Effects of endocrine disrupting chemicals on gonad development: Mechanistic insights from fish and mammals.

Over the past century, evidence has emerged that endocrine disrupting chemicals (EDCs) have an impact on reproductive health. An increased frequency of reproductive disorders has been observed worldwide in both wildlife and humans that is correlated with accidental exposures to EDCs and their increased production. Epidemiological and experimental studies have highlighted the consequences of early exposures and the existence of key windows of sensitivity during development. Such early in life exposures can have an immediate impact on gonadal and reproductive tract development, as well as on long-term reproductive health in both males and females. Traditionally, EDCs were thought to exert their effects by modifying the endocrine pathways controlling reproduction. Advances in knowledge of the mechanisms regulating sex determination, differentiation and gonadal development in fish and rodents have led to a better understanding of the molecular mechanisms underlying the effects of early exposure to EDCs on reproduction. In this manuscript, we review the key developmental stages sensitive to EDCs and the state of knowledge on the mechanisms by which model EDCs affect these processes, based on the roadmap of gonad development specific to fish and mammals.

Impacts of endocrine disrupting chemicals on reproduction in wildlife and humans.

Endocrine disrupting chemicals (EDCs) are ubiquitous in aquatic and terrestrial environments. The main objective of this review was to summarize the current knowledge of the impacts of EDCs on reproductive success in wildlife and humans. The examples selected often include a retrospective assessment of the knowledge of reproductive impacts over time to discern how the effects of EDCs have changed over the last several decades. Collectively, the evidence summarized here within reinforce the concept that reproduction in wildlife and humans is negatively impacted by anthropogenic chemicals, with several altering endocrine system function. These observations of chemicals interfering with different aspects of the reproductive endocrine axis are particularly pronounced for aquatic species and are often corroborated by laboratory-based experiments (i.e. fish, amphibians, birds). Noteworthy, many of these same indicators are also observed in epidemiological studies in mammalian wildlife and humans. Given the vast array of reproductive strategies used by animals, it is perhaps not surprising that no single disrupted target is predictive of reproductive effects. Nevertheless, there are some general features of the endocrine control of reproduction, and in particular, the critical role that steroid hormones play in these processes that confer a high degree of susceptibility to environmental chemicals. New research is needed on the implications of chemical exposures during development and the potential for long-term reproductive effects. Future emphasis on field-based observations that can form the basis of more deliberate, extensive, and long-term population level studies to monitor contaminant effects, including adverse effects on the endocrine system, are key to addressing these knowledge gaps.

Impact of activated sludge configuration and operating conditions on in vitro and in vivo responses and trace organic compound removal.

This study tested municipal sewage effluents generated at the pilot scale using conventional activated sludge (CAS), nitrifying activated sludge (CAS-N) and biological nutrient removal (BNR) in terms of the removal of trace organic compounds (TrOCs) and final effluent quality as indicated by yeast estrogenicity screening (YES), short term zebrafish reproduction and fathead minnow life-cycle tests. Under cold weather conditions (extended SRTs), the BNR configuration reduced the concentrations of the largest number of TrOCs while under warm weather conditions (reduced SRTs) the CAS-N was most effective. By comparison, YES test results indicated statistically lower responses in the BNR effluent in the warm weather tests and no difference between the effluents of CAS-N and BNR in the cold weather tests. Short term tests with adult zebrafish revealed no impact of the BNR and CAS-N effluents on egg production. By contrast egg production and gene expression in the CAS-exposed zebrafish were substantially less than that of control exposures and were similar to that of exposures to ammonia at similar concentrations as the CAS exposures. In fathead minnow life-cycle tests, exposures to CAS effluent (70-50% v/v) resulted in considerable mortality, reduced growth and reduced egg production that was likely due to the elevated ammonia concentrations. The CAS-N effluent (100% v/v) also resulted in some mortality and reduced growth and egg production in the fathead minnows. By contrast, the BNR effluent (100% v/v) had no effect on mortality, growth or egg production. The results suggest that enhancements to wastewater treatment plants that are associated with improved nitrogen removal can result in enhanced removal of TrOCs and can reduce the harmful effects of the effluents on aquatic biota.

