Neuroendocrine disruption of organizational and activational hormone programming in poikilothermic vertebrates.

In vertebrates, sexual differentiation of the reproductive system and brain is tightly orchestrated by organizational and activational effects of endogenous hormones. In mammals and birds, the organizational period is typified by a surge of sex hormones during differentiation of specific neural circuits; whereas activational effects are dependent upon later increases in these same hormones at sexual maturation. Depending on the reproductive organ or brain region, initial programming events may be modulated by androgens or require conversion of androgens to estrogens. The prevailing notion based upon findings in mammalian models is that male brain is sculpted to undergo masculinization and defeminization. In absence of these responses, the female brain develops. While timing of organizational and activational events vary across taxa, there are shared features. Further, exposure of different animal models to environmental chemicals such as xenoestrogens such as bisphenol A-BPA and ethinylestradiol-EE2, gestagens, and thyroid hormone disruptors, broadly classified as neuroendocrine disrupting chemicals (NED), during these critical periods may result in similar alterations in brain structure, function, and consequently, behaviors. Organizational effects of neuroendocrine systems in mammals and birds appear to be permanent, whereas teleost fish neuroendocrine systems exhibit plasticity. While there are fewer NED studies in amphibians and reptiles, data suggest that NED disrupt normal organizational-activational effects of endogenous hormones, although it remains to be determined if these disturbances are reversible. The aim of this review is to examine how various environmental chemicals may interrupt normal organizational and activational events in poikilothermic vertebrates. By altering such processes, these chemicals may affect reproductive health of an animal and result in compromised populations and ecosystem-level effects.

Exposure to the Contraceptive Progestin, Gestodene, Alters Reproductive Behavior, Arrests Egg Deposition, and Masculinizes Development in the Fathead Minnow (Pimephales promelas).

Endogenous progestogens and pharmaceutical progestins enter the environment through wastewater treatment plant effluent and agricultural field runoff. Lab studies demonstrate strong, negative exposure effects of these chemicals on aquatic vertebrate reproduction. Behavior can be a sensitive, early indicator of exposure to environmental contaminants associated with altered reproduction yet is rarely examined in ecotoxicology studies. Gestodene is a human contraceptive progestin and a potent activator of fish androgen receptors. Our objective was to test the effects of gestodene on reproductive behavior and associated egg deposition in the fathead minnow. After only 1 day, males exposed to ng/L of gestodene were more aggressive and less interested in courtship and mating, and exposed females displayed less female courtship behavior. Interestingly, 25% of the gestodene tanks contained a female that drove the male out of the breeding tile and displayed male-typical courtship behaviors toward the other female. Gestodene decreased or arrested egg deposition with no observed gonadal histopathology. Together, these results suggest that effects on egg deposition are primarily due to altered reproductive behavior. The mechanisms by which gestodene disrupts behavior are unknown. Nonetheless, the rapid and profound alterations of the reproductive biology of gestodene-exposed fish suggest that wild populations could be similarly affected.

Aqueous exposure to the progestin, levonorgestrel, alters anal fin development and reproductive behavior in the eastern mosquitofish (Gambusia holbrooki).

