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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]

A classroom activity to introduce students to the intrinsic and economic values of biodiversity.

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]

The natural world is very different today than it was 10,000 or even 1,000 years ago. Every natural ecosystem on the globe has been altered, many severely, as a consequence [...]

Author(s): Tiffany M. Knight (corresponding author) [1, 2]; Michael W. McCoy [1]; Jonathan M. Chase [2]; Krista A. McCoy [1]; Robert D. Holt [1] Predation can be intense, creating strong [...]

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]

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]

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]

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]

Nearly all aquatic ecosystems are affected by sublethal levels of anthropogenic chemical contamination, but other agents of large‐scale anthropogenic disruption of ecosystems have received more attention. Consequently, ecologists do not fully appreciate how sublethal contaminant exposure affects ecosystems. Sublethal contaminants can affect ecological systems directly via their impacts on an organism's fitness or indirectly by changing the strengths of species interactions. This study investigated how an emerging class of contaminants—pharmaceuticals and personal care products (PPCPs)—influences food webs by affecting the biology of organisms and by interfering with predator–prey interactions. Specifically, we investigated how three common PPCPs—caffeine, DEET (N, N‐diethyl‐meta‐toluamide), and triclosan—affect the strength of the interaction between a common mosquito predator (i.e., mosquito fish) and mosquito larvae as well as how these PPCPS affect mosquito survival, life history traits, and oviposition site choices. We found that all three PPCPs, individually and combined as a mixture, reduced predator consumption rates. Relative to a contaminant‐free control, the presence of predator cues reduced mosquito oviposition and larval abundance for all PPCP treatments except for DEET. Predator cues reduced mosquito adult emergence across PPCP treatments; however, mosquitoes that were exposed to caffeine did not emerge as adults even in the absence of predator cues. This study shows that the effects of PPCPs are diverse and can interact with mosquitoes and their predators in ways that cannot be predicted by their individual effects. In a contaminated world, ecologists need to better understand how sublethal concentrations of ubiquitous, biologically active pollutants might challenge what we think we know about how ecological systems function. [ABSTRACT FROM AUTHOR]

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]

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]

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]

Endocrine disrupting chemicals (EDCs) are pollutants found throughout the environment that disrupt normal endocrine processes. In mice, penis development is thought to be most susceptible to EDCs during a critical developmental window occurring on embryonic days (E) 15.5-17.5. However, androgen signaling begins on E13.5 when androgen receptor (AR) protein is found in the genitalia and testosterone is circulating. We hypothesize that disrupting androgen signaling prior to the established critical window sensitizes the penis to future androgen disruption. To test this hypothesis, CD1 dams were exposed to vinclozolin or a corn oil solvent control on E13.5 and E14.5 and AR levels were measured with immunohistochemistry on E14.5. Early antiandrogen exposure reduced AR within nuclei and decreased intensity of AR expression within E14.5 genitalia. To evaluate the influence of antiandrogen exposure before the known critical window of penis development, two groups of pregnant dams (n = 3) were exposed to vinclozolin starting at either E13.5 or E14.5 and continued exposure through E16.5. Histology and M.O.U.S.E. scoring were used to quantify penis abnormalities. To account for differences in total doses mice experienced due to differences in length of dosing time, we compared animals that received the same total doses. Exposure to antiandrogens on E13.5 exacerbated malformations when exposure was continued through sexually dimorphic development. Both exposure time and vinclozolin dose are important for severity of vinclozolin-induced penis abnormalities in mice. This work shows that antiandrogen exposure prior to sensitive periods can exacerbate the effects of later antiandrogen exposure on reproductive development.

Background: Congenital abnormalities vary in presentation, yet studies using model organisms tend to focus on occurrence rather than severity of the defect. Scoring severity of abnormalities in model systems allows explicit hypothesis testing during basic, translational, and reverse translational studies. We developed and validated a protocol to quantify severity of male urogenital feminization (hypospadias) in the mouse model. Hypospadias is one of the most common birth defects in the world., Methods: To induce genital feminization, pregnant mice were exposed to different concentrations of the antiandrogen vinclozolin. Genitalia were photographed at gestational age 18.5. A dichotomous scoring system to evaluate genital feminization was developed, and validated against histological measurements of urethral length. A training protocol was developed for novice scorers, and criteria were defined to evaluate precision and accuracy of scores., Results: Vinclozolin induced variation in hypospadias severity. Severity scores were tightly correlated with histologically determined urethral length and both techniques showed similar dose-response relationships. Novice observers were trained to precisely and accurately score hypospadias severity., Conclusion: This standardized scoring system advances the mouse as a model to study urogenital development, and will facilitate research on the mechanisms driving genital feminization in males, and aid translational hypospadias research.

Tradeoffs between time to and size at metamorphosis occur in many organisms with complex life histories. The ability to accelerate metamorphosis can increase survival to the next life stage, but the resulting smaller size at metamorphosis is often associated with lower post-metamorphic survival or reduced fecundity of adults. Reduced fecundity is thought to be because of reduced energy reserves, longer time to maturity, or reduced capacity to carry eggs or compete for mates. This pattern could also be explained by a shift in allocation to somatic growth that further retards the growth or development of reproductive tissues. The main goal of this study was to determine if the relationship between growth and development of somatic and gonadal tissues depends on environmental conditions. We address this question through two experiments in which we quantify the development and growth of the body and gonads of Xenopus laevis reared in different resource environments. First, tadpoles were reared communally and development and growth were evaluated over time. Restricted food reduced somatic and gonadal growth rate, but did not affect the developmental rate of either tissue type. Second, tadpoles were reared individually and evaluated at metamorphosis. Restricted food reduced somatic development and growth, but only influenced size, and not developmental stage of testes at metamorphosis. This work demonstrates that environmental conditions influence tradeoffs between growth and development of somatic and gonadal tissues, apparently in a sex-specific manner. These tradeoffs may contribute to phenotypic correlations between small size and reduced fitness., ((c) 2007 Wiley-Liss, Inc.)