Male primer endocrine responses to preovulatory female cyprinids under natural conditions in Sweden.

This study investigated two related aspects of male-female reproductive interactions in the family Cyprinidae: (1) whether ovulating female rudd Scardinius erythrophthalmus (subfamily Leuciscinae) induce endocrine and gonadal priming responses in conspecific males, a phenomenon which has been described only in species from the subfamily Cyprininae such as goldfish, Carassius auratus, crucian carp Carassius carassius and common carp, Cyprinus carpio and (2) whether the stimuli mediating these responses are species-specific. Field studies of three sympatric European cyprinids, two leuciscins (S. erythrophthalmus and white bream Blicca bjoerkna) and one cyprinin (C. carassius), were conducted on fishes captured in Sweden in the spawning season and held in net pens under natural conditions. As previously reported in C. carassius, male S. erythrophthalmus increased milt (sperm and seminal fluid) volume and plasma concentrations of the sperm maturation hormone 4-pregnen-17,20ß-diol-3-one (17,20ß-P) when they were held with female S. erythrophthalmus induced to ovulate by injection of Ovaprim (GnRH analogue plus dopamine antagonist). Male S. erythrophthalmus had larger milt volumes than male C. carassius prior to and following exposure to ovulatory conspecifics, but exhibited a smaller proportional milt increase in response to stimulation, suggesting species differences in sperm allocation at spawning. The presence of female S. erythrophthalmus and B. bjoerkna did not affect milt volumes of C. carassius under two experimental conditions: (1) ovulating S. erythrophthalmus and B. bjoerkna did not increase the milt volumes of C. carassius and (2) S. erythrophthalmus and B. bjoerkna did not interfere with the milt volume increase induced in male C. carassius by ovulating conspecifics. These results suggest that, as in C. auratus, C. carassius and C. carpio (subfamily Cyprininae), female S. erythrophthalmus (subfamily Leuciscinae) release a preovulatory pheromone that exerts priming effects on male hormones and sperm allocation. The findings also indicate that C. carassius discriminate between the reproductive odours of conspecifics and heterospecifics.

Testing the synergistic effects of GnRH and testosterone on the reproductive physiology of pre-adult pink salmon Oncorhynchus gorbuscha.

To test the hypothesis that the hypothalmic gonadotropin-releasing hormone (GnRH) and testosterone (T) co-treatment stimulates both the hypothalmo-pituitary-gonadal (HPG) and hypothalmo-pituitary-interrenal axes, the reproductive and osmoregulatory responses of pre-adult pink salmon Oncorhynchus gorbuscha were compared after GnRH and T administration either alone or in combination. Relative to controls, neither GnRH nor T treatment resulted in significantly greater ovarian or testicular growth, but co-treatment significantly increased ovarian growth after 5 months. Interestingly, the stimulation was undetectable after 3 months. However, once daily photoperiod began shortening after the summer solstice, c. 2 months before the natural spawning date, GnRH+T-treated females were stimulated to produce larger ovaries. Final fish body length and the size of individual eggs did not differ among treatment groups. GnRH+T eggs, however, showed signs of advanced vitellogenesis relative to GnRH-treated and control eggs, whereas T-treated eggs became atretic. Testis size increased significantly from initial values and most males were spermiating, but this growth and development were independent of hormone treatments. Final plasma ion, metabolite and cortisol concentrations did not differ among treatment groups. It is concluded that GnRH+T co-treatment was effective in stimulating female but not male maturation. GnRH and T treatment, however, presumably had little effect on the hypothalmo-pituitary-interrenal axis as observed by ionoregulatory status.

