Ng EE Research Articles

Comparative Breeding and Behavioral Responses to Ethinylestradiol Exposure in Wild and Laboratory Maintained Zebrafish (Danio rerio) Populations

Genetic variation has a significant effect on behavior, fitness, and response to toxicants; however, this is rarely considered in ecotoxicological studies. We compared fitness-related behavioral traits, breeding activity, and the effects of exposure to the environmental estrogen ethinylestradiol (EE(2)) on reproduction in a laboratory (Wild Indian Karyotype, WIK) strain and a wild-caught population (Bangladesh, BLD01) of Danio rerio (zebrafish). In WIK fish, males with higher observed heterozygocity were more active reproductively and more successful in securing parentage, but these relationships were not apparent in the BLD01 fish. The frequency of reproductive behaviors increased in WIK zebrafish for exposure to 0.4 ng/L EE(2), which was not apparent in the BLD01 zebrafish. The different strains showed the same threshold for hepatic vitellogenin gene (vtg) induction (2.2 ng EE(2)/L), but results suggested an elevated response level in the BLD01. There were no effects on total egg production up to 2.2 ng EE(2)/L in either population; however, there was reduced egg fertilization rate at 2.2 ng EE(2)/L in the BLD01 fish. These results show consistency in the general responses to EE(2) between these two genetically divergent strains of zebrafish, but also illustrate differences in their breeding biology and response sensitivities. These findings highlight the need for due consideration of the source (and genetics) of populations used in ecological risk assessment for accurate comparisons among studies.

Impacts of Early Life Exposure to Estrogen on Subsequent Breeding Behavior and Reproductive Success in Zebrafish

Impacts of exposure to environmental estrogens on reproductive development are well documented, but recently wider concern has been raised due to evidence that such exposures can disrupt normal patterns of reproductive behavior, dominance, and parentage, with potential population level implications. It is fundamental therefore to understand any such effects for effective risk assessment. This study investigated the impact of a transient exposure to ethinylestradiol (EE(2)) during early life (from 20-60 days post fertilization), including at a dosing level within the environmental range, on the subsequent reproductive behavior and success in both male and female zebrafish (Danio rerio) in competitive breeding scenarios. There were no obvious effects of the early life EE(2) exposures on the subsequent gonadal phenotypes in either mature males or females. In fact, reproductive success in males exposed to 2.76 ng EE(2)/L was increased in competitive spawning scenarios. In contrast, exposure of females to EE(2) (9.86 ng/L) during early life reduced their subsequent reproductive success in competitive spawning scenarios. Mate choice experiments suggested this was a consequence of the females' diminished courting behavior toward males, rather than any male preference for unexposed females. Reproductive capability of females is generally considered a key determinant in population demographics and dynamics, and therefore the effect of exposure to EE(2) on female reproductive success may have significant implications for exposed fish populations.

Environmentally relevant exposure to 17α-ethinylestradiol affects the telencephalic proteome of male fathead minnows

Estrogens are key mediators of neuronal processes in vertebrates. As such, xenoestrogens present in the environment have the potential to alter normal central nervous system (CNS) function. The objectives of the present study were (1) to identify proteins with altered abundance in the male fathead minnow telencephalon as a result of low-level exposure to17α-ethinylestradiol (EE 2), and (2) to better understand the underlying mechanisms of 17β-estradiol (E 2) feedback in this important neuroendocrine tissue. Male fathead minnows exposed to a measured concentration of 5.4 ng EE 2/L for 48 h showed decreased plasma E 2 levels of approximately 2-fold. Of 77 proteins that were quantified statistically, 14 proteins were down-regulated after EE 2 exposure, including four histone proteins, ATP synthase, H+ transporting subunits, and metabolic proteins (lactate dehydrogenase B4, malate dehydrogenase 1b). Twelve proteins were significantly induced by EE 2 including microtubule-associated protein tau (Mapt), astrocytic phosphoprotein, ependymin precursor, and calmodulin. Mapt showed an increase in protein abundance but a decrease in mRNA expression after EE 2 exposure , suggesting there may be a negative feedback response in the telencephalon to decreased mRNA transcription with increasing Mapt protein abundance. These results demonstrate that a low, environmentally relevant exposure to EE 2 can rapidly alter the abundance of proteins involved in cell differentiation and proliferation, neuron network morphology, and long-term synaptic potentiation. Together, these findings provide a better understanding of the molecular responses underlying E 2 feedback in the brain and demonstrate that quantitative proteomics can be successfully used in ecotoxicology to characterize affected cellular pathways and endocrine physiology.