Endogenous progestogens are important regulators of vertebrate reproduction. Synthetic progestins are components of human contraceptive and hormone replacement pharmaceuticals. Both progestogens and progestins enter the environment through a number of sources, and have been shown to cause profound effects on reproductive health in various aquatic vertebrates. Progestins are designed to bind human progesterone receptors, but they also have been shown to strongly activate androgen receptors in fish. Levonorgestrel (LNG) activates fish androgen receptors and induces development of male secondary sex characteristics in females of other species. Although behavior has been postulated to be a sensitive early indicator of exposure to certain environmental contaminants, no such research on the reproductive behavior of gestagen-exposed fish has been conducted to date. The goal of our study was to examine the exposure effects of a human contraceptive progestin, LNG, on the reproductive development and behavior of the viviparous eastern mosquitofish (Gambusia holbrooki). Internal fertilization is a requisite characteristic of viviparous species, and is enabled by an androgen driven elongation of the anal fin into the male gonopodium (i.e., phallus). In this study, we exposed adult mosquitofish to ethanol (EtOH control), 10ng/L, and 100ng/L LNG for 8d using a static replacement exposure design. After 8d, a subset of males and females from each treatment were examined for differences in the 4:6 anal fin ratio. In addition, paired social interaction trials were performed using individual control males and control females or females treated 10ng/L or 100ng/L LNG. Female mosquitofish exposed to LNG were masculinized as evidenced by the elongation of the anal fin rays, a feature normal to males and abnormal to females. LNG caused significant increases in the 4:6 anal fin ratios of female mosquitofish in both the 10ng/L and 100ng/L treatments, although these differences were not significant between the two treatments. LNG caused significant increases in the 4:6 anal fin ratio of males exposed to 100ng/L, with no effects observed in the 10ng/L treatment. In addition, the reproductive behavior of control males paired with female mosquitofish exposed to 100ng/L LNG was also altered, for these males spent more time exhibiting no reproductive behavior, had decreased attending behavior, and a lower number of gonopodial thrusts compared to control males paired to control female mosquitofish. Given the rapid effects on both anal fin morphology and behavior observed in this study, the mosquitofish is an excellent sentinel species for the detection of exposure to LNG and likely other 19-nortestosterone derived contraceptive progestins in the environment.

Environmental gestagens activate fathead minnow (Pimephales promelas) nuclear progesterone and androgen receptors in vitro.

Gestagen is a collective term for endogenous and synthetic progesterone receptor (PR) ligands. In teleost fishes, 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) and 17α,20ß,21-trihydroxy-4-pregnen-3-one (20ß-S) are the predominant progestogens, whereas in other vertebrates the major progestogen is progesterone (P4). Progestins are components of human contraceptives and hormone replacement pharmaceuticals and, with P4, can enter the environment and alter fish and amphibian reproductive health. In this study, our primary objectives were to clone the fathead minnow (FHM) nuclear PR (nPR), to develop an in vitro assay for FHM nPR transactivation, and to screen eight gestagens for their ability to transactivate FHM nPR. We also investigated the ability of these gestagens to transactivate FHM androgen receptor (AR). Fish progestogens activated FHM nPR, with DHP being more potent than 20ß-S. The progestin drospirenone and P4 transactivated the FHM nPR, whereas five progestins and P4 transactivated FHM AR, all at environmentally relevant concentrations. Progestins are designed to activate human PR, but older generation progestins have unwanted androgenic side effects in humans. In FHMs, several progestins proved to be strong agonists of AR. Here, we present the first mechanistic evidence that environmental gestagens can activate FHM nPR and AR, suggesting that gestagens may affect phenotype through nPR- and AR-mediated pathways.

Sources, concentrations, and exposure effects of environmental gestagens on fish and other aquatic wildlife, with an emphasis on reproduction.

Fish and other aquatic wildlife, including frogs, turtles, and alligators, have been used as vertebrate sentinels for the effects of endocrine disrupting and other emerging chemicals of concern found in aquatic ecosystems. Research has focused on the effects of estrogenic, androgenic, and thyroidogenic compounds, but there is a growing body of literature on the reproductive health exposure effects of environmental gestagens on aquatic wildlife. Gestagens include native progestogens, such as progesterone, and synthetic progestins, such as gestodene and levonorgestrel, which bind progesterone receptors and have critically important roles in vertebrate physiology, especially reproduction. Roles for progestogen include regulating gamete maturation and orchestrating reproductive behavior, both as circulating hormones and as secreted pheromones. Gestagens enter the aquatic environment through paper mill effluent, wastewater treatment plant effluent, and agricultural runoff. A number of gestagens have been shown to negatively affect reproduction, development, and behavior of exposed fish and other aquatic wildlife at ng/L concentrations, and these compounds have been measured in the environment at single to 375 ng/L. Given the importance of endogenous progestogens in the regulation of gametogenesis, secondary sex characteristics, and reproductive behavior in vertebrates and the documented exposure effects of pharmaceutical progestins and progesterone, environmental gestagens are an emerging class of contaminants that deserve increased attention from researchers and regulators alike. The potential for environmental gestagens to affect the reproductive health of aquatic vertebrates seems evident, but there are a number of important questions for researchers to address in this nascent field. These include identifying biomarkers of gestagen exposure; testing the effects of environmentally relevant mixtures; and determining what other physiological endpoints and taxa might be affected by exposure to environmental gestagens.

