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We describe a label‐free proteomics protocol for the interrogation of the placental proteome. Step‐by‐step directions, including tissue cleanup and preparation, proteolytic digestion, nanoLC–MS/MS data collection and data analysis, are provided. The workflow has been applied toward exploring differential protein expression patterns in placentas from women who have been exposed to drugs during pregnancy relative to those who have not. We collected 20 tissue specimens, each representing a combination of spatially diverse sections across the placenta. These specimens were analyzed in the work described here, to survey information across the entire organ. This protocol can be scaled up or down as needed. [ABSTRACT FROM AUTHOR]

We describe a label‐free proteomics protocol for the interrogation of the placental proteome. Step‐by‐step directions, including tissue cleanup and preparation, proteolytic digestion, nanoLC–MS/MS data collection and data analysis, are provided. The workflow has been applied toward exploring differential protein expression patterns in placentas from women who have been exposed to drugs during pregnancy relative to those who have not. We collected 20 tissue specimens, each representing a combination of spatially diverse sections across the placenta. These specimens were analyzed in the work described here, to survey information across the entire organ. This protocol can be scaled up or down as needed. [ABSTRACT FROM AUTHOR]

Across diverse taxa, germ cell development is controlled by an intricate cascade of processes that are tightly controlled by the hypothalamic-pituitary-gonadal axis. Endocrine disturbances, such as those induced by endocrine disrupting chemicals (EDCs) can negatively affect spermatogenesis. Here, we investigate whether spermatogenesis is altered in the giant toad, Rhinella marina , living in agricultural areas where EDCs are used relative to suburban areas. We also ask if reductions in spermatogenesis were associated with developmental gonadal abnormalities (intersex) found in the same frogs. We found that toads in agricultural areas exhibited reduced spermatogenesis relative to non-agricultural animals, and that those reductions were not associated with gross gonadal abnormalities. All toads living in agricultural areas had reduced spermatogenesis relative to those living in non-agricultural areas regardless of whether they had gonadal abnormalities originating during development. Similarities in reproductive dysfunction among diverse taxa living in agricultural areas, including humans, suggest that many vertebrate taxa living in agricultural areas around the globe are likely experiencing some level of reproductive dysfunction. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Early social behaviors like juvenile play are important for normal cognitive and social development. Deficits in these behaviors are associated with neurodevelopmental disorders, such as autism. Rat juvenile rough-and-tumble play is a useful behavioral biomarker of neurodevelopment, and is sensitive to chemical factors such as pre and neonatal hormones. Despite a rich body of literature characterizing hormonal programming of rodent juvenile play, the physiological mechanisms that regulate the organization of play behavior are not well characterized. Synthesizing results to understand the role of endocrine signaling in the development of play behavior remains difficult due to methodological inconsistency across studies. In this review, we synthesize what is known about hormonal mechanisms programming play, advocate standardized protocols for investigating rat play, and identify key areas where future research is needed. A synthetic understanding of the relationship between endocrine signaling and behavioral programming will improve our ability to understand the development and onset of neurodevelopmental disorders in humans and ultimately will help prevent these devastating conditions. [ABSTRACT FROM AUTHOR]

[Display omitted] • ' Candidatus Mellornella promiscua' is described. • This new bacterial species is a pathogen of the mud crab, Eurypanopeus depressus. • The bacterial pathogen causes hepatopancreatic necrosis. • A genome, phylogenetic trees, pathology, and ultrastructure are provided. • The discovery builds our understanding of crustacean disease ecology. Bacterial pathogens are a long-standing threat to the longevity and survival of crustacean hosts. Their presence and continuing emergence require close monitoring to understand their impact on fished, cultured, and wild crustacean populations. We describe a new bacterial pathogen belonging to the Anaplasmataceae family (Alphaproteobacteria: Rickettsiales), providing pathological, ultrastructural, phylogenetic, and genomic evidence to determine a candidate genus and species (' Candidatus Mellornella promiscua'). This bacterium was found to infect the mud crab, Eurypanopeus depressus, on the North Carolina coastline (USA) at a prevalence of 10.8%. ' Candidatus Mellornella promiscua' was often observed in co-infection with the rhizocephalan barnacle, Loxothylacus panopaei. The bacterium was only found in the hepatopancreas of the mud crab host, causing cytoplasmic hypertrophy, tubule necrosis, large plaques within the cytoplasm of the host cell, and an abundance of sex-pili. The circular genome of the bacterium is 1,013,119 bp and encodes 939 genes in total. Phylogenetically, the new bacterium branches within the Anaplasmataceae. The genome is dissimilar from other described bacteria, with 16S gene similarity observed at a maximum of 85.3% to a Wolbachia endosymbiont. We explore this novel bacterial pathogen using genomic, phylogenetic, ultrastructural, and pathological methods, discussing these results in light of current bacterial taxonomy, similarity to other bacterial pathogens, and the potential impact upon the surrounding disease ecology of the host and benthic ecosystem. [ABSTRACT FROM AUTHOR]