Altered Sexual Development in Roach (Rutilus rutilus) Exposed to Environmental Concentrations of the Pharmaceutical 17α-Ethinylestradiol and Associated Expression Dynamics of Aromatases and Estrogen Receptors

Wild roach (Rutilus rutilus) inhabiting UK rivers contaminated with estrogenic effluents from wastewater treatment works show altered sexual development, including intersex, and this can impact negatively on their reproductive capabilities. The molecular events underlying these disruptions in gender assignment, however, are still poorly understood. In this study, two isoforms of aromatase (cyp19a1a and cyp19a1b) were cloned from the roach, and effects of exposure to 17alpha-ethinylestradiol (EE(2)) during early life were determined on the expression of both aromatases and on the estrogen receptors (ERs) (subtypes esr1 and esr2b) and analyzed against effects on the progression of gonadal sex differentiation. Exposure to environmentally relevant concentrations of EE(2) during the critical period of sex differentiation resulted in gonadal feminization and all roach exposed to 4 ng EE(2)/l were females. These effects on gonadal development were associated with alterations in the expression of both esr and cyp19a1 genes in bodies and heads of exposed fish with the most marked effects on the expression of esr1 and cyp19a1b. Our findings show that both aromatase isoforms and both ER subtypes are associated with sexual differentiation in roach, and alterations in their expression can signal for disruptions in sexual development.

Gonadal transcriptome responses and physiological consequences of exposure to oestrogen in breeding zebrafish ( Danio rerio)

Environmental oestrogens are widespread in the aquatic environment and cause alterations in sexual development and function in vertebrates. The molecular pathways underpinning these effects, however, remain poorly understood. In this study, we aimed at generating a mechanistic understanding of the disruptive effects of exposure to environmentally relevant concentrations of 17α-ethinyloestradiol (EE 2) on reproduction in zebrafish, by anchoring the transcriptomic alterations induced with the physiological consequences of exposure. Breeding colonies of zebrafish were exposed for a 21-day period to three concentrations of EE 2 (0.05, 0.5 and 5 ng/L) and the gonadal transcriptomic alterations induced (determined using a 17,000 oligonucleotide microarray) were analysed together with physiological effects seen on reproductive output of both males and females. Exposure to 5 ng EE 2/L resulted in reproductive impairment characterised by a decrease in egg production, alterations in sperm quality and reduced fertilisation success. The effects seen were associated with altered expression of 114 and 131 genes in the gonads of males and females, respectively. The biological processes most affected by the exposure were protein metabolism in males and mitochondria organisation and biogenesis in females. Genes involved in the regulation of cell cycle progression, the ubiquitin system and glutathione peroxidase were affected by the EE 2 exposure and associated with the changes observed in gamete quality in both genders. In summary, we demonstrated that EE 2 exposure compromised the reproductive health of breeding zebrafish at environmentally relevant concentrations. The molecular mechanisms mediating some of these effects were identified and included those impacting processes central to gametogenesis in both males and females.