Current concepts in neuroendocrine disruption.

In the last few years, it has become clear that a wide variety of environmental contaminants have specific effects on neuroendocrine systems in fish, amphibians, birds and mammals. While it is beyond the scope of this review to provide a comprehensive examination of all of these neuroendocrine disruptors, we will focus on select representative examples. Organochlorine pesticides bioaccumulate in neuroendocrine areas of the brain that directly regulate GnRH neurons, thereby altering the expression of genes downstream of GnRH signaling. Organochlorine pesticides can also agonize or antagonize hormone receptors, adversely affecting crosstalk between neurotransmitter systems. The impacts of polychlorinated biphenyls are varied and in many cases subtle. This is particularly true for neuroedocrine and behavioral effects of exposure. These effects impact sexual differentiation of the hypothalamic-pituitary-gonadal axis, and other neuroendocrine systems regulating the thyroid, metabolic, and stress axes and their physiological responses. Weakly estrogenic and anti-androgenic pollutants such as bisphenol A, phthalates, phytochemicals, and the fungicide vinclozolin can lead to severe and widespread neuroendocrine disruptions in discrete brain regions, including the hippocampus, amygdala, and hypothalamus, resulting in behavioral changes in a wide range of species. Behavioral features that have been shown to be affected by one or more these chemicals include cognitive deficits, heightened anxiety or anxiety-like, sociosexual, locomotor, and appetitive behaviors. Neuroactive pharmaceuticals are now widely detected in aquatic environments and water supplies through the release of wastewater treatment plant effluents. The antidepressant fluoxetine is one such pharmaceutical neuroendocrine disruptor. Fluoxetine is a selective serotonin reuptake inhibitor that can affect multiple neuroendocrine pathways and behavioral circuits, including disruptive effects on reproduction and feeding in fish. There is growing evidence for the association between environmental contaminant exposures and diseases with strong neuroendocrine components, for example decreased fecundity, neurodegeneration, and cardiac disease. It is critical to consider the timing of exposures of neuroendocrine disruptors because embryonic stages of central nervous system development are exquisitely sensitive to adverse effects. There is also evidence for epigenetic and transgenerational neuroendocrine disrupting effects of some pollutants. We must now consider the impacts of neuroendocrine disruptors on reproduction, development, growth and behaviors, and the population consequences for evolutionary change in an increasingly contaminated world. This review examines the evidence to date that various so-called neuroendocrine disruptors can induce such effects often at environmentally-relevant concentrations.

An inter-laboratory study on the variability in measured concentrations of 17ß-estradiol, testosterone, and 11-ketotestosterone in white sucker: implications and recommendations.

Endocrine-disrupting chemicals are exogenous substances that can impact the reproduction of fish, potentially by altering circulating concentrations of 17ß-estradiol (E2), testosterone (T), and 11-ketotestosterone (11-KT). Common methods to measure steroids in plasma samples include radioimmunoassays (RIAs) and enzyme-linked immunosorbant assays (ELISAs). The present study examines variability in E2, T, and 11-KT across 8 laboratories measuring reference and pulp mill effluent-exposed white sucker (Catostomus commersoni) plasma. We examine the contribution of assay type (RIA vs ELISA), standardized hormone extraction, location of values on the standard curve (upper and lower limits), and other variables on the ability to distinguish hormone levels between reference and exposed fish and the impact of these variables on quantitation of hormones in different laboratories. Of the 8 participating laboratories, 7 of 8 and 7 of 7 identified differences between sites for female E2 and female T, respectively, and 7 of 7 and 4 of 5 identified no differences between male T and male 11-KT. Notably, however, the ng/mL concentration of steroids measured across laboratories varied by factors of 10-, 6-, 14-, and 10-fold, respectively. Within laboratory intra-assay variability was generally acceptable and below 15%. Factors contributing to interlaboratory variability included calculation errors, assay type, and methodology. Based on the interlaboratory variability detected, we provide guidelines and recommendations to improve the accuracy and precision of steroid measurements in fish ecotoxicology studies.

Creating females? Developmental effects of 17α-ethynylestradiol on the mangrove rivulus' ovotestis.

