A new ELISA for the three-spined stickleback (Gasterosteus aculeatus L.) spiggin, using antibodies against synthetic peptide

The aim of this study was to develop an enzyme-linked immunosorbent (ELISA) assay to quantify spiggin in the three-spined stickleback. Spiggin is a glue protein produced in the kidney of male three-spined stickleback under the control of androgens during the breeding period. Disturbances of spiggin production in male fish and abnormal induction of spiggin in female fish are considered as valuable biomarkers of exposure to (anti-)androgenic chemicals. Polyclonal antibodies against a peptide sequence of spiggin (HRD-16) were used and the specificity of the antibodies was verified by Western blotting and direct ELISA experiments. By using HRD-16 antibodies and spiggin standard preparation, a competitive ELISA was set-up and validated. This assay appears sensitive, with a detection limit of 0.5 U/mL, and specific, as shown by the competition curves, obtained by serial dilution of male and female kidney homogenates, that were parallel to the spiggin standard curves. The ability of the spiggin ELISA to quantify spiggin induction was achieved by exposing male and female three-spined sticklebacks to 0.1 and 1 microg/L of methyltestosterone. The results show a significant dose-dependent induction of spiggin in methyltestosterone-exposed female fish compared to controls.     

Molecular cloning and characterization of a nuclear androgen receptor activated by 11-ketotestosterone

Although 11-ketotestosterone is a potent androgen and induces male secondary sex characteristics in many teleosts, androgen receptors with high binding affinity for 11-ketotestosterone or preferential activation by 11-ketotestosterone have not been identified. So, the mechanism by which 11-ketotestosterone exhibits such high potency remains unclear. Recently we cloned the cDNA of an 11-ketotestosterone regulated protein, spiggin, from three-spined stickleback renal tissue. As spiggin is the only identified gene product regulated by 11-ketotestosterone, the stickleback kidney is ideal for determination of the mechanism of 11-ketotestosterone gene regulation. A single androgen receptor gene with two splicing variants, belonging to the androgen receptor-β subfamily was cloned from stickleback kidney. A high affinity, saturable, single class of androgen specific binding sites, with the characteristics of an androgen receptor, was identified in renal cytosolic and nuclear fractions. Measurement of ligand binding moieties in the cytosolic and nuclear fractions as well as to the recombinant receptor revealed lower affinity for 11-ketotestosterone than for dihydrotestosterone. Treatment with different androgens did not up-regulate androgen receptor mRNA level or increase receptor abundance, suggesting that auto-regulation is not involved in differential ligand activation. However, comparison of the trans-activation potential of the stickleback androgen receptor with the human androgen receptor, in both human HepG2 cells and zebrafish ZFL cells, revealed preferential activation by 11-ketotestosterone of the stickleback receptor, but not of the human receptor. These findings demonstrate the presence of a receptor preferentially activated by 11-ketotestosterone in the three-spined stickleback, so far the only one known in any animal.     

Development of methods for extraction and in vitro quantification of estrogenic and androgenic activity of wastewater samples

Chemicals released into the environment by anthropogenic activities have been linked to estrogenic or androgenic effects in exposed wildlife, and there is a need to develop and validate rapid and cost-effective methods to quantify the total estrogenic and androgenic activity of environmental water samples. In this study, estrogen receptors (ER) were isolated from sheep (Ovis aries) uteri and rainbow trout (Oncorhynchus mykiss) livers and androgen receptors (AR) were isolated from rainbow trout brains. The isolated receptors were used in competitive receptor binding assays to test the affinity of known estrogenic and androgenic chemicals for the receptor binding site, and results were compared with literature values for the rat uterine ER binding assay and the E-Screen. The relative binding affinities of the tested compounds to ER from different species were similar, and binding to the ER was a more responsive endpoint than the cellular effect measured in the E-Screen. Using the sheep ER binding assay in combination with solid-phase extraction, the estrogenic activity in a raw sewage sample from a municipal treatment plant in Brisbane (Queensland, Australia) was measured at 51-73 ng/L estradiol equivalents (EEq).     

Parallel evolution? Microsatellite variation of recently isolated marine and freshwater three-spined stickleback

Variation at nine microsatellite loci, four of which are linked to phenotypic traits (spine length and lateral plate morphology) in Canadian three-spined stickleback Gasterosteus aculeatus , are used to test for selection on marine and freshwater three-spined stickleback morphs in Iceland. There are indications of strong selection on loci linked to dorsal spine length, providing another potential example of parallel divergence at the genomic level in three-spined stickleback.     

