Vitellogenin Response Research Articles

Survival, Growth, and Reproduction Responses in a Three-Generation Exposure of the Zebrafish (Danio rerio) to Perfluorooctane Sulfonate.

A prior multigenerational perfluorooctane sulfonic acid (PFOS) exposure investigation in zebrafish reported adverse effects at 0.734 µg/L, among the lowest aquatic effect levels for PFOS reported to date. The present three-generation PFOS exposure quantified survival, growth, reproduction, and vitellogenin (VTG; egg yolk protein) responses in zebrafish, incorporating experimental design and procedural improvements relative to the earlier study. Exposures targeting 0.1, 0.6, 3.2, 20, and 100 µg/L in parental (P) and first filial (F1) generations lasted for 180 days post fertilization (dpf) and the second filial generation (F2) through 16 dpf. Survival decreased significantly in P and F2 generation exposures, but not in F1, at the highest PFOS treatment (100 µg/L nominal, 94-205 µg/L, measured). Significant adverse effects on body weight and length were infrequent, of low magnitude, and occurred predominantly at the highest exposure treatment. Finally, PFOS had no significant effects on P or F1 egg production and survival or whole-body VTG levels in P or F1 male fish. Overall, the predominance and magnitude of adverse PFOS effects at <1 µg/L reported in prior research were largely nonrepeatable in the present study. In contrast, the present study indicated a threshold for ecologically relevant adverse effects in zebrafish at 117 µg/L (SE 8 µg/L, n = 10) for survival and 47 µg/L (SE 11 µg/L, n = 19) for all statistically significant negative effects observed. Environ Toxicol Chem 2024;43:115-131. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

An international cross-laboratory survey on fish vitellogenin analysis: Methodological challenges and opportunities for best practice

Vitellogenin (VTG) is a biomarker for possible endocrine activity of chemicals acting via the estrogen, androgen, or steroidogenesis pathways. VTG is assessed in standardised fish guideline studies conducted for regulatory safety assessment of chemicals. VTG data can be highly variable leading to concerns for potential equivocal, false positive and/or negative outcomes. Consequently, additional fish testing may be required to address uncertainties in the VTG response, and possibly erroneous/missed identification of endocrine activity. To better understand the technical challenges of VTG assessment and reporting for regulatory purposes, a survey was sent to 27 testing laboratories performing these analyses. The survey results from 16 respondents (6 from the UK, 3 from the USA, and 7 from the EU) were analysed and discussed in a follow-up webinar. High variability in background VTG concentrations was widely acknowledged and thought to be associated with fish batch, husbandry, laboratory practices, and several methodological aspects. These include sample collection and storage, VTG quantification, data handling, and the benchmarks used for data acceptability. Information gathered in the survey provides a basis for improving and harmonizing the measurement of VTG in fish, and an opportunity to reassess the suitability of current acceptability criteria in test guidelines.

The comparative toxicity of rainfall overflows and a municipal effluent plume in Elliptio complanata using a novel qPCR array approach

Municipal effluents have the potential to disrupt the endocrine system involved in reproduction in aquatic organisms. The purpose of this study was to develop a novel a quantitative polymerase chain reaction (qPCR) array for the freshwater mussel Elliptio complanata to compare the toxic properties of municipal effluents and rainfall overflows. Mussels were caged for 3 months at upstream and downstream sites of a municipal effluent discharge point and 2 rainfall overflow sites in the Saint-Lawrence River. The data revealed that exposure to municipal effluents and overflow sites lead to increased expression of vitellogenin (VTG) in male mussels, altered sexual differentiation in females, gene expression involved in oxidative stress (superoxide dismutase, glutathione S-transferase) and DNA damage (chromosome mismatch and repair of covalently-bound DNA adducts). Mussels at the downstream site accumulated also large amounts of heterotrophic bacteria but not at the overflow sites. However, mussels at the overflow sites had decreased expression in ABC transporter gene expression. Scaling analysis revealed that the following gene expression were related to effects at higher level of biological organization such as total RNA levels, gonad mass and gonad somatic index: DNA repair of covalent DNA adducts, mistmach chromosomal recombination during meiosis and undifferentiated cells in gonads. In conclusion, exposure to rainfall overflow sites have similar effects to municipal effluents based on VTG and oxidative stress responses at the transcriptomic levels only but the effects at the DNA integrity and sexual differentiation were involved in adverse outcome pathways of urban pollution in Elliptio complanata mussels.