Interest in the occurrence and fate of trace organic contaminants in the aquatic environment and their potential effects on all organisms has increased over the past two decades. Researches on contaminants have included both natural and synthetic estrogenic contaminants, neuroactive pharmaceuticals, and other endocrine disrupting chemicals that are mediated by the androgen and progesterone receptors. Exposure to very low concentrations (ng/L or parts per trillion) of compounds such as 17α-ethynylestradiol (EE(2)), a synthetic estrogen, can affect gonadal development, viability and production of eggs, fertilization rate, and sexual differentiation in fishes. Researchers and aquaculturists have used exposures to relatively higher concentrations of androgens and estrogens, for example 17α-methyltestosterone and EE(2), respectively, to direct sexual differentiation in a number of fishes. Rivulus is an androdioecious teleost that in nature exists mostly as selfing, simultaneous hermaphrodites as well as a small number of males that outcross with hermaphrodites. No one has either collected females in the wild or created functional females in the laboratory. This study had two goals: (1) to develop a reliable protocol to produce female rivulus to enable downstream technologies such as embryo injections and (2) to investigate developmental effects of EE(2) on the sexual outcome, reproductive health, and relevant gene expression in rivulus. With these goals in mind, we exposed newly hatched rivulus to nominal concentrations of 0.1, 0.5, or 1.0 parts per million (ppm) EE(2) for 4 weeks, grew them to maturity in control water, and then compared egg production; production and viability of embryos; age of reproductive maturity; and gene expression in the brain, gonad, and liver. Expression levels of seven genes with known relevance to gonadal development and function (cyp19a1b, cyp19a1a, dmrt1, figα, ERα, ERß, and vtg) were measured using quantitative polymerase chain reaction (PCR). There was a significant decrease in cyp19a1a gene expression in the brain, corresponding to increased exposure to EE(2). Gonadal gene expression for cyp19a1a, ERα, and dmrt1 also decreased in response to EE(2). Vtg expression in the liver was unaffected. Our hypothesis that exposure to EE(2) during gonadal differentiation would direct female development was not supported by the data. However, treated fish exhibited impaired reproductive health that included reduced expression of relevant genes and, importantly, decreased fertility, increased sterility, and delay of age of reproductive maturity. The results of this study suggest that the development and maintenance of a simultaneous hermphrodite ovotestis may be particularly sensitive to its hormonal milieu.

"Mangrove 'killifish': an exemplar of integrative biology": introduction to the symposium.

The mangrove rivulus, Kryptolebias marmoratus, (hereafter, rivulus) is one of the only two vertebrates known to self-fertilize, with the other being a recently named close relative, Kryptolebias hermaphroditus (Tatarenkov et al. 2012). Rivulus is also the first fish species found to have environmental sex determination, whereby lower temperatures inhibit ovarian development, thus providing one potential route that avoids inbreeding depression (Harrington 1967; Conover 2004). Wild rivulus exist as androdioecious populations in which both hermaphrodites and, although relatively rare, males are found (Taylor 2000). In the laboratory, individual adult rivulus can produce isogenic embryos. Under as yet unknown environmental conditions, males develop and outcrossing between the hermaphrodites and males occurs (Taylor 2000; Mackiewicz et al. 2006a, 2006b). It is intriguing to consider the behavioral, neurological, and endocrinological control necessary to accommodate this reproductive strategy (Sakakura et al. 2006; Orlando et al. 2006; Earley et al. 2008). In addition to environmental sex determination and androdioecious reproduction, rivulus is also known to emerge from its aquatic surroundings and assume a transitory, terrestrial existence (Ong et al. 2007; Taylor et al. 2008; Cooper et al. 2012). Rivulus is an emerging and potentially powerful model for integrative and comparative biological research and, in part, this emergence has been catalyzed by this first symposium on its biology. The well-attended symposium comprised 11 speakers, which included four women and seven men, with academic ranks ranging from postdoctoral fellow to full professor, who came from four countries. This symposium will help drive future research within this taxon and will facilitate collaborations among researchers. It has already facilitated networking between heads of laboratories and current and potential future postdoctoral fellows and students. The organizing committee looks forward to the next rivulus symposium with great anticipation.

A graphical systems model and tissue-specific functional gene sets to aid transcriptomic analysis of chemical impacts on the female teleost reproductive axis.