Effects of 17α-ethynylestradiol on EROD activity, spiggin and vitellogenin in three-spined stickleback ( Gasterosteus aculeatus)

The three-spined stickleback ( Gasterosteus aculeatus) has quantifiable biomarkers of exposure to estrogens (vitellogenin), androgens (spiggin) and aryl hydrocarbon receptor (AhR) agonists (EROD activity) and is therefore a promising test species for biomonitoring of reprotoxic chemicals in aquatic environments. In this study we evaluated the effects of 17α-ethynylestradiol (EE 2) on EROD activity, induction of vitellogenin and spiggin, hepatosomatic index (HSI), ovarian somatic index (OSI) and nephrosomatic index (NSI). Adult male and female three-spined sticklebacks were exposed to concentrations of 0–170 ng EE 2/l (measured concentrations) in a flow-through system for 21 days. Exposure to 170 ng EE 2/l resulted in a significant 8- and 9-fold induction of gill EROD activity in males and females, respectively. In livers, EROD activity expressed in relation to microsomal protein content was suppressed due to a significant increase in microsomal protein content. Hepatic EROD activity per se expressed as picomol/min was not affected by exposure to EE 2. The lowest observed effect concentration for induction of vitellogenin in males was 53.7 ng EE 2/l. In females, vitellogenin levels were significantly higher in those exposed to170 ng EE 2/l compared to controls. Spiggin production was significantly inhibited and NSI lower in males exposed to 170 ng EE 2/l. In both females and males LSI was significantly higher in fish exposed to 170 ng EE 2/l than in controls. In females exposed to 170 ng EE 2/l, OSI was significantly lower and NSI higher than controls. The observed results from this study show that a synthetic estrogen can affect the well-known biomarker of exposure for dioxin-like compounds, EROD activity, and further that this response can differ between tissues. These findings are important for interpretation of biomonitoring data.

An enzyme-linked immunosorbent assay for rare minnow ( Gobiocypris rarus) vitellogenin and comparison of vitellogenin responses in rare minnow and zebrafish ( Danio rerio)

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to determine vitellogenin (Vtg) in rare minnow ( Gobiocypris rarus) based on the separation and purification of rare minnow Vtg (r-Vtg) as well as the production of polyclonal antibody against r-Vtg in rabbits. Three different ELISAs for measuring r-Vtg were then compared: (1) indirect ELISA with the antibody against carp ( Cyprinus carpio) Vtg (c-Vtg) (IC-ELISA); (2) competitive ELISA with the antibody against c-Vtg, and using r-Vtg for coating the plates and preparing standard curve (CC-ELISA); (3) competitive ELISA with the antibody against r-Vtg, and using r-Vtg for coating the plates and preparing standard curve (CR-ELISA). The result showed that the homologous CR-ELISA was the most sensitive among the three assays for quantifying r-Vtg. The sensitivities to 17α-ethinylestradiol (EE 2) of rare minnow and zebrafish ( Danio rerio) were compared upon the establishment of homologous competitive ELISA. The lowest observed effect concentrations (LOECs) to induce Vtg were found to be 0.8 ng EE 2 l − 1 for rare minnow and 4 ng EE 2 l − 1 for zebrafish respectively. Afterwards, CR-ELISA was applied to measure Vtg concentration in whole body homogenate (WBH) of juvenile rare minnow fed by three diets (tubifex from wastewater treatment plant, Artemia nauplii and commercial pellet food), and the agreements between bioassay and GC–MS analysis demonstrated that rare minnow was a sensitive fish model for assessing estrogenic effects of endocrine disrupting compounds in aquatic environment.

Life-stage-dependent sensitivity of zebrafish ( Danio rerio) to estrogen exposure