Conflicts of interest, situations where personal or organizational considerations have compromised or biased professional judgment and objectivity, can weaken scientific credibility, pose threats to biodiversity and ecosystem services, and are often precursors to corruption. Here, we review historical and international examples of conflicts of interest and their impacts on global biodiversity. We present a contemporary example of a conflict of interest that might have implications for the U.S. Environmental Protection Agency's re-evaluation of the safety of the herbicide atrazine. To help scientists, natural resource managers, policy makers, and judicial officials identify and thwart conflicts of interest, we review strategies used by individuals and organizations with conflicts of interest to evade environmental and public health decisions, discuss the role of the scientific and governmental review processes in maintaining scientific integrity, and offer recommendations to reduce bias and facilitate sound and swift decision making for enhanced environmental health. [ABSTRACT FROM AUTHOR]

Objective: The biological effects of the herbicide atrazine on freshwater vertebrates are highly controversial. In an effort to resolve the controversy, we conducted a qualitative meta-analysis on the effects of ecologically relevant atrazine concentrations on amphibian and fish survival, behavior, metamorphic traits, infections, and immune, endocrine, and reproductive systems. Data sources: We used published, peer-reviewed research and applied strict quality criteria for inclusion of studies in the meta-analysis. Data synthesis: We found little evidence that atrazine consistently caused direct mortality of fish or amphibians, but we found evidence that it can have indirect and sublethal effects. The relationship between atrazine concentration and timing of amphibian metamorphosis was regularly nonmonotonic, indicating that atrazine can both accelerate and delay metamorphosis. Atrazine reduced size at or near metamorphosis in 15 of 17 studies and 14 of 14 species. Atrazine elevated amphibian and fish activity in 12 of 13 studies, reduced antipredator behaviors in 6 of 7 studies, and reduced olfactory abilities for fish but not for amphibians. Atrazine was associated with a reduction in 33 of 43 immune function end points and with an increase in 13 of 16 infection end points. Atrazine altered at least one aspect of gonadal morphology in 7 of 10 studies and consistently affected gonadal function, altering spermatogenesis in 2 of 2 studies and sex hormone concentrations in 6 of 7 studies. Atrazine did not affect vitellogenin in 5 studies and increased aromatase in only 1 of 6 studies. Effects of atrazine on fish and amphibian reproductive success, sex ratios, gene frequencies, populations, and communities remain uncertain. Conclusions: Although there is much left to learn about the effects of atrazine, we identified several consistent effects of atrazine that must be weighed against any of its benefits and the costs and benefits of alternatives to atrazine use. [ABSTRACT FROM AUTHOR]