Social hierarchy modulates responses of fish exposed to contaminants of emerging concern.

Many organisms, including the fathead minnow (Pimephales promelas), a toxicological model organism, establish social hierarchies. The social rank of each male in a population is under the control of the hypothalamic-pituitary-gonadal (HPG) axis mainly through regulation of circulating androgen concentrations, which in turn drive the expression of secondary sex characteristics (SSCs). As dominant and subordinate males in an exposure study are initially under different physiological conditions (i.e., differing plasma androgen concentrations), we proposed that they belong to different subpopulations in the context of exposure to compounds that may interact with the HPG axis. Using a meta-analysis of our data from several previously published studies, we corroborated the hypothesis that social status, as indicated by SSCs, results in distinct clusters (eigenvalues >0.8 explaining >80% of variability) with differential expression of plasma vitellogenin, a commonly used biomarker of exposure to contaminants of emerging concern (CEC). Furthermore, we confirmed our predictions that exposure to estrogenic CECs would homogenize plasma vitellogenin response (E1: cluster mean SSC values decreased to 4.33 and 4.86 relative to those of control; E2: decreased to 4.8 and 5.37) across the social hierarchy. In contrast, serotonin-specific reuptake inhibitors expand this response range (cluster mean SSC increased to 5.21 and 6.5 relative to those of control). Our results demonstrated that social hierarchies in male fathead minnows result in heterogeneous responses to chemical exposure. These results represent a cautionary note for the experimental design of single-sex exposure studies. We anticipate our study to be a starting point for the re-evaluation of toxicological data analyses in single sex exposure experiments.

Pathogenic infection attenuates the response of the estrogenic biomarker, vitellogenin, in rainbow trout

In the environment, aquatic organisms are exposed multiple instead of single stressors. During recent years, considerable progress has been achieved in assessing combination effects of chemical mixtures. For instance, it has been demonstrated that the induction of vitellogenin (VTG) in (male) fish – an established biomarker of exposure to estrogen-active compounds – responds additively to combinations of estrogen-active compounds, while simultaneous exposure to ligands of the estrogen receptor and of the arylhydrocarbon receptor has an antagonistic effect on VTG induction. In contrast to the progress in understanding combination effects of chemical mixtures, approaches to assess the combined effects of chemical and physical and/or biological stressors are less developed. Here, we study the combined impact of a chemical and a biological stressor on the biomarker VTG in rainbow trout. To this end, juvenile fish were exposed to two doses of the prototypic estrogen-active compound, 17-beta-estradiol (E2) in combination with the fish-pathogenic parasite Tetracapsuloides bryosalmonae, the etiological agent of the proliferative kidney disease (PKD). The VTG response was assessed at the mRNA level using qRT-PCR; in parallel, the liver transcriptomic response to the single stressors and their combinations was examined. Combined chemical/biological exposure resulted in a significant inhibition of both the rate and the magnitude of the VTG response, while neither the parasite nor the fish response to the parasite was affected by the presence of E2. The observed attenuation of the VTG biomarker response in diseases may have implications for the utility of this marker in environmental scenarios with low estrogenic exposure.