Physiological condition differentially affects the behavior and survival of two populations of sockeye salmon during their freshwater spawning migration.

Recently, a segment of the Adams-Shuswap sockeye salmon (Oncorhynchus nerka) population initiated freshwater migration several weeks earlier than historically recorded, resulting in high mortality rates. The comigrating Chilko population maintained their historic river entry timing and did not experience elevated mortality. To test the hypothesis that population-specific differences in physiological condition would differentially influence behavior and survival when exposed to fisheries capture stress, we physiologically sampled individuals from both populations at the onset of the freshwater phase of their reproductive migration and tracked the remainder of their migrations using radio telemetry. Adams-Shuswap individuals had slower migration rates and were less likely to reach natal subwatersheds relative to Chilko individuals. Metabolic and osmoregulatory impairment was related to mortality for Adams-Shuswap individuals but not for Chilko individuals. Similarly, physiological condition correlated with migration rate for Adams-Shuswap but not Chilko fish. Survival to natal subwatersheds was 1.9 times higher for Chilko relative to Adams-Shuswap, a result that did not emerge until individuals approached natal subwatersheds several days after the stressor was applied. We conclude that physiological condition differentially affects the behavior and survival of these two populations, which may be a consequence of the early-entry phenomenon by a segment of the Adams-Shuswap population.

Effects of experimental manipulations of salinity and maturation status on the physiological condition and mortality of homing adult sockeye salmon held in a laboratory.

Relatively little is known about the physiological response and mortality consequences of the return of anadromous fish to freshwater (FW). We explored the consequences of the return to FW by collecting maturing sockeye salmon from the marine waters off the mouth of the Fraser River and holding approximately 50 sockeye in each of five treatments: saltwater (SW; salinity = 28 ppt), iso-osmotic water (ISO; 13 ppt), FW (0 ppt), SW + gonadotropin-releasing hormone (SW + GnRH), and FW + GnRH. Exogenous GnRH treatments were intended to accelerate maturation. Results demonstrate that gill Na(+),K(+) ATPase activity, sex steroid concentrations, and cortisol levels were highly responsive to experimental manipulations and followed predicted trajectories (i.e., FW + GnRH sockeye were the most mature and FW adapted). There were few among-treatment differences in hematocrit and plasma concentrations of lactate, glucose, Na(+), Cl(-), and plasma osmolality among sockeye that survived to the end of treatments, indicating that sockeye rigorously maintain internal homeostatic conditions while alive. There were large among-treatment differences in mortality (SW+GnRH > SW> FW+GnRH > FW=ISO), and each treatment experienced a notable increase in mortality rate around the fifth day of treatment. Our results indicate that salinity represented a modestly larger challenge to the experimental sockeye than did the artificially accelerated sexual maturation. Our results also suggest that maturing sockeye either successfully acclimate to FW within 5 d of exposure or perish. These findings are consistent with the predictions of the theory of anadromy, in suggesting that the return of adults to FW can be physiologically challenging and can represent a period of significant natural mortality.

An investigation into the role of prostaglandins in zebrafish oocyte maturation and ovulation.