Comparative study of the olfactory epithelium of the three-spined stickleback (Gasterosteus aculeatus) and the nine-spined stickleback (Pungitius pungitius)

Summary The olfactory epithelium of the three-spined stickleback (Gasterosteus aculeatus) and the nine-spined stickleback (Pungitius pungitius) has been studied with a conventional histochemical and a novel immunological staining technique. In both species, the sensory epithelium is arranged in folds separated by non-sensory epithelial tissue. In the nine-spined stickleback, intrinsic folds consisting of non-sensory cells are found in the apical part of the sensory epithelium where they divide the surface of the sensory epithelium into small islets. These non-sensory cells are non-ciliated, flattened and piled on top of each other; they contain numerous electron-translucent vesicles. The intrinsic folds are absent from the sensory epithelium of the three-spined stickleback. In both species, axons of receptor cells form a layer of fibers in the sensory epithelium immediately above the basal cells. In the three-spined stickleback, thick branches of the olfactory nerve are frequently found in this layer. These branches are only occasionally observed in the sensory epithelium of the nine-spined stickleback. Thus, the three-spined stickleback and the nine-spined stickleback show considerable differences in the organization of the sensory regions of the olfactory epithelium.     

Rapid bioconcentration of steroids in the plasma of three-spined stickleback Gasterosteus aculeatus exposed to waterborne testosterone and 17β-oestradiol

The relationship over time between the concentrations of two steroids, singly and in combination, in a static exposure system and in the blood of three-spined stickleback Gasterosteus aculeatus , held within the exposure system was investigated. Groups of three-spined stickleback were exposed (nominally) to either 1000 ng l⁻¹ 17β-oestradiol (E2), testosterone (T) or E2 and T in combination at the same concentrations for 6 days. Both water and fish were sampled at intervals and steroid concentrations in both compartments were determined. The plasma steroid time profile revealed a rapid bioconcentration within the first 6 h of exposure. The plasma steroid levels attained at this time point (20–90 ng ml⁻¹) were up to 50-fold (E2) and 200-fold (T) greater than the actual levels of steroid measured in the exposure water, while levels in the blood of control fish did not exceed 4 ng ml⁻¹. The substantial elevation of plasma steroid levels relative to the concentrations of steroid to which the fish were exposed in the ambient water gives scope for delivery of the steroids to target endocrine tissues at levels far in excess of what might be predicted on the basis of passive branchial uptake alone. These results are discussed in relation to endocrine disruption, and in particular the occurrence of effects in fish exposed to levels of endocrine active substances that are seemingly physiologically irrelevant.     

Monitoring Endocrine Disrupter Compounds in the Tunisian Hamdoun River using In Vitro Bioassays

Anthropogenic chemicals occurring in the environment, namely endocrine-disrupting chemicals (EDCs), have generated growing concern over their potential adverse effects on human wildlife health and ecosystem processes. This interest resulted particularly from their abilities to mimic the effect of endogenous hormones. In this study, we used stable transfected reporter cell lines to investigate the endocrine-disrupting profile of water as well as sediment samples. Samples are collected from up- and downstream of an industrial wastewater discharge point at the Hamdoun River in the vicinity of an industrial zone located at the center of Tunisia. The analysis of estrogen, androgen, and xenobiotic (pregnane X and dioxin) ligands receptors expressed by chimeric cell lines indicated that while the water and sediment samples from upstream sites have lower levels of estrogenic activity, those from downstream exhibited stronger estrogenic, aryl hydrocarbon receptor (AhR), and Pregnane X Receptor (PXR) activities. Moreover, collected samples have shown hormonal activity in terms of all tested receptors except the androgenic ones. In vitro recombinant estrogen receptor competitive binding assays revealed that while the estrogenic activities of the downstream water sample compounds had a strong affinity for estrogen receptor α (ERα), those present in the sediment samples showed a weaker one. These findings were consolidated by subsequent chemical analysis (high-performance liquid chromatography with UV detectors). Our results indicate that the water and sediment discharges at the Hamdoun River represent a major sink for EDCs from natural and industrial effluents, particularly those of the textile industry, with pernicious potential to disrupt normal endocrine functions.     