The aim of this study was to identify periods in zebrafish ( Danio rerio) development when estrogen exposure has long-term consequences on reproductive capabilities at the adult stage. To this end, zebrafish were exposed to 10 ng/L ethynylestradiol (EE 2) during three stages of gonadal differentiation: (i) the juvenile hermaphroditic stage when gonads display the morphology of an immature ovary (in our zebrafish colony this lasted from 15 to 42 days post-fertilization [dpf]), (ii) the gonad transition stage when the hermaphroditic gonad differentiates into either testes or ovary (from 43 to day 71 dpf), and (iii) the premature stage of testicular and ovarian development (from 72 to 99 dpf). The consequences of stage-specific exposure to EE 2 were assessed by determining time to first spawning, fecundity (number of eggs per female per day), fertilization success (percentage of fertilized eggs) and sex ratio of the adults. Exposure during the gonad transition period induced a delay in the onset of spawning and a significant reduction of fecundity and fertilization success, whereas exposure during the hermaphroditic stage or during the premature stage had no significant impact on the reproductive parameters of adult fish. The results from this experiment pointed to the gonad transition stage as being most susceptible to persistent effects of developmental estrogen exposure. In a second experiment, the concentration dependency of the EE 2effects was evaluated by exposing zebrafish during the gonad transition stage (43–71 dpf) to 1.67, 3 or 10 ng EE 2/L. Significant effects of EE 2 on adult reproduction were found with 3 and 10 ng EE 2/L, but not with 1.67 ng/L. Histological examination of the gonads revealed that at termination of EE 2 exposure (71 dpf), all individuals in the 3 and 10 ng EE 2/L treatment possessed ovaries. However, this feminising effect appeared to be reversible since at the adult stage (190 dpf), both fish with ovaries and with testes were found. Thus, EE 2 exposure during the gonad transition stage seems to have no persistent effect on gonad histology but on reproductive capabilities.

Induction of vitellogenesis in 17α-ethinylestradiol-exposed rainbow trout ( Oncorhynchus mykiss): a method comparison

Juvenile rainbow trout, Oncorhynchus mykiss, were exposed to the synthetic estrogen 17α-ethinylestradiol (EE 2) through injection (1, 10, 25 and 50 μg EE 2/g fish/week) and via water exposure (1, 10 and 100 ng EE 2/l). After seven (injection and water exposure) and 14 days (only for water exposure), blood and plasma vitellogenin concentrations were quantified using indirect endpoints, i.e. plasma alkaline-labile phosphorus (ALP), plasma protein and plasma calcium. In addition, the relative gonad (GSI) and liver weight (HSI) were recorded. Actual plasma vitellogenin concentrations were measured with an enzyme immunoassay. Only fish injected with 50 μg EE 2/g fish had a significantly higher gonad weight. No concentration-dependent changes in the HSI were detected in fish exposed via the water, but a significant dose-dependent increase of the HSI was observed in fish injected with EE 2. Exposure of rainbow trout to EE 2 had a significant effect on all tested plasma parameters. Plasma protein, phosphoprotein and calcium concentrations were significantly higher after two weeks exposure to 100 ng EE 2/l. Fish injected with 10, 25 and 50 μg EE 2/g fish exhibited increased plasma protein concentrations after 1 week. Compared to the controls, plasma ALP and calcium levels were significantly higher in all injected fish. A significant and positive correlation was observed between all three plasma parameters and between these indirect parameters and the actual plasma vitellogenin concentrations. These findings indicate that both the plasma ALP and the plasma calcium assay have a similar sensitivity as that of available antibody-based assays (EIA), at least in EE 2 exposure studies, and thus these assays can provide a rapid, simple and cost-effective alternative to available immunoassays.

Development of an ELISA for vitellogenin in whole body homogenate of zebrafish ( Danio rerio)

The yolk protein, lipovitellin (Lv) was purified from ovaries of mature female zebrafish ( Danio rerio) by gel filtration and anion exchange chromatography. Polyclonal antibodies against Lv were raised in rabbits. Anti-Lv IgG was purified by affinity chromatography. SDS-PAGE followed by Western blotting was performed to analyse the specificity of the antibody and the immunological similarities between Lv and vitellogenin (Vtg). Anti-Lv IgG was used to develop a direct non-competitive sandwich ELISA to measure Vtg concentrations of whole body homogenate (WBH) in zebrafish. The intra- and interassay variabilities were 5.8% and 10.4%, respectively. The sensitivity was 0.2 ng Vtg ml −1 and the practical detection limit was 40 ng Vtg g −1 fish (wet weight). Adult male zebrafish were exposed to a nominal water concentration of 10 ng l −1 of ethinylestradiol (EE 2) in a semi-static exposure system for 7 days. Compared with the control group, exposure to 10 ng EE 2 l −1 induced a 200-fold increase in Vtg levels.