Exposure to 17α-ethynylestradiol causes dose and temporally dependent changes in intersex, females and vitellogenin production in the Sydney rock oyster

Although mounting evidence suggests exposure to estrogenic contaminants increases vitellogenin production in molluscs, demonstration of dose-response relationships and knowledge of the temporal nature of the vitellogenin response with continual exposure is currently lacking for biomarker utility. To address this knowledge gap, adult Sydney rock oysters, Saccostrea glomerata, were exposed to a range of environmentally relevant concentrations of 17α-ethynylestradiol (EE2) (0, 6.25, 12.5, 25 or 50 ng/l) in seawater under laboratory conditions. Vitellogenin induction and gonadal development was assessed following 4, 21 and 49 days exposure to EE2. Vitellogenin was found to increase in a dose dependent manner with EE2 exposure for females (4 and 49 days) and males (4 and 21 days). Histological examination of gonads revealed a number of individuals exhibited intersex (ovotestis) in 50 ng/l EE2 (after 21 days) and in 6.25 and 12.5 ng/l EE2 (after 49 days). Furthermore, a significant shift towards females was observed following 49 days exposure at 50 ng/l EE2 suggesting estrogenic exposure is capable of facilitating a progression for protandric males from male-intersex-female gametal status. Increases in female vitellogenin (4 days) were predictive of later increases in female developmental stages at 21 days and increases in oocyte area following 49 days. Male vitellogenin (4 days) was predictive of decreased male percentages and lower male developmental stages at 49 days. Vitellogenin in S. glomerata is a predictive biomarker of estrogenic exposure and effect if sampled soon after exposure and at the commencement of a gonadal development cycle.

Variable Vitellogenin Response of Japanese Medaka (Oryzias latipes) to Weekly Estrogen Exposure

Japanese medaka (Oryzias latipes) is a valuable model organism in reproductive and developmental toxicity testing. The purpose of this experiment is to assess the response of medaka to aquatic estrogen exposure over the course of 1 year. Each week, three pairs of adult male medaka were exposed separately for 4 days (100% static renewal daily) to 17beta-estradiol at a nominal level of 25 microg/l, with a fourth pair of fish exposed separately to an ethanol control. Vitellogenin (VTG) induction was observed each week, with hepatic and plasma VTG levels significantly higher (P < 0.001) than reported for ethanol control specimens. A significant (P < 0.001) increasing trend was observed for plasma VTG results over the duration of the study, whereas a decreasing trend (P = 0.030) of hepatic VTG was evident. A Durbin-Watson test, however, did not demonstrate any serial autocorrelation of hepatic (d = 1.180) or plasma (d = 1.311) VTG levels over the duration of the study. Time-series transformations of the hepatic and plasma VTG data did not reveal any significant seasonal or behavioral patterns. However, significant intermittent peaks in VTG production were observed in both tissue types during the study. These data indicate that some consideration must be taken to time long-term medaka exposures (>20 weeks) in order to eliminate any influence of cyclic changes on plasma VTG response. Alternatively, hepatic cytosolic measurement of VTG appears to show a more sensitive response to aquatic estrogen exposure.

Occurrence and implications of estrogens and xenoestrogens in sewage effluents and receiving waters from South East Queensland

We report a survey on the occurrence of estrogens (estrone, E1; 17 β-estradiol, E2; 17 α-ethynylestradiol, EE2) and xenoestrogens (bisphenol-A, BPA; 4- t-octylphenol, 4- t-OP; 4-nonylphenols, 4-NP; and nonylphenol mono- and di-ethoxylates, NPE1 and NPE2) in effluents from five wastewater treatment plants and their receiving waters in South East Queensland. The total xenoestrogen concentrations in effluent ranged between 2446 ng/L and 6579 ng/L, with 4-NP and NPE1-2 having much higher concentration levels than BPA and 4- t-OP. The estrogen levels in effluent varied from 9.12 to 32.22 ng/L for E1, from 1.37 ng/L to 6.35 ng/L for E2 and from 0.11 ng/L to 1.20 ng/L for EE2. No significant differences ( p < 0.05) in the concentrations of the selected estrogenic compounds were found for the effluents from the five sewage treatment plants. The estrogens and xenoestrogens were also found in the receiving waters at relatively lower concentration levels due to dilution of effluents in the rivers. Based on the chemical analysis data and relative potency of the compound from in vitro and in vivo bioassays from the literature, the calculated in vitro EEQ values (estrogen equivalents) in the receiving river waters downstream of the effluent discharge points ranged from 1.32 to 11.79 ng/L, while the in vivo EEQ values (vitellogenin response in rainbow trout) ranged from 2.48 to 21.18 ng/L. The three estrogens accounted for the majority of the EEQ in the water samples. This study indicates that the rivers of South East Queensland are at potential risk.