A variety of human land uses involve the release of toxins into the environment. Wildlife live alongside humans across this array of land uses and thus, are exposed to varying chemical milieus. Kidneys are the principle excretory organs for vertebrates and excessive or chronic exposure to exogenous toxins can lead to renal pathology and renal failure. Although studies have linked chemical exposure to specific renal diseases across diverse taxa, none compare renal lesions occurring in wildlife living in different types of human-modified landscapes. We identify lesions characteristic of renal stress, including toxin exposure, in 82 giant toad (Bufo marinus) males living in habitats ranging from suburban to agricultural. In a previous study [McCoy K.A., Bortnick L.J., Campbell C.M., Hamlin H.J., Guillette L.J., Jr., St. Mary C.M. Agriculture Alters Gonadal Form and Function in Bufo marinus. Environ Health Persp; in press.], these individuals were examined for gonadal abnormalities, which were significantly and positively associated with percentage of agriculture at the collection site. Thus, we hypothesized the same association for renal abnormalities. We scored gross anatomical abnormalities and used light microscopy to identify tubular and interstitial lesions that have been associated with toxicant exposure in other organisms, including humans. Renal lesions indicative of tubular disease were observed at one suburban and two agricultural sites, whereas interstitial lesions were most severe at one agricultural site. Although there was no relationship between frequency of renal disease and proportion of agriculture in the collection vicinity, the renal lesions we identify are consistent with toxin exposure and are similar to those found in human drug abusers and patients suffering medication-induced nephropathy. This is the first study to describe renal lesions in a wild amphibian species and investigate the distribution of renal lesions... [Copyright &y& Elsevier]

BACKGROUND: Many agricultural contaminants disrupt endocrine systems of wildlife. However, evidence of endocrine disruption in wild amphibians living in agricultural areas has been controversial. Typically, studies on the effects of pollutants on wildlife attempt to compare polluted with unpolluted sites. OBJECTIVES: We took a novel approach to address this question by explicitly quantifying the relationship between gonadal abnormalities and habitats characterized by differing degrees of agricultural activity. METHODS: We quantified the occurrence of gonadal abnormalities and measures of gonadal function in at least 20 giant toads (Bufo marinus) from each of five sites that occur along a gradient of increasing agricultural land use from 0 to 97%. RESULTS: The number of abnormalities and frequency of intersex gonads increased with agriculture in a dose-dependent fashion. These gonadal abnormalities were associated with altered gonadal function. Testosterone, but not 17β-estradiol, concentrations were altered and secondary sexual traits were either feminized (increased skin mottling) or demasculinized (reduced forearm width and nuptial pad number) in intersex toads. Based on the end points we examined, female morphology and physiology did not differ across sites. However, males from agricultural areas had hormone concentrations and secondary sexual traits that were intermediate between intersex toads and nonagricultural male toads. Skin coloration at the most agricultural site was not sexually dimorphic; males had female coloration. CONCLUSIONS: Steroid hormone concentrations and secondary sexual traits correlate with reproductive activity and success, so affected toads likely have reduced reproductive success. These reproductive abnormalities could certainly contribute to amphibian population declines occurring in areas exposed to agricultural contaminants. [ABSTRACT FROM AUTHOR]

Abstract: Aquatic nitrate contamination has escalated over the past 50 years, primarily due to intensified fertilizer application and sewage production worldwide. Nitrate''s role in the decline of amphibian populations remains unclear, although studies suggest that nitrate exposure affects larval development. We exposed Bufo terrestris tadpoles to environmentally relevant nitrate concentrations from Gosner stage 25 through forelimb emergence. Tadpoles were exposed to fluctuating (0–30mg/L NO3-N, alternated three times per week) or constant nitrate concentrations (0, 5, 15, or 30mg/L NO3-N), and effects were compared in two water types: natural spring water and reverse-osmosis filtered water, fortified with electrolytes (ROe). We measured growth and developmental rates, survival, time to metamorphosis, metamorph body size, hepatosomatic index, and whole-body thyroxine (T4) concentrations at forelimb emergence. Based on our observations, we reached three main conclusions: (1) in constant nitrate, tadpoles in ROe water grew faster, and were generally larger with higher or similar T4 at metamorphosis than tadpoles raised in spring water, irrespective of nitrate concentration, (2) in fluctuating nitrate (flux), there were no differences in time to or size at metamorphosis in either water type relative to controls; however, mean T4 concentrations in the flux treatment showed a reversed pattern in the two water types (SP>ROe) compared to the general pattern observed with constant nitrate exposure (ROe >SP), and (3) the effect of nitrate on growth and development depended on water type. In ROe water with high nitrate (ROe-30mg/L NO3-N), tadpoles metamorphosed an average of 5 days (13%) earlier than control animals, but were similar in size to controls. However, spring water tadpoles reared in high nitrate (30mg/L) delayed metamorphosis by 7 days (18%) compared to animals reared in SP-0, and 11 days (32%) compared to tadpoles raised in ROe-30. This delayed development allowed SP-30 tadpoles to reach a larger size that was more similar to metamorphs raised in ROe water. Based on information from other studies, we conclude that, in ROe water, tadpoles exhibited an expected stress response to nitrate (e.g. metamorphosed earlier as nitrate concentration increased). However, we suggest that, in spring water, tadpoles were exposed to additional stressors that decreased growth rate and thyroxine concentrations, and that this effect was modified by nitrate. [Copyright &y& Elsevier]