An in vivo model fish system to test chemical effects on sexual differentiation and development: exposure to ethinyl estradiol

A model system was characterized which may be used as an in vivo screen for effects of chemicals or environmental mixtures on sexual differentiation and development of reproductive organs and gametes. We evaluated the effects of a model environmental estrogen, ethinyl estradiol (EE 2), on the d-rR strain of medaka, Oryzias latipes, using a nano-injection exposure. Gonad histopathology indicated that a single injection of 0.5–2.5 ng EE 2/egg can cause phenotypic sex-reversal of genetic males to females. Sex-reversals could be detected as early as 7 days post-hatch. Sex-reversed males had female-typical duct development and the secondary sex characteristics we measured were generally consistent with phenotype, with the exception of a few EE 2-exposed XX and XY females which possessed ambiguous anal fins. Using discriminant analysis, we determined that the presence or absence of the secondary sex characteristic, a dorsal fin notch, was a very reliable indicator of gonadal sex. No instances of gonadal intersexes were observed. Ethinyl estradiol also appeared to reduce growth but not condition (weight-at-length) and exposed XX females appeared to have a higher incidence of atretic follicles relative to controls. Our results suggest that estrogenic chemicals may influence sexual differentiation and development and that the medaka model is well suited to assessing these effects.

Sexual behavior in Japanese quail as a test end point for endocrine disruption: effects of in ovo exposure to ethinylestradiol and diethylstilbestrol.

Chemicals having a capacity to disturb the endocrine system have attracted considerable interest during recent years. There is a shortage of well-characterized in vivo tests with which to study such disturbances in different classes of vertebrates. In the present study, test end points related to reproduction in the Japanese quail were used to examine the estrogenic activity of chemicals. The synthetic estrogens ethinylestradiol (EE(2)) and diethylstilbestrol (DES), used as model compounds, were injected into the yolk of embryonated eggs. After the birds had been raised to sexual maturity, we examined sexual behavior, plasma testosterone concentrations, and testis morphology in adult males. The lowest doses resulting in a significantly depressed male sexual behavior were 6 ng/g egg for EE(2) and 19 ng/g egg for DES. Testis weight asymmetry was increased at 6 ng EE(2)/g egg, but DES had no effect at any treatment level. The area of the androgen-dependent cloacal gland was significantly reduced at 57 ng DES/g egg. No effects on plasma testosterone concentration or body weight following exposure to EE(2) or DES were observed at any dose level. Depressed male sexual behavior was the most sensitive of the end points studied, and we suggest that this ecologically relevant end point be included in avian in vivo testing for neuroendocrine disruptors.

The avian egg as a test system for endocrine disrupters: effects of diethylstilbestrol and ethynylestradiol on sex organ develo

Many environmental contaminants are known or suspected to interfere with hormonal function in animals. In vivo test methods to detect and characterize chemicals that disrupt the endocrine system are therefore urgently needed. In this study, we assessed the usefulness of abnormalities of the reproductive organs as test endpoints for estrogenic activity of xenobiotics in Japanese quail embryos. Two synthetic estrogens, diethylstilbestrol (DES) and ethynylestradiol (EE 2), were injected into the yolks of embryonated eggs. At a dose as low as 2 ng EE 2/g egg, all male embryos became feminized, containing ovary-like tissue in the left testis. The extent of feminization of the testes was determined by measuring the relative area of the ovary-like component. Persistent Müllerian ducts (oviducts) in male embryos, and malformations of the Müllerian ducts in females occurred at 2 ng EE 2/g egg and higher doses. DES was approximately one-third to one-tenth as potent as EE 2. The morphological changes studied were dose-dependent, indicating that they are useful as test endpoints for estrogenic activity. Feminization of the left testis in males proved to be the most sensitive endpoint. We propose the quail egg as a simple in vivo test system for estrogenic compounds.