19 degrees C, respectively), but also at temperature cycles of 12-19 degrees C in order to simulate the field situation. The EE(2) exposure caused a 7-74-fold increase of hepatic Vtg mRNA. The synthesis of Vtg mRNA was clearly stimulated in fish held at higher water temperatures (12-19 degrees C and 19 degrees C, respectively). On the protein level, Vtg showed a similar pattern; the higher the temperature, the higher the concentration of Vtg in the plasma. The experiment further revealed a temperature-dependent increasing amount of hepatic estrogen receptor alpha mRNA (ERalpha) after exposure to waterborne EE(2). The gene expression of estrogen receptor beta-1 (ERbeta-1) and the glucocorticoid receptor (GR) in the liver of EE(2) exposed fish, however, showed no treatment-related alterations. In line with observed constant bile cortisol concentrations, our data do not indicate corresponding stress related effects on hepatic Vtg production. The present survey, however, clearly demonstrates that increased temperature significantly elevates the estrogen-induced expression of Vtg and therefore has to be considered when interpreting environmental monitoring studies.

Steroid hormones are essential for the normal function of most organ systems in vertebrates. Reproductive activities in females and males, such as the differentiation, growth and maintenance of the reproductive system, require signaling by sex steroid hormones. Although extensively studied in mammals and a few fish and bird species, the evolution and molecular mechanisms associated with the nuclear steroid hormone receptors are still poorly understood in amphibians and reptiles. Given our interest in environmental signaling of sex determination as well as a major interest in environmental contaminants that can mimic steroid hormone signaling, we have established an approach to study the molecular function (ligand binding and trans-activation) of steroid hormone receptors cloned from reptiles. This approach involves molecular cloning and sequencing of steroid hormone receptors, phylogenic analysis and in vitro trans-activation assays using endogenous or exogenous ligands. Comparing the in vitro trans-activation induced by different ligands with receptors cloned from different species would develop additional functional relationships (classification) among steroid hormone receptors. This approach can provide insight into understanding why each species could have different responses to exogenous ligands. Further, we have developed a novel and less invasive approach to obtaining mRNA for molecular cloning and sequencing of steroid hormone receptors in reptiles and other non-mammalian species, using blood cells as a source of genetic material. For example, white blood cells (WBCs) and red blood cells (RBCs) of the American alligator both express steroid hormone receptors and have adequate amounts of mRNA for molecular cloning. This approach would allow us to analyze components of endocrine function of steroid hormones without sacrificing animals. Especially in endangered species, this approach could provide an understanding of endocrine functions, elucidate the phylogenic relationships of various receptors in vitro, such as the steroid hormone receptors, and determine possible effects of environmental contaminants in a minimally invasive manner.

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-17beta (E(2)) 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 beta) 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 E(2) 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.

A previous study from our laboratory examining development in neonatal alligators from polluted Lake Apopka, Florida, found numerous differences relative to neonates from a reference site, Lake Woodruff National Wildlife Refuge. We postulated that the differences were the result of organizational changes derived from embryonic exposure to environmental contaminants and are related to the poor reproductive success reported in alligators from Lake Apopka. In this study we examine differences in alligators collected as eggs from these two populations and raised under similar conditions for 1 yr. Egg hatch rates did not differ between lake populations; however, posthatching mortality was much higher among Lake Apopka hatchlings. Snout-vent length and body mass were greater in Lake Apopka hatchlings, but no differences were detected between lake populations in thyroid, liver, and spleen mass corrected for body size or in plasma concentrations of testosterone and estradiol. Males from Lake Woodruff exhibited greater relative expression of gonadal mRNA for steroidogenic factor 1 (Nr5a1) and steroidogenic acute regulatory protein (Star) than males from Lake Apopka. Alligators from Lake Woodruff also expressed all genes examined in a sexually dimorphic pattern. In contrast, mRNA expression did not differ between males and females from Lake Apopka for Nr5a1, Star, cytochrome P450 11A1 (Cyp11a1), and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1). Our results document persistent differences in development, survivorship, and gene expression in alligators from a contaminated environment. Because these animals were raised under similar laboratory conditions, the differences are most likely of embryonic origin and organizational in nature.

Steroid hormones are essential for the normal function of many organ systems in vertebrates. Reproductive activity in females and males, such as the differentiation, growth, and maintenance of the reproductive system, requires signaling by the sex steroids. Although extensively studied in mammals and a few fish, amphibians, and bird species, the molecular mechanisms of sex steroid hormone (estrogens, androgens, and progestins) action are poorly understood in reptiles. Here we evaluate hormone receptor ligand interactions in a freshwater turtle, the red-belly slider (Pseudemys nelsoni), after the isolation of cDNAs encoding an estrogen receptor alpha (ERalpha), an androgen receptor (AR), and a progesterone receptor (PR). The full-length red-belly slider turtle (t)ERalpha, tAR, and tPR cDNAs were obtained using 5' and 3' rapid amplification cDNA ends. The deduced amino acid sequences showed high identity to the chicken orthologs (tERalpha, 90%; tAR, 71%; tPR, 71%). Using transient transfection assays of mammalian cells, tERalpha protein displayed estrogen-dependent activation of transcription from an estrogen-responsive element-containing promoter. The other receptor proteins, tAR and tPR, also displayed androgen- or progestin-dependent activation of transcription from androgen- and progestin-responsive murine mammary tumor virus promoters. We further examined the transactivation of tERalpha, tAR and tPR by ligands using a modified GAL4-transactivation system. We found that the GAL4-transactivation system was not suitable for the measurement of tAR and tPR transactivations. This is the first report of the full coding regions of a reptilian AR and PR and the examination of their transactivation by steroid hormones.