Do suspended sediments modulate the effects of octylphenol on rainbow trout?

A system was devised which allows particles to remain in suspension in a conventional 60 L aquarium without undue disturbance to resident fish. Using this system, juvenile rainbow trout were exposed for one week to 4-tert-octylphenol (OP, 10–1000 μg/L) with or without the presence of suspended sediments (10–20 mg/L of natural suspended sediments from the River Calder, UK). About 8% of the added OP partitioned to the solid phase. Vitellogenin levels were determined in the plasma of the exposed rainbow trout and showed a dose-dependent increase with regards to OP exposure concentration. Considerable variation in the vitellogenin response was observed between separate runs with the same OP concentration. There was no statistically significant (at P < 0.05) difference in plasma VTG levels between the OP treatments with or without suspended sediments. This suggests that the dissolved concentration is the key factor and natural suspended sediment neither protects against, nor exacerbates, the endocrine disrupting effect of OP on fish.

Hypoxia Does Not Influence the Response of Fish to a Mixture of Estrogenic Chemicals

Chemical risk assessment procedures assign a major role to standardized toxicity tests, in which the response of a particular organism to a single test substance is determined under otherwise constant and favorable conditions in the laboratory. This approach fails to consider the potential for chemical interactions, as well as failing to consider how the toxicological response varies, depending on the conditions of exposure. As yet, the issue of confounding factors on chemically mediated effects in wildlife has received little attention, despite the fact that a range of physicochemical parameters, including temperature, water quality, and pH, are known to modify chemical toxicity. Here, we consider how the estrogenic response of fish varies with regard to hypoxia. Fathead minnows (Pimephales promelas) were exposed to a mixture of estrogenic chemicals under hypoxic or normoxic conditions. Their estrogenic response was characterized using an in vivo assay, involving the analysis of the egg yolk protein, vitellogenin (VTG). The results revealed that there was no effect of hypoxia on the VTG response in either treatment group at the end of the exposure period. This suggests that this end point is robust and relatively insensitive to the effects of any physiological changes that arise as a result of hypoxia. The implications of these negative findings are discussed in terms of their relevance with regard to the development of risk assessment policy.

Evidence of temperature-dependent effects on the estrogenic response of fish: Implications with regard to climate change

Chemical risk assessment is fraught with difficulty due to the problem of accounting for the effects of mixtures. In addition to the uncertainty arising from chemical-to-chemical interactions, it is possible that environmental variables, such as temperature, influence the biological response to chemical challenge, acting as confounding factors in the analysis of mixture effects. Here, we investigate the effects of temperature on the response of fish to a defined mixture of estrogenic chemicals. It was anticipated that the response to the mixture may be exacerbated at higher temperatures, due to an increase in the rate of physiological processing. This is a pertinent issue in view of global climate change. Fathead minnows ( Pimephales promelas) were exposed to the mixture in parallel exposure studies, which were carried out at different temperatures (20 and 30 °C). The estrogenic response was characterised using an established assay, involving the analysis of the egg yolk protein, vitellogenin (VTG). Patterns of VTG gene expression were also analysed using real-time QPCR. The results revealed that there was no effect of temperature on the magnitude of the VTG response after 2 weeks of chemical exposure. However, the analysis of mixture effects at two additional time points (24 h and 7 days) revealed that the response was induced more rapidly at the higher temperature. This trend was apparent from the analysis of effects both at the molecular and biochemical level. Whilst this indicates that climatic effects on water temperature are not a significant issue with regard to the long-term risk assessment of estrogenic chemicals, the relevance of short-term effects is, as yet, unclear. Furthermore, analysis of the patterns of VTG gene expression versus protein induction gives an insight into the physiological mechanisms responsible for temperature-dependent effects on the reproductive phenology of species such as roach. Hence, the data contribute to our understanding of the implications of global climate change for wild fish populations.

