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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.

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. [ABSTRACT FROM AUTHOR]

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. Key Points: Contaminants of emerging concern (CECs) are widespread in Great Lakes tributaries surface water, posing a potential hazard to fish health.We compare results from three independently developed tools to assess the congruence in findings related to potential hazard of CEC exposure to fish in select Great Lakes tributaries.Multiple lines of evidence provide useful information for resource managers to make decisions regarding management activities to reduce exposure of aquatic biota to CECs and for researchers to prioritize studies focused on understanding hazards of CEC exposure to fish. [ABSTRACT FROM AUTHOR]

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. Research Highlights: Sufficient mRNAs of steroid hormone receptors and related co‐factors in blood cells.Stress altered levels of corticosterone and several metabolites in plasma.Stress altered mRNA levels of glucocorticoid receptor and JUN in blood cells. [ABSTRACT FROM AUTHOR]

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. Corexit 9500, a chemical dispersant used during the Deepwater Horizon oil spill, displayed significant estrogenicity at 100 ppm via both alligator nuclear estrogen receptors 1 and 2 in a transactivation assay in vitro. However, exposure to Corexit at 0.25‐25 ppm in ovo before sex determination did not skew the sex ratio or impact testicular mRNA abundances at 1 week of age. Further investigations will provide a better understanding of potential sex determination‐associated endocrine activities. [ABSTRACT FROM AUTHOR]