Oligonucleotide microarrays and other 'omics' approaches are powerful tools for unsupervised analysis of chemical impacts on biological systems. However, the lack of well annotated biological pathways for many aquatic organisms, including fish, and the limited power of microarray-based analyses to detect low level differential expression of individual genes can hinder the ability to infer and understand chemical effects based on transcriptomic data. Here we report on the supervised assembly of a series of tissue-specific functional gene sets intended to aid transcriptomic analysis of chemical impacts on the female teleost reproductive axis. Gene sets were defined based on an updated graphical systems model of the teleost brain-pituitary-gonadal-hepatic axis. Features depicted in the model were organized into gene sets and mapped to specific probes on three zebrafish (Danio rerio) and two fathead minnow (Pimephales promelas) microarray platforms. Coverage of target genes on the microarrays ranged from 48% for the fathead minnow arrays to 88% for the most current zebrafish platform. Additionally, extended fathead minnow gene sets, incorporating first degree neighbors identified from a Spearman correlation network derived from a large compendium of fathead minnow microarray data, were constructed. Overall, only 14% of the 78 genes queried were connected in the network. Among those, over half had less than five neighbors, while two genes, cyclin b1 and zona pellucida glycoprotein 3, had over 100 first degree neighbors, and were neighbors to one another. Gene set enrichment analyses were conducted using microarray data from a zebrafish hypoxia experiment and fathead minnow time-course experiments conducted with three different endocrine-active chemicals. Results of these analyses demonstrate the utility of the approach for supporting biological inference from ecotoxicogenomic data and comparisons across multiple toxicogenomic experiments. The graphical model, gene mapping, and gene sets described are now available to the scientific community as tools to support ecotoxicogenomic research.

A computational model of the hypothalamic: pituitary: gonadal axis in female fathead minnows (Pimephales promelas) exposed to 17α-ethynylestradiol and 17ß-trenbolone.

BACKGROUND: Endocrine disrupting chemicals (e.g., estrogens, androgens and their mimics) are known to affect reproduction in fish. 17α-ethynylestradiol is a synthetic estrogen used in birth control pills. 17ß-trenbolone is a relatively stable metabolite of trenbolone acetate, a synthetic androgen used as a growth promoter in livestock. Both 17α-ethynylestradiol and 17ß-trenbolone have been found in the aquatic environment and affect fish reproduction. In this study, we developed a physiologically-based computational model for female fathead minnows (FHM, Pimephales promelas), a small fish species used in ecotoxicology, to simulate how estrogens (i.e., 17α-ethynylestradiol) or androgens (i.e., 17ß-trenbolone) affect reproductive endpoints such as plasma concentrations of steroid hormones (e.g., 17ß-estradiol and testosterone) and vitellogenin (a precursor to egg yolk proteins). RESULTS: Using Markov Chain Monte Carlo simulations, the model was calibrated with data from unexposed, 17α-ethynylestradiol-exposed, and 17ß-trenbolone-exposed FHMs. Four Markov chains were simulated, and the chains for each calibrated model parameter (26 in total) converged within 20,000 iterations. With the converged parameter values, we evaluated the model's predictive ability by simulating a variety of independent experimental data. The model predictions agreed with the experimental data well. CONCLUSIONS: The physiologically-based computational model represents the hypothalamic-pituitary-gonadal axis in adult female FHM robustly. The model is useful to estimate how estrogens (e.g., 17α-ethynylestradiol) or androgens (e.g., 17ß-trenbolone) affect plasma concentrations of 17ß-estradiol, testosterone and vitellogenin, which are important determinants of fecundity in fish.

Effects of estrogens and antiestrogens on gene expression of fathead minnow (Pimephales promelas) early life stages.