This study explored the potential for ovarian-derived prostaglandins (PGs) to be involved in the regulation of oocyte maturation and ovulation in zebrafish. It was demonstrated that cultured vitellogenic follicles have the capacity to produce prostaglandin E(2) (PGE(2)) and PGF(2alpha) in response to arachidonic acid (AA) in a concentration-dependent manner, and that AA stimulates the in vitro production of 17beta-estradiol (E(2)). The production of AA-stimulated PGF(2alpha) was significantly reduced by treatment with the non-selective cyclooxygenase (COX) inhibitor, indomethacin (INDO). Treatment of full-grown follicles with AA did not induce oocyte maturation as assessed by germinal vesicle breakdown, but INDO significantly decreased the rate of spontaneous maturation. Using Real-Time PCR, it was shown that follicles of different developmental size classes (primary growth and pre-vitellogenic, early-vitellogenic, and mid- to full-grown vitellogenic) express enzymes that release (cytosolic phospholipase A(2) (cPLA(2)); phospholipase Cgamma1) or metabolize (COX-1, COX-2, and prostaglandin synthase-2) AA to PG metabolites. The expression of cPLA(2) was found to be significantly greater in full-grown follicles compared to follicles of the pre- and early-vitellogenic stages. In vivo studies demonstrated that breeding groups of zebrafish exposed to 100 microg/L INDO exhibited reduced spawning rates and clutch sizes compared with control and 1 microg/L INDO exposed fish. In other studies, it was shown that naturally spawning groups of females exhibit increased ovarian levels of PGF(2alpha), E(2), and 17alpha,20beta-dihydroxy-4-pregnen-3-one (a maturation-inducing hormone in zebrafish) near the time of ovulation compared with non-breeding females. Collectively, these experiments indicate that the AA pathway in zebrafish ovaries is involved in the regulation of oocyte maturation and ovulation and a non-selective inhibitor of COX disrupts these processes.

Reproductive and stress hormone levels in goldfish (Carassius auratus) exposed to oil sands process-affected water.

Athabasca oil sands mining in northern Alberta produces process-affected waters that are characterized by the presence of naphthenic acids, polycyclic aromatic hydrocarbons, and high salinity. The purpose of this study was to examine the impact of these process-affected waters on reproductive and stress related endpoints in mature goldfish, Carassius auratus. In two separate studies, testosterone and 17beta-estradiol levels in the plasma were significantly reduced in both male and female goldfish caged for 19 days in process-affected waters relative to controls. This effect was most pronounced in goldfish caged at a site containing mature fine tailing and tailings pond water (P5). Ovarian and testicular tissues from fish in the caging studies were incubated in vitro to evaluate potential differences in basal steroid production levels and responsiveness to human chorionic gonadotropin (hCG). Basal levels of testosterone were reduced significantly in males and females from P5 compared with the control pond (P1) demonstrating that the gonads from exposed fish had a diminished steroidogenic capacity. Gonadal tissues of fish from all ponds responded similarly to hCG suggesting that the steroid biosynthetic pathway remained functionally intact. Plasma cortisol levels were significantly higher in male goldfish caged in a pond containing mature fine tailings and capped with uncontaminated water (P3) and in P5 compared with P1. Collectively, these studies suggest that waste products of oil sands mining have the potential to disrupt the normal endocrine functioning in exposed fish through alterations to both reproductive and glucocorticoid hormone biosynthesis. In additional laboratory studies, exposure of goldfish to a naphthenic acid extract for 7 days failed to replicate the effects of processes-affected waters on plasma steroid levels and the causative agent(s) responsible for the effects on steroid biosynthesis remains to be identified.

Sediment TCDD-EQs and EROD and MROD activities in Ranid frogs from agricultural and nonagricultural sites in Michigan (USA).