Determining the mechanisms of toxicity induced by pollutants has long been a research priority in lieu of considering the mechanisms of resilience that prevent deleterious impacts. Protective mechanisms in many taxa can be therapeutically targeted to enhance resilience to synthetic toxicants. For example, the environmental sensor, Nuclear factor (erythroid-derived 2)-like 2 (Nfe2l2 or Nrf2), a transcription factor, facilitates transcription of many protective genes. Hypospadias is a common malformation of the penis. The risk of being born with hypospadias increases with pollutant exposure. We use vinclozolin-induced hypospadias in the mouse as a model to test the hypothesis that pollutant-induced birth defects can be prevented and reduced in severity by augmenting natural mechanisms of resilience. Pregnant mice were exposed to the demasculinizing toxicant, vinclozolin, in combination with increasing doses of the NRF2 activator, sulforaphane. The sulforaphane dose that most effectively increased masculinization (anogenital distance) was identified and used to test the hypothesis that sulforaphane reduces the hypospadias-inducing potency of vinclozolin. Finally, a Nrf2 knockout study was conducted to test whether NRF2 was required for the sulforaphane-induced rescue effects. Sulforaphane supplementation to vinclozolin exposed embryos increased anogenital distance in a nonlinear fashion typical of Nrf2 activators. The most effective dose of sulforaphane (45 mg/kg) reduced the occurrence and severity of vinclozolin-induced hypospadias and corrected penis morphogenesis. The sulforaphane-induced rescue effect was dependent on the presence of Nrf2. Nrf2 plays a critical role in protecting the fetus from vinclozolin and reduces the incidence and severity of hypospadias, the most common birth defect in boys in many countries. This work lays a foundation for developing prenatal supplements that will protect the fetus from pollutant-induced hypospadias. Studying the protective mechanisms that drive resilience to toxicants will facilitate innovation of protective therapies. [Display omitted] • Sulforaphane (SFN) increases anogenital distance in antiandrogen exposed fetuses • SFN reduces the potency of vinclozolin to alter urogenital development • SFN reduces hypospadias incidence and severity induced by vinclozolin • Nrf2 a master regulator of cytoprotective genes controls SFN-induced rescue effects [ABSTRACT FROM AUTHOR]

Emerging concepts in developmental biology, such as facilitated variation and dynamical patterning modules, address a major shortcoming of the Modern Synthesis in Biology: how genotypic variation is transduced into functional yet diverse phenotypic variation. Still, we lack a theory to explain how variation at the cellular and tissue level is coordinated into variation at the whole-organism level, especially as priority of cellular and tissue functions change over an individual’s lifetime and are influenced by environmental variation. Here, we propose that interactions among a limited subset of physiological factors that we call, integrators, regulate most phenotypic variation at the organismal level. Integrators are unique among physiological factors in that they have the propensity to coordinate the expression of conserved gene modules of most types of tissues because they participate as nodes in a hierarchical network. In other words, integrator networks impose physiological epistasis, meaning that whole-organism phenotypic responses will be influenced by previous experiences, current environmental conditions, and fitness priorities as encoded by individual integrators. Below, we provide examples of how integrator networks are responsible for both profound and irreversible phenotypic changes (i.e., metamorphosis, sexual differentiation) as well as subtler, transient (e.g., pelage color, seasonal fluctuations in lymphoid and reproductive tissues) variation. The goal of this article is not to describe completely how integrator networks function, but to stimulate discussion about the role of physiology in linking genetic to phenotypic variation. To generate useful data sets for understanding integrator networks and to inform whole-organism physiology generally, we describe several useful tools including vector-field editing, response-surface regression, and experiments of life-table responses. We then close by highlighting some implications of integrator networks for conservation and biomedicine. [ABSTRACT FROM AUTHOR]