Endocrine disrupting chemicals (EDCs) are known to contaminate aquatic environments and alter the growth and reproduction of organisms. The objective of this study was to evaluate the sensitivity and utility of fathead minnow (Pimephales promelas) early life-stages as a model to measure effects of estrogenic and antiestrogenic EDCs on physiological and gene expression endpoints relative to growth and reproduction. Embryos (<24-h postfertilization, hpf) were exposed to a potent estrogen (17α-ethinyl estradiol, EE(2) , 2, 10, and 50 ng L(-1)); a weak estrogen (mycotoxin zearalenone, ZEAR, same concentrations as above); an antiestrogen (ZM 189, 154; 40, 250, and 1000 ng L(-1)); and to mixtures of EE(2) and ZM until swim-up stage (∼170 hpf). Exposure to all concentrations of ZEAR and to the lowest concentration of ZM resulted in increased body sizes, whereas high concentrations of EE(2) decreased body sizes. There was a significant increase in the frequency of abnormalities (mostly edema) in larvae exposed to all concentrations of EE(2), and high ZEAR, and EE(2) + ZM mixture groups. Expression of growth hormone was upregulated by most of the conditions tested. Exposure to 50 ng L(-1) ZEAR caused an induction of insulin-like growth factor 1, whereas exposure to 40 ng L(-1) ZM caused a downregulation of this gene. Expression of steroidogenic acute regulatory protein gene was significantly upregulated after exposure to all concentrations of EE(2) and luteinizing hormone expression increased significantly in response to all treatments tested. As expected, EE(2) induced vitellogenin expression; however, ZEAR also induced expression of this gene to similar levels compared to EE(2). Overall, exposure to EE(2) + ZM mixture resulted in a different expression pattern compared to single exposures. The results of this study suggest that an early life stage 7-day exposure is sufficient to recognize and evaluate effects of estrogenic compounds on gene expression in this fish model.

Gene expression responses in male fathead minnows exposed to binary mixtures of an estrogen and antiestrogen.

BACKGROUND: Aquatic organisms are continuously exposed to complex mixtures of chemicals, many of which can interfere with their endocrine system, resulting in impaired reproduction, development or survival, among others. In order to analyze the effects and mechanisms of action of estrogen/anti-estrogen mixtures, we exposed male fathead minnows (Pimephales promelas) for 48 hours via the water to 2, 5, 10, and 50 ng 17alpha-ethinylestradiol (EE2)/L, 100 ng ZM 189,154/L (a potent antiestrogen known to block activity of estrogen receptors) or mixtures of 5 or 50 ng EE(2)/L with 100 ng ZM 189,154/L. We analyzed gene expression changes in the gonad, as well as hormone and vitellogenin plasma levels. RESULTS: Steroidogenesis was down-regulated by EE(2) as reflected by the reduced plasma levels of testosterone in the exposed fish and down-regulation of genes in the steroidogenic pathway. Microarray analysis of testis of fathead minnows treated with 5 ng EE(2)/L or with the mixture of 5 ng EE(2)/L and 100 ng ZM 189,154/L indicated that some of the genes whose expression was changed by EE(2) were blocked by ZM 189,154, while others were either not blocked or enhanced by the mixture, generating two distinct expression patterns. Gene ontology and pathway analysis programs were used to determine categories of genes for each expression pattern. CONCLUSION: Our results suggest that response to estrogens occurs via multiple mechanisms, including canonical binding to soluble estrogen receptors, membrane estrogen receptors, and other mechanisms that are not blocked by pure antiestrogens.

Expression signatures for a model androgen and antiandrogen in the fathead minnow (Pimephales promelas) ovary.

Trenbolone, an anabolic androgen, and flutamide, an antiandrogen, are prototypical model compounds for agonism and antagonism of the androgen receptor. We hypothesized that 48 h exposures of female fathead minnows (Pimephales promelas) to environmentally relevant concentrations of these chemicals would alter genes regulated by the androgen receptor and that a mixture of the two compounds would block the effects. Gene expression in the ovaries was analyzed using a fathead minnow-specific 22,000-gene microarray. Flutamide altered abouttwicethe number of genes astrenbolone, most of which appeared to be through pathways not associated with the androgen receptor. A group of 70 genes, of which we could identify 37, were reciprocally regulated by trenbolone and flutamide. These are candidates for specific biomarkers for androgen receptor mediated gene expression. Four genes stand out as specifically related to reproduction: sperm associated antigen 8 (SPAG8), CASP8 and FADD-like apoptosis regulator (CFLAR), corticotropin releasing hormone (CRH), and 3beta-hydroxysteroid dehydrogenases (3beta-HSD). Three notable transcriptional regulators including myelocytomatosis viral oncogene homologue (MYC), Yin Yang 1 (YY1), and interferon regulator factor 1 (IRF1) may function as early molecular switches to control phenotypic changes in ovary tissue architecture and function in response to androgen or antiandrogen exposure.