Effects of 4-nonylphenol and 17α-ethynylestradiol exposure in the Sydney rock oyster, Saccostrea glomerata: Vitellogenin induction and gonadal development

Adult Saccostrea glomerata were exposed to environmentally relevant concentrations of 4-nonylphenol (1 μg/L and 100 μg/L) and 17α-ethynylestradiol (5 ng/L and 50 ng/L) in seawater over 8 weeks. Exposures were performed to assess effects on vitellogenin induction and gonadal development during reproductive conditioning. Chronic direct estrogenicity within gonadal tissue was assessed via an estrogen receptor-mediated, chemical-activated luciferase reporter gene-expression assay (ER-CALUX ®). Estradiol equivalents (EEQ) were greatest in the 100 μg/L 4-nonylphenol exposure (28.7 ± 2.3 ng/g tissue EEQ) while 17α-ethynylestradiol at concentrations of 50 ng/L were 2.2 ± 1.5 ng/g tissue EEQ. Results suggest 4-nonylphenol may be accumulated in tissue and is partly resistant to biotransformation; maintaining its potential for chronic estrogenic action, while 17α-ethynylestradiol, although exhibiting greater estrogenic potency on biological endpoints possibly exerts its estrogenic action before being rapidly metabolised and/or excreted. A novel methodology was developed to assess vitellogenin using high-performance liquid chromatography (HPLC). Exposure to both 17α-ethynylestradiol (50 ng/L) and 4-nonylphenol (100 μg/L) produced increases in vitellogenin for females, whereas males exhibited increases in vitellogenin when exposed to 50 ng/L 17α-ethynylestradiol only. Females exhibited greater vitellogenin responses than males at 50 ng/L 17α-ethynylestradiol only. Histological examination of gonads revealed a number of individuals exhibiting intersex (ovotestis) in 50 ng/L 17α-ethynylestradiol exposures. Male individuals in 1 μg/L and 100 μg/L 4-nonylphenol exposures and 5 ng/L 17α-ethynylestradiol were at earlier stages of spermatogenic development than corresponding controls.

Sensitive and robust gene expression changes in fish exposed to estrogen – a microarray approach

BackgroundVitellogenin is a well established biomarker for estrogenic exposure in fish. However, effects on gonadal differentiation at concentrations of estrogen not sufficient to give rise to a measurable vitellogenin response suggest that more sensitive biomarkers would be useful. Induction of zona pellucida genes may be more sensitive but their specificities are not as clear. The objective of this study was to find additional sensitive and robust candidate biomarkers of estrogenic exposure.ResultsHepatic mRNA expression profiles were characterized in juvenile rainbow trout exposed to a measured concentration of 0.87 and 10 ng ethinylestradiol/L using a salmonid cDNA microarray. The higher concentration was used to guide the subsequent identification of generally more subtle responses at the low concentration not sufficient to induce vitellogenin. A meta-analysis was performed with data from the present study and three similar microarray studies using different fish species and platforms. Within the generated list of presumably robust responses, several well-known estrogen-regulated genes were identified. Two genes, confirmed by quantitative RT-PCR (qPCR), fulfilled both the criteria of high sensitivity and robustness; the induction of the genes encoding zona pellucida protein 3 and a nucleoside diphosphate kinase (nm23).ConclusionThe cross-species, cross-platform meta-analysis correctly identified several robust responses. This adds confidence to our approach used for identifying candidate biomarkers. Specifically, we propose that analyses of an nm23 gene together with zona pellucida genes may increase the possibilities to detect an exposure to low levels of estrogenic compounds in fish.