Abstract: Atrazine is the most commonly detected pesticide contaminant of ground water, surface water, and precipitation. Atrazine is also an endocrine disruptor that, among other effects, alters male reproductive tissues when animals are exposed during development. Here, we apply the nine so-called “Hill criteria” (Strength, Consistency, Specificity, Temporality, Biological Gradient, Plausibility, Coherence, Experiment, and Analogy) for establishing cause–effect relationships to examine the evidence for atrazine as an endocrine disruptor that demasculinizes and feminizes the gonads of male vertebrates. We present experimental evidence that the effects of atrazine on male development are consistent across all vertebrate classes examined and we present a state of the art summary of the mechanisms by which atrazine acts as an endocrine disruptor to produce these effects. Atrazine demasculinizes male gonads producing testicular lesions associated with reduced germ cell numbers in teleost fish, amphibians, reptiles, and mammals, and induces partial and/or complete feminization in fish, amphibians, and reptiles. These effects are strong (statistically significant), consistent across vertebrate classes, and specific. Reductions in androgen levels and the induction of estrogen synthesis – demonstrated in fish, amphibians, reptiles, and mammals – represent plausible and coherent mechanisms that explain these effects. Biological gradients are observed in several of the cited studies, although threshold doses and patterns vary among species. Given that the effects on the male gonads described in all of these experimental studies occurred only after atrazine exposure, temporality is also met here. Thus the case for atrazine as an endocrine disruptor that demasculinizes and feminizes male vertebrates meets all nine of the “Hill criteria”. [Copyright &y& Elsevier]

Predation can be intense, creating strong direct and indirect effects throughout food webs. In addition, ecologists increasingly recognize that fluxes of organisms across ecosystem boundaries can have major consequences for community dynamics. Species with complex life histories often shift habitats during their life cycles and provide potent conduits coupling ecosystems. Thus, local interactions that affect predator abundance in one ecosystem (for example a larval habitat) may have reverberating effects in another (for example an adult habitat). Here we show that fish indirectly facilitate terrestrial plant reproduction through cascading trophic interactions across ecosystem boundaries. Fish reduce larval dragonfly abundances in ponds, leading to fewer adult dragonflies nearby. Adult dragonflies consume insect pollinators and alter their foraging behaviour. As a result, plants near ponds with fish receive more pollinator visits and are less pollen limited than plants near fish-free ponds. Our results confirm that strong species interactions can reverberate across ecosystems, and emphasize the importance of landscape-level processes in driving local species interactions. [ABSTRACT FROM AUTHOR]

Endocrine disrupting compounds (EDCs) are ubiquitous environmental pollutants that alter endocrine system function, induce birth defects, and a myriad of other negative health outcomes. Although the mechanism of toxicity of many EDCs have been studied in detail, little work has focused on understanding the mechanisms through which pregnant dams and fetuses protect themselves from EDCs, or if those protective mechanisms are sexually dimorphic in fetuses. In this study, we examined proteomic alterations in the livers of mouse dams and their male and female fetuses induced by vinclozolin, a model antiandrogenic EDC. Dam livers upregulated nine phase I and phase II detoxification pathways and pathway analysis revealed that more pathways are significantly enriched in dam livers than in fetal livers. Phase I and II detoxification proteins are also involved in steroid and steroid hormone biosynthesis and vinclozolin likely alters steroid levels in both the dam and the fetus. The response of the fetal liver proteome to vinclozolin exposure is sexually dimorphic. Female fetal livers upregulated proteins in xenobiotic metabolism pathways, whereas male fetal livers upregulated proteins in oxidative phosphorylation pathways. These results suggest that female fetuses increase protective mechanisms, whereas male fetuses increase ATP production and several disease pathways that are indicative of oxidative damage. Females fetuses upregulate proteins and protective pathways that were similar to the dams whereas males did not. If this sexually dimorphic pattern is typical, then males might generally be more sensitive to EDCs. • Dam livers respond to vinclozolin exposure by upregulating protective mechanisms • Fetal livers have a sexually dimorphic response to vinclozolin exposure. • Female fetal livers upregulate protective mechanisms. • Male fetal livers upregulate mechanisms associated with stress and disease. [ABSTRACT FROM AUTHOR]