A computational model of the hypothalamic-pituitary-gonadal axis in male fathead minnows exposed to 17alpha-ethinylestradiol and 17beta-estradiol.

Estrogenic chemicals in the aquatic environment have been shown to cause a variety of reproductive anomalies in fish including full sex reversal, intersex, and altered population sex ratios. Two estrogens found in the aquatic environment, 17alpha-ethinylestradiol (EE(2)) and 17beta-estradiol (E(2)), have been measured in wastewater treatment effluents and have been shown to cause adverse effects in fish. To further our understanding of how estrogen exposure affects reproductive endpoints in the male fathead minnow (FHM, Pimephales promelas), a physiologically based computational model was developed of the hypothalamic-pituitary-gonadal (HPG) axis. Apical reproductive endpoints in the model include plasma steroid hormone and vitellogenin concentrations. Using Markov chain Monte Carlo simulation, the model was calibrated with data from unexposed FHM, and FHM exposed to EE(2) and E(2). Independent experimental data sets were used to evaluate model predictions. We found good agreement between our model predictions and a variety of measured reproductive endpoints, although the model underpredicts unexposed FHM reproductive endpoint variances, and overpredicts variances in estrogen-exposed FHM. We conclude that this model provides a robust representation of the HPG axis in male FHM.

Characterization of ontogenetic changes in gene expression in the fathead minnow (Pimephales promelas).

Recently, researchers have begun looking at changes in gene expression in the fathead minnow (Pimephales promelas) after contaminant exposure as a way to develop biomarkers of exposure and effects. However, the bulk of this research has been conducted on adults, with few studies focusing on early life stages. Expression of selected genes important in growth, development, and reproduction in teleosts was quantified by quantitative polymerase chain reaction during different developmental time periods (from 0 to 28 d postfertilization [dpf]). Over the developmental period studied, there was a significant up-regulation of growth hormone mRNA and no significant changes in the expression of insulin-like growth factor 1. Thyroid hormone receptors A and B were detected in 4 dpf embryos and their expression stayed relatively constant. The variation in cytochrome P45019A mRNA expression was large during the first week of development, returning to 0 dpf expression levels thereafter. Estrogen receptor 2B was up-regulated during the first three weeks postfertilization, returning to prehatch values by 28 dpf. Expression of hydroxysteroid dehydrogenase 3B and steroidogenic acute regulatory protein increased after the third or fourth week postfertilization, respectively. Vitellogenin exhibited a large degree of variation within time points, especially after day 15, and a significant up-regulation for this gene was observed at 7 and 10 dpf. Knowledge of the normal changes in gene expression during embryo and larval development will allow for better experimental design and selection of suitable biomarkers when testing the potential toxicological effects of contaminants in this model fish species.

Endocrine disrupting chemicals in fish: developing exposure indicators and predictive models of effects based on mechanism of action.

Knowledge of possible toxic mechanisms (or modes) of action (MOA) of chemicals can provide valuable insights as to appropriate methods for assessing exposure and effects, thereby reducing uncertainties related to extrapolation across species, endpoints and chemical structure. However, MOA-based testing seldom has been used for assessing the ecological risk of chemicals. This is in part because past regulatory mandates have focused more on adverse effects of chemicals (reductions in survival, growth or reproduction) than the pathways through which these effects are elicited. A recent departure from this involves endocrine-disrupting chemicals (EDCs), where there is a need to understand both MOA and adverse outcomes. To achieve this understanding, advances in predictive approaches are required whereby mechanistic changes caused by chemicals at the molecular level can be translated into apical responses meaningful to ecological risk assessment. In this paper we provide an overview and illustrative results from a large, integrated project that assesses the effects of EDCs on two small fish models, the fathead minnow (Pimephales promelas) and zebrafish (Danio rerio). For this work a systems-based approach is being used to delineate toxicity pathways for 12 model EDCs with different known or hypothesized toxic MOA. The studies employ a combination of state-of-the-art genomic (transcriptomic, proteomic, metabolomic), bioinformatic and modeling approaches, in conjunction with whole animal testing, to develop response linkages across biological levels of organization. This understanding forms the basis for predictive approaches for species, endpoint and chemical extrapolation. Although our project is focused specifically on EDCs in fish, we believe that the basic conceptual approach has utility for systematically assessing exposure and effects of chemicals with other MOA across a variety of biological systems.