Nesting, courtship and kidney hypertrophy in Schistocephalus-infected male three-spined stickleback from an upland lake

The effect of Schistocephalus solidus infection on reproductive development and behaviour of male three-spined stickleback Gasterosteus aculeatus from an upland lake (Llyn Frongoch, Wales, U.K.) was investigated. Uninfected and infected males were collected on two separate occasions during the 2005 breeding season and encouraged to build nests under favourable laboratory conditions. Male nuptial colouration, courtship levels and nesting activity were recorded daily over two separate 21 day studies. Completed nests were removed, encouraging males to build multiple nests. On termination of each study, males were dissected and the kidney and any S. solidus plerocercoids were removed and weighed. In contrast to uninfected males, which readily built multiple nests, no infected males completed a nest during either study. Infected males also exhibited significantly reduced courtship levels, nesting activity, nuptial colouration, kidney development and body condition, compared with uninfected fish. Among infected three-spined stickleback, courtship and nesting behaviours tended to be more severely impacted in fish harbouring heavier plerocercoid burdens. In contrast to other populations, male three-spined stickleback from Llyn Frongoch appear to be unable to overcome the negative impacts of infections on reproductive development and behaviour when provided with short-term favourable conditions. Discrepancies between populations might have been related to the trophic status at the site of capture, emphasizing the importance of studying the biological effects of infections under different environmental conditions.     

Endocrine disrupters with (anti)estrogenic and (anti)androgenic modes of action affecting reproductive biology of Xenopus laevis: I. Effects on sex steroid levels and biomarker expression

Adult Xenopus laevis were exposed in vivo to ethinylestradiol, tamoxifen, methyldihydrotestosterone and flutamide as (anti)estrogenic and (anti)androgenic compounds, respectively, for four weeks at a concentration of 10(-8) M and to Lambro river water, a polluted river from Italy. Effects of the treatments were analysed by mRNA expression of retinol-binding protein (RBP), transferrin (TF), transthyretin (TTR) and vitellogenin (VTG) in the liver of male and female X. laevis, to analyse the potential of these genes to detect endocrine disrupting compounds (EDC) with different modes of action. In addition, plasma VTG and sex steroid levels, estradiol-17beta (E(2)) and testosterone (T), were analysed. Sex steroids were depressed by ethinylestradiol in both sexes whereas tamoxifen increased E(2) in females. The induction of VTG protein plasma levels was more pronounced at the protein level compared to hepatic VTG mRNA expression in response to estrogenic treatment but VTG mRNA expression detected both, estrogenic and antiestrogenic EDC. The mRNA expression of TF was decreased by estrogenic and increased by antiestrogenic treatment while TTR mRNA expression was down-regulated and RBP mRNA up-regulated by estrogenic exposure. The other treatments did not affect the mRNA expression of the examined genes.     

Endocrine disruptors in bottled mineral water: estrogenic activity in the E-Screen

Human exposure to endocrine disruptors is well documented by biomonitoring data. However, this information is limited to few chemicals like bisphenol A or phthalate plasticizers. To account for so-far unidentified endocrine disruptors and potential mixture effects we employ bioassays to detect endocrine activity in foodstuff and consequently characterize the integrated exposure to endocrine active compounds. Recently, we reported a broad contamination of commercially available bottled water with estrogenic activity and presented evidence for the plastic packaging being a source of this contamination. In continuation of that work, we here compare different sample preparation methods to extract estrogen-like compounds from bottled water. These data demonstrate that inappropriate extraction methods and sample treatment may lead to false-negative results when testing water extracts in bioassays. Using an optimized sample preparation strategy, we furthermore present data on the estrogenic activity of bottled water from France, Germany, and Italy: eleven of the 18 analyzed water samples (61.1%) induced a significant estrogenic response in a bioassay employing a human carcinoma cell line (MCF7, E-Screen). The relative proliferative effects ranged from 19.8 to 50.2% corresponding to an estrogenic activity of 1.9-12.2 pg estradiol equivalents per liter bottled water. When comparing water of the same spring that is packed in glass or plastic bottles made of polyethylene terephthalate (PET), estrogenic activity is three times higher in water from plastic bottles. These data support the hypothesis that PET packaging materials are a source of estrogen-like compounds. Furthermore, the findings presented here conform to previous studies and indicate that the contamination of bottled water with endocrine disruptors is a transnational phenomenon.     