Quantification and associated variability of induced vitellogenin gene transcripts in fathead minnow (Pimephales promelas) by quantitative real‐time polymerase chain reaction assay

Ecological risk assessors have a growing need for sensitive and rapid indicators of environmental exposures in aquatic ecosystems resulting from natural and synthetic estrogen-like compounds. Investigators developing subcellular exposure markers in traditional sentinel organisms must be vigilant about inherent variability of analyses, especially regarding regulatory and policy statements. Here, we report a quantitative real-time polymerase chain reaction (QPCR) assay for the detection of vitellogenin transcripts environmentally triggered in fathead minnows (Pimephales promelas). We demonstrate that our QPCR assay exhibits little inter- or intra-assay variability (21.7 and 11.9%, respectively). This method appears to be robust in terms of variability stemming from extrinsic sources, indicating that it may be readily transferable to laboratories having the requisite equipment. Our primary focus in development of this method derived from the observation that transcriptional responses of the vitellogenin gene (vtg) in fathead minnows demonstrated high biological variability between identically treated individuals, even under controlled laboratory conditions (coefficient of variation, > 100%). This variability was not seen in other genes from the same RNA preparations that we examined, suggesting that it is specific to the vitellogenin response. Our data and those of others suggest that variability in vtg expression is common to a number of aquatic vertebrates, which is indicative of genetic causation. Despite a relatively high degree of variability in vtg transcription, this method is sensitive enough to detect exposures of 5.0 ng 17alpha-ethinylestradiol (EE2)/L within 24 h of exposure, and it has the ability to discriminate 10.0 and 5.0 ng EE2/L within 48 h. The vitellogenin QPCR assay is a highly sensitive, comparatively rapid, and inexpensive method for the detection and characterization of exposure to environmental estrogens and estrogen mimics.

Vitellogenin synthesis in primary cultures of fish liver cells as endpoint for in vitro screening of the (anti)estrogenic activity of chemical substances

Concern over possible adverse effects of endocrine-disrupting compounds on fish has caused the development of appropriate testing methods. In vitro screening assays may provide initial information on endocrine activities of a test compound and thereby may direct and optimize subsequent testing. Induction of vitellogenin (VTG) is used as a biomarker of exposure of fish to estrogen-active substances. Since VTG induction can be measured not only in vivo but also in fish hepatocytes in vitro, the use of VTG induction response in isolated fish liver cells has been suggested as in vitro screen for identifying estrogenic-active substances. The main advantages of the hepatocyte VTG assay are considered its ability to detect effects of estrogenic metabolites, since hepatocytes in vitro remain metabolically competent, and its ability to detect both estrogenic and anti-estrogenic effects. In this article, we critically review the current knowledge on the VTG response of cultured fish hepatocytes to (anti)estrogenic substances. In particular, we discuss the sensitivity, specificity, and variability of the VTG hepatocyte assay. In addition, we review the available data on culture factors influencing basal and induced VTG production, the response to natural and synthetic estrogens as well as to xenoestrogens, the detection of indirect estrogens, and the sources of assay variability. The VTG induction in cultured fish hepatocytes is clearly influenced by culture conditions (medium composition, temperature, etc.) and culture system (hepatocyte monolayers, aggregates, liver slices, etc.). The currently available database on estrogen-mediated VTG induction in cultured teleost hepatocytes is too small to support conclusive statements on whether there exist systematic differences of the VTG response between in vitro culture systems, VTG analytical methods or fish species. The VTG hepatocyte assay detects sensitively natural and synthetic estrogens, whereas the response to xenoestrogens appears to be more variable. The detection of weak estrogens can be critical due to the overshadow with cytotoxic concentrations. Moreover, the VTG hepatocyte assay is able to detect antiestrogens as well as indirect estrogens, i.e substances which require metabolic activation to induce an estrogenic response. Nevertheless, more chemicals need to be analysed to corroborate this statement. It will be necessary to establish standardized protocols to minimize assay variability, and to develop a set of pass-fail criteria as well as cut-offs for designating positive and negative responses.