In vitro studies have demonstrated atrazine-mediated induction of 7-ethoxyresorufin O-deethylase (EROD) activity. EROD is an enzyme active in the metabolism of many compounds, including many xenobiotics. These studies have suggested that atrazine may affect reproductive function by altering steroid metabolism. The goal of this study was to determine whether relationships could be detected between measured atrazine concentrations in surface waters and the liver-somatic index (LSI) and EROD and 7-methoxyresorufin O-deethylase (MROD) activities in the livers of ranid frogs. In addition, sediment dioxin toxic equivalents (TCDD-EQs) were determined using the H4IIE-luc cell bioassay. Adult and juvenile green frogs (Rana clamitans), bullfrogs (R. catesbeiana), and Northern leopard frogs (R. pipiens) were collected from areas with extensive corn cultivation and areas where there was little agricultural activity in south central Michigan in the summer of 2003. Atrazine concentrations at nonagricultural sites ranged from less than the limit of quantification (0.17 microg atrazine/L) to 0.23 microg atrazine/L and did not exceed 1.2 microg atrazine/L at agricultural sites. Sediment TCDD-EQs were measurable only at one agricultural site. Of the measured parameters, only LSI values in adult male frogs differed significantly between agricultural and nonagricultural sites, with greater values observed at agricultural sites. In green frogs, EROD and MROD activities were measurable in both adult and juvenile frogs and were similar among sites. Median EROD activities ranged from 13 to 21 pmol/min/mg protein in adult male green frogs and from 5 to 13 pmol/min/mg protein in adult female green frogs. Juvenile frogs had greater EROD and MROD activities than adult frogs. Bullfrogs and leopard frogs had greater activities than did green frogs. Atrazine concentrations were significantly and negatively correlated with MROD activity in adult male green frogs (Spearman R = -0.800). LSI and EROD and MROD activities of adult female or juvenile green frogs were not significantly correlated with atrazine concentrations. These results suggest that atrazine does not appear to have a consistent association with EROD or MROD activities in wild-caught green frogs.

Plasma steroid hormone concentrations, aromatase activities and GSI in ranid frogs collected from agricultural and non-agricultural sites in Michigan (USA).

The triazine herbicide atrazine has been hypothesized to disrupt sexual development in frogs by up-regulating aromatase activity, resulting in greater estradiol (E2) concentrations and causing feminization in males. The goal of this study was to collect native ranid frogs from atrazine-exposed ponds and determine whether relationships exist between measured atrazine concentrations and the gonadosomatic index (GSI), plasma concentrations of testosterone (T), E2 or 11-ketotestosterone (KT), or with aromatase activity. In the summer of 2002 and 2003, adult and juvenile green frogs (Rana clamitans), bullfrogs (R. catesbeiana) and Northern leopard frogs (R. pipiens) were collected from areas with extensive corn cultivation and areas where there was little agricultural activity in south-central Michigan. Atrazine concentrations were below the limit of quantification at non-agricultural sites. Atrazine concentrations did not exceed 2 microg/L at most agricultural sites, but a concentration of 250 microg atrazine/L was measured in one sample from one site in 2002. Plasma steroid concentrations varied among locations. Aromatase activity was measurable in less than 11% of testes in adult males, and in less than 4% of testes in juvenile males. Median aromatase activities in ovaries of adult females ranged from 3 to 245 pmol/h/mg protein, and maximum activities were 2.5-fold greater in juveniles than in adults. Atrazine concentrations were not significantly correlated with any of the parameters measured in this study. These results indicate that atrazine does not up-regulate aromatase in green frogs in the wild, and does not appear to affect plasma steroid hormone concentrations.

Gill and liver histopathological changes in yellow perch (Perca flavescens) and goldfish (Carassius auratus) exposed to oil sands process-affected water.

The extraction of bitumen from the Athabasca oil sands (Alberta, Canada) produces significant volumes of process-affected water containing elevated levels of naphthenic acids (NAs), ions, and polycyclic aromatic hydrocarbons (PAHs). The sublethal response of aquatic organisms exposed to oil sands constituents in experimental aquatic environments that represent possible reclamation options has been studied. In this study, the effects of process-affected waters on gill and liver tissues in yellow perch (Perca flavescens) and caged goldfish (Carassius auratus) held in several reclamation ponds at Syncrude's Mildred Lake site have been assessed. Following a 3-week exposure, significant gill (epithelial cell necrosis, mucous cell proliferation) and liver (hepatocellular degeneration, inflammatory cell infiltration) histopathological changes were noted in fish held in waters containing high levels of oil sands process-affected water. In addition, measurements of gill dimensions (gill morphometrical indices) proved sensitive and provided evidence of a physiological disturbance (gas exchange) with exposure to oil sands materials. Due to the complexity of oil sands process-affected water, the cause of the alterations could not be attributed to specific oil sands constituents. However, the histopathological parameters were strong indicators of exposure to oil sands process-affected water and morphometrical data were sensitive indicators of pathological response, which can be used to identify the interactive effects of ionic content, NAs, and PAHs in future laboratory studies.