Comparative Study of the Lateral-line System of the Three-spined Stickleback (Gasterosteus aculeatus) and the Nine-spined Stickleback (Pungitius pungitius)

Abstract The morphology of the lateral-line system of the nine-spined stickleback (Pungitius pungitius) and the three-spined stickleback (Gasterosteus aculeatus) has been studied. In the nine-spined stickleback, a preopercular, infraorbital, supraorbital, postotic and peduncular canal can be identified on both sides of the body. Replacement lines are found as a continuation of the preopercular and infraorbital canal. In addition, lines of free neuromasts are found on the mandible and trunk. An accessory line is present above and below the peduncular canal. The presence of both canals and accessory lines on the peduncle suggests that the peduncle in this species has important sensory functions. No canals are found in the three-spined stickleback. Instead, replacement lines corresponding to the canals can be identified on the head. Accordingly, the lateral-line system of the three-spined and the nine-spined stickleback has a different structure. The lateral-line system of both species shows signs of specialization but the three-spined stickleback has a more specialized lateral-line system than the nine-spined stickleback.     

Cytochrome P450 1A, 1B, and 1C mRNA induction patterns in three-spined stickleback exposed to a transient and a persistent inducer

Cytochrome P450 1 (CYP1) mRNA induction patterns in three-spined stickleback (Gasterosteus aculeatus) were explored for use in environmental monitoring of aryl hydrocarbon receptor (AHR) agonists. The cDNAs of stickleback CYP1A, CYP1B1, CYP1C1, and CYP1C2 were cloned and their basal and induced expression patterns were determined in the brain, gill, liver and kidney. Also, their induction time courses were compared after waterborne exposure to a transient (indigo) or a persistent (3,3',4,4',5-pentacholorbiphenyl PCB 126) AHR agonist. The cloned stickleback CYP1s exhibited a high amino acid sequence identity compared with their zebrafish orthologs and their constitutive tissue distribution patterns largely agreed with those reported in other species. PCB 126 (100 nM) induced different CYP1 expression patterns in the four tissues, suggesting tissue-specific regulation. Both indigo (1 nM) and PCB 126 (10 nM) induced a strong CYP1 expression in gills. However, while PCB 126 gave rise to a high and persistent induction in gills and liver, induction by indigo was transient in both organs. The number of putative dioxin response elements found in each CYP1 gene promoter roughly reflected the induction levels of the genes. The high responsiveness of CYP1A, CYP1B1, and CYP1C1 observed in several organs suggests that three-spined stickleback is suitable for monitoring of pollution with AHR agonists.     

Interaction of Staphylococcal Alpha Toxin and the Estrogenic Hormone Diethylstilbestrol

Previous work in this laboratory showed that diethylstilbestrol was capable of suppressing induced furunculosis in rabbits. The present study indicates that the synthetic estrogenic hormone diethylstilbestrol which is used for acne, estrogen deficiency, cancer, and other disorders, can reduce the cytolytic action of staphylococcal alpha toxin. The cytotoxic action of purified alpha toxin for tissue cultures was evaluated by use of such parameters as total and viable cell counts, glucose, and protein determination, and cytopathic effects (CPE) in the presence and absence of steroids. To 3-day-old primary rabbit baby kidney tissue cultures, 1 to 5 μg of diethylstilbestrol per ml was added; growth of tissue cultures in Eagles medium was continued till the 6th day, and then one tissue cytopathic dose per milliliter of alpha toxin was added, and the subsequent fate of tissue cultures was assayed. Such cultures yielded higher total and viable cell counts, utilized more glucose, and contained more protein than the control cultures. In control cultures, CPE was observed on the 3rd hr after the addition of alpha toxin, and it was complete in 24 hr, whereas in tissue cultures treated with diethylstilbestrol, the CPE was significantly reduced. The data presented in this study made possible the availability of a suppressor of the cytolytic action of alpha toxin and might be useful in assaying the action of alpha toxin in an in vitro inexpensive test system.     

Parasite fauna in sticklebacks (Gasterosteidae) from bodies of water of the Kola region

The results of a parasitological study of the two species of sticklebacks (Pungitius pungitius and Gasterosteus aculeatus) inhabiting the Kola region are presented. The 42 and 14 species of parasite were found in the nine-spined and three-spined stickleback, respectively. The paucity of the parasite fauna in the three-spined stickleback is observed. Distribution of parasites in Kola waterbodies and ecological peculiarities in system "parasite-host" are presented.     