Effects of nonylphenol on juveniles and adults in the grey mullet, Liza aurata

The aim of this study was to investigate the effects of nonylphenol (NP), an environmental pollutant known to have estrogenic activity, on grey mullets. Juvenile and adult physiology was monitored by the expression of vitellogenin (VTG), which is commonly induced by estrogenic pollutants, and cytochrome P4501A1(CYP1A1) as a first signal of detoxification. The dose–response estrogenic effects of NP (25, 100, 1000 μg/l) on hepatic VTG transcript and plasma protein levels, as well as on CYP1A1 transcription and its associated ethoxyresorufin O-deethylase (EROD) activity, were assessed in juvenile Liza aurata; 17β-estradiol (E2; 2 μg/l) treatment for 1 week served as positive control. In addition, we investigated VTG and CYP1A1 expression in adult males injected with two NP at 0.25 or 250 mg/kg body weight relative to 0.07 mg/kg of E2 as a positive control. Juvenile exposure to NP failed to induce a measurable VTG response. In the adult fish, NP exerted estrogenic effects only at the highest dose injected. E2 treatment elicited VTG induction only in adults in a time-related manner. In contrast, NP treatment induced a dose-dependent decrease in CYP1A1 response in both juveniles and adults. An inhibitory effect of E2 on CYP1A1 was evident in all treatment groups as well. These data suggest that, in biomonitoring studies, testing the expression of different biomarkers may provide a more realistic picture of the environmental conditions.

Gonadotropin and estrogen responses in freshwater turtle ( Chrysemys picta) from Cape Cod, Massachusetts

As a result of chemical waste disposal on the Massachusetts Military Reservation, a Superfund site on Cape Cod, MA, contaminated groundwater plumes have formed. These plumes are of concern due to the widespread use of groundwater wells as a drinking water source by the local population. Prior observations on a sentinel species Chrysemys picta field-trapped from ponds on Cape Cod suggested deficits in reproductive processes including lower levels of vitellogenin, estradiol-17β, oviduct weights, and oocyte numbers in females and lower testicular weight and sperm count in males. Possible loci in the hypothalamic–pituitary–gonadal–liver axis at which xenobiotics may act were determined in turtles trapped from Moody Pond (a test site) and Washburn Pond (a reference site). Specifically, gonadotropin and estrogen responses were assessed using plasma steroids and vitellogenin as markers. Basal vitellogenin levels were significantly lower in Moody Pond females; however, vitellogenin responses to estradiol-17β were the same in both groups, indicating a normal hepatic response to estrogen. In contrast, estradiol-17β secretion was not stimulated by gonadotropin in Moody Pond females, compared to Washburn animals. Basal plasma testosterone and the response to gonadotropin in males were similar, although steroid levels in Moody Pond animals were slower to return to baseline after gonadotropin injection. The results suggest that a low-level mixture of xenobiotic contaminants may interfere with the steroid metabolic pathways in turtles exposed to the test site, but not the reference site, environment.

Evaluation of the Model Anti-androgen Flutamide for Assessing the Mechanistic Basis of Responses to an Androgen in the Fathead Minnow (Pimephales promelas)

In this study, we characterized the effects of flutamide, a model mammalian androgen receptor (AR) antagonist, on endocrine function in the fathead minnow (Pimephales promelas), a small fish species that is widely used for testing endocrine-disrupting chemicals (EDCs). Binding assays with whole cells transiently transfected with cloned fathead minnow AR indicated that flutamide binds competitively to the receptor. However, as is true in mammalian systems, a 2-hydroxylated metabolite of flutamide binds to the AR with a much higher affinity than the parent chemical. Mixture experiments with flutamide and the androgen 17beta-trenbolone demonstrated that the anti-androgen effectively blocked trenbolone-induced masculinization (nuptial tubercle production) of female fathead minnows, indicating antagonism of an AR receptor-mediated response in vivo. Conversely, reductions in vitellogenin in trenbolone-exposed females were not blocked by flutamide, suggesting that the vitellogenin response is not directly mediated through the AR. The results of these studies provide data demonstrating the validity of using the fathead minnow as a model species for detecting EDCs that exert toxicity through interactions with the AR.