Altered development and reproduction in mosquitofish exposed to pulp and paper mill effluent in the Fenholloway River, Florida USA.

Female mosquitofish exposed to pulp and paper mill effluent (PME) in the Fenholloway River, Florida, USA have masculinized secondary sex characteristics and altered aromatase enzyme activity. We and others have shown that the Fenholloway River PME contains androgenic and progestogenic substance(s). The present study was designed to test the hypothesis that the development and reproductive health of PME-exposed Fenholloway River mosquitofish are altered compared to mosquitofish living in Econfina River, which is the reference site. Fish were collected on a single day from both sites in June and August 1999 and January and June 2000. We compared standard length, anal fin length and segment number; body, liver, and gonad mass; and number of eggs and embryos from Fenholloway and Econfina River mosquitofish. The data were analyzed collectively for generalized site effect, for site effects during reproductive and nonreproductive seasons, and for repeatability of site effects between years. Mosquitofish exposed to PME in the Fenholloway River were generally smaller in length and mass, anal fin segment number was greater, and the number of embryos, but not oocytes, was significantly decreased compared to the reference site fish. Anal fin length and segment number and liver and testis masses were generally greater in Fenholloway compared to the Econfina River males. The importance of this study is that we have documented masculinized development and decreased embryo production in PME-exposed mosquitofish and that these site effects are generally consistent across seasons and between years.

What is normal? A characterization of the values and variability in reproductive endpoints of the fathead minnow, Pimephales promelas.

Jensen et al. [Jensen, K.M., Korte, J.J., Kahl, M.D., Pasha, M.S., Ankley, G.T., 2001. Aspects of basic reproductive biology and endocrinology in the fathead minnow (Pimephales promelas). Comp. Biochem. Physiol. C 128, 127-141.] investigated aspects of the normal reproductive biology of the fathead minnow (FHM, P. promelas), and subsequent studies have generated a large amount of additional reproductive data for endpoints such as plasma steroid hormone and vitellogenin concentrations, spawn interval, and secondary sex characteristics (i.e., nuptial tubercle score and fat pad weight). These data were analyzed and fitted with statistical distributions to improve understanding of the variability in normal, unexposed, adult male (n=154) and female (n=186) FHM. Summary statistics for most endpoints were consistent with results from other more limited studies of FHMs. Male fat pad weight, and in both sexes gonad and liver weights were found to be proportional to body weight. Multiple statistical distributions were found to characterize each endpoint with the exception of spawn interval. Based on one of the largest datasets ever compiled for controlled studies, results presented herein provide a robust point of reference for the quantitative assessment of reproductive processes in the fathead minnow.

Reproductive seasonality of the female Florida gar, Lepisosteus platyrhincus.

Our objective was to characterize the reproductive seasonality of a wild population of female Florida gar, Lepisosteus platyrhincus. We measured the gonadosomatic index, plasma estradiol, testosterone, and vitellogenin concentrations, and follicle diameters of fish collected from Orange Lake, Florida (USA). Additionally, the reproductive stage of the ovary was resolved histologically. We observed a seasonal pattern in reproduction. Following a quiescent period in the summer, there was an increase in sex steroid hormones during the fall, associated with the onset of vitellogenesis and active oogenesis. During the February collection, we directly observed ovulation in several females. This observation was supported by a sharp decrease in the gonadosomatic index between February and March as well as a decline in plasma hormones to basal levels during the summer months. Our data suggest the Florida gar has a group-synchronous ovary and the majority of females spawned during the early spring, followed by a general decrease in reproductive parameters to summer levels. While hormone and vitellogenin peaks may vary annually and variations in these reproductive patterns are expected for different collection sites, this study is important as it is the first characterization of the seasonal morphological and endocrinological reproductive pattern in a female holostean species. Moreover, it increases our understanding of basic reproductive biology of semitropical fishes in general, and nonteleost, bony fishes in particular.