Changes in the diet of the coastal cod <Emphasis Type="Italic">Gadus morhua marisalbi</Emphasis> in the Kandalaksha Gulf of the White Sea under conditions of increased abundance of three-spined stickleback <Emphasis Type="Italic">Gasterosteus aculeatus</Emphasis>

The data on feeding of coastal cod Gadus morhua marisalbi in the area of the Chupa Bay (Kandalaksha Gulf of the White Sea) are presented. In the food spectrum of cod, over 25 cm long fish and their eggs dominated (77.5% by frequency of occurrence and 91.7% by weight). Other groups of food organisms occupied a secondary place. Among fish, three-spined stickleback Gasterosteus aculeatus was the most prominent food item in the diet of cod (48.3% by weight). The published data on long-term changes of the diet of cod are considered, which are mostly related to considerable fluctuations of the abundance of three-spined stickleback in the White Sea. It is shown that, at present, three-spined stickleback again plays an important role in the diet of the White Sea cod     

Purification and characterization of vitellin from the estuarine mysid Neomysis integer (Crustacea; Mysidacea)

Invertebrates account for roughly 95% of all animals, yet surprisingly, little effort has been invested to understand their value in signaling potential environmental endocrine disruption. There has been, however, much recent attention on vitellogenin induction in egg-laying invertebrates and vertebrates as indicators of exposure to estrogenic xenobiotics. Mysid shrimp (Crustacea: Mysidacea) have been put forward by several researchers and regulatory bodies (e.g., US-EPA) as suitable test organisms for the evaluation of environmental endocrine disruption. In view of developing sensitive assays to study endocrine disruption in the estuarine mysid Neomysis integer, we isolated and characterized vitellin, the major yolk protein in eggs. Vitellin was purified using gel filtration and characterized by electrophoresis using different staining procedures. Specific (as shown by Western blotting) polyclonal antibodies were produced in rabbit against the purified vitellin of N. integer. These antisera will be used to develop immunoassays to study vitellogenesis in mysids and to detect potential stimulatory or inhibitory effects of endocrine disruptors on the production of vitellin.     

Acute exposure to offshore produced water has an effect on stress- and secondary stress responses in three-spined stickleback Gasterosteus aculeatus

Pollution is one of today's greatest problems, and the release of contaminants into the environment can cause adverse changes in vitally important biological pathways. In this study, we exposed three-spined stickleback Gasterosteus aculeatus to produced water (PW), i.e. wastewater from offshore petroleum production. PW contains substances such as alkylphenols (APs) and aromatic hydrocarbons (PAHs) known to induce toxicant stress and endocrine disruption in a variety of organisms. Following exposure to PW, a standardized confinement treatment was applied as a second stressor (PW-stress), testing how fish already under stress from the pollutant would respond to an additional stressor. The endpoint for analysis was a combination of blood levels of cortisol and glucose, in addition to transcribed levels of a set of genes related to toxicant stress, endocrine disruption and general stress. The findings of this study indicate that low doses of PW do not induce vitellogenin in immature female stickleback, but do cause an upregulation of cytochrome (CYP1A) and UDP-glucuronsyltransferase (UDP-GT), two biomarkers related to toxicant stress. However, when the second stressor was applied, both genes were downregulated, indicating that the confinement exposure had a suppressive effect on the expression of toxicant biomarkers (CYP1A and UDP-GT). Further, two of the stress related genes, heat shock protein 90 (HSP90) and stress-induced phosphoprotein (STIP), were upregulated in both PW- and PW-stress-treatment, but not in the water control confinement treatment, indicating that PW posed as a larger stress-factor than confinement for these genes. The confinement stressor caused an increased level of glucose in both control and PW-treated fish, indicating hyperglycemia, a commonly reported stress response in fish.     

Naringenin-type flavonoids show different estrogenic effects in mammalian and teleost test systems

The estrogenic activity of several intermediary plant compounds has raised concern about possible risks of unwanted interference with endocrine regulation, but on the other hand there are potential medical benefits, in particular in treatment of menopausal symptoms or cancer. In the present study, we compare the estrogenic effects of phytoestrogens naringenin, 8-prenylnaringenin, 6-(1,1-dimethylallyl)naringenin, and the synthetic 4'-acetyl-7-prenyloxynaringenin. Two mammalian in vitro systems and a fish in vivo system were used to study the estrogenic properties with reference to genistein, 17-beta-estradiol or ethynylestradiol. Strong differences were observed between the mammalian in vitro and the fish in vivo test system. In the medaka sex reversal/vtg gene expression assay no estrogenic effects of the naringenin-type flavonoids were observed, while mammalian in vitro systems showed a similar and graded response to the test compounds.