Regulation of hepatic progesterone and estrogen receptors in the female turtle, Chrysemys picta: relationship to vitellogenesis

Previous studies using the fresh water turtle Chrysemys picta have demonstrated the differential expression of the two progesterone receptor isoforms (PRA and PRB) in the liver during the turtle seasonal cycle, correlating with ER mRNA levels and hepatic vitellogenesis. During the inter-vitellogenic periods the ratio of PRB:PRA favors PRA, suggesting that the PRB:PRA ratio may be important in the regulation of vitellogenesis. Based on these and other studies we hypothesize that progesterone may have differential effects on the estrogen (E)-induced vitellogenin response (inhibitory or stimulatory), depending on the PRB:PRA ratio. In this study, we determined the expression pattern and the hormonal regulation of progesterone receptor (PR) isoforms in the liver, during the estrogen-induced vitellogenin response in female C. picta. Northern blot analysis was used to evaluate the changes in vitellogenin mRNA, estrogen receptor (ER) mRNA, and PR mRNA expression; Western blot to determine changes in PR isoform expression and a homologous ELISA for measurement of plasma vitellogenin. The expression of PR isoforms in the liver of female turtles at the mRNA and protein levels was differentially regulated by estradiol and progesterone. Estradiol treatment enhanced the transcription of PR mRNA isoforms and possibly translation and/or increased stability of PRB protein in the female turtle liver. In contrast, this hormone decreased PRA protein levels. Progesterone alone down-regulated progesterone receptor isoform A (PRA) and progesterone receptor isoform B (PRB) proteins equally, but did not affect PR mRNA levels. Estradiol markedly increased hepatic ER mRNA, vtg mRNA, and vtg in plasma, but this effect was not impacted significantly by progesterone. The results presented here indicate that in female turtles hepatic PRB:PRA ratios are markedly changed by estradiol treatment due primarily to a decrease in PRA. The change in the PRB:PRA ratio after hormonal treatment confirms that progesterone and estrogen exposure will be a determinant in the regulation of vitellogenesis, and, in turn, that the regulation of vitellogenesis will be determined by the ratio of PR isoforms and the physiological levels of steroid hormones.

The Effects of the Soy Isoflavone Genistein on the Reproductive Development of Striped Bass

AbstractThe aquaculture industry has made great strides toward developing diets for finfish that provide adequate nutrition for the animals being maintained while minimizing the cost to the farmer. Alternative, plant‐derived protein sources are used to minimize cost. Soybean meal is the plant protein most commonly used because it maintains adequate growth. However, soy is rich in phytoestrogens, the most abundant of which is genistein. Studies have shown variable physiological effects associated with exposure to genistein. The objective of this research was to investigate the effects of feeding genistein to juvenile striped bass Morone saxatilis at varying concentrations in the diet. The first experiment was designed to determine whether juvenile striped bass are capable of responding to estrogen exposure. Juvenile striped bass were given weekly injections of estradiol benzoate for 3 weeks and showed a significant response to the estrogen via expression of vitellogenin. In two additional experiments, genistein was fed to striped bass fingerlings (approximately 120 d old and 60–100 d old) in varying concentrations (0, 2, 4, and 8 mg/g of diet) to determine whether a similar response would be elicited. No significant differences were found among the growth variables and histological endpoints, although a significant vitellogenin response was observed at the 2‐mg/g and 8‐mg/g doses. These results show that the juvenile striped bass did respond to the estrogen‐like function of genistein and that the genistein induced a U‐shaped response curve that is characteristic of the low‐dose effects of some endocrine‐disrupting chemicals.