Atrazine concentrations, gonadal gross morphology and histology in ranid frogs collected in Michigan agricultural areas.

The triazine herbicide atrazine has been suggested to be a potential disruptor of normal sexual development in male frogs. The goals of this study were to collect native ranid frogs from sites in agricultural and non-agricultural areas and determine whether hypothesised atrazine effects on the gonads could be observed at the gross morphological and histological levels. Juvenile and adult green frogs (Rana clamitans), bullfrogs (R. catesbeiana) and leopard frogs (R. pipiens) were collected in the summers of 2002 and 2003. Atrazine concentrations were below the limit of quantification at non-agricultural sites, and concentrations did not exceed 2 microg/L at most agricultural sites. One concentration greater than 200 microg atrazine/L was measured once at one site in 2002. Hermaphroditic individuals with both male and female gonad tissue in either one or both gonads, were found at a low incidence at both non-agricultural and agricultural sites, and in both adults and juveniles. Testicular oocytes (TO) were found in male frogs at most of the sites, with the greatest incidence occurring in juvenile leopard frogs. TO incidence was not significantly different between agricultural and non-agricultural sites with the exception of juveniles collected in 2003. Atrazine concentrations were not significantly correlated with the incidence of hermaphroditism, but maximum atrazine concentrations were correlated with TO incidence in juvenile frogs in 2003. However, given the lack of a consistent relationship between atrazine concentrations and TO incidence, it is more likely the TOs observed in this study result from natural processes in development rather than atrazine exposure.

Sex steroid regulation of brain glutamic acid decarboxylase (GAD) mRNA is season-dependent and sexually dimorphic in the goldfish Carassius auratus.

GABA, the major inhibitory neurotransmitter of the vertebrate brain, has been shown to play an important role in vertebrate reproduction by regulating LH release and sexual behavior. We have studied the expression of the GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD), in goldfish throughout the reproductive cycle in May (mature), November (early gonadal recrudescence) and February (late gonadal recrudescence) and in response to implanted sex steroids. Levels of GAD67 and GAD65 mRNA levels in the hypothalamus of both males and females were highest in the early stages of gonadal recrudescence. In the telencephalon, a different seasonal pattern of GAD expression was evident. The telencephalic expression GAD67, GAD65 and a novel isoform, GAD3, were highest in sexually mature fish in May. Five-day intraperitoneal implantation of gonad-intact fish with testosterone (T), estradiol (E2) or progesterone (P4) did not affect GAD expression in November and February. This is in contrast to results in May when sex differences in steroid responsiveness were evident. Progesterone decreased hypothalamic GAD67 and GAD65 in females and was without effect in males. All other treatments did not alter GAD67, GAD65 or GAD3 expression in the hypothalamus. Both T and P4 decreased GAD67 and GAD65 levels in the telencephalon of male goldfish but had no effect in females. Serum sex steroid levels in control and implanted mature males and females in May were similar so it is unlikely that sex differences in the GAD responses were a result of differences in serum sex steroid levels. These contrasting effects of sex steroids on males and females suggest important sex differences in the regulation of the GADs in sexually mature goldfish.

Seasonal exposures to triazine and other pesticides in surface waters in the western Highveld corn-production region in South Africa.

The objective of this study was to characterize concentrations of atrazine, terbuthylazine, and other pesticides in amphibian habitats in surface waters of a corn-production area of the western Highveld region (North-West Province) of South Africa. The study was conducted from November 2001 to June 2002, coinciding with the corn-production season. Pesticide residues were measured at regular intervals in surface water from eight ponds, three in a non-corn-growing area (NCGA) and five within the corn-growing area (CGA). Measured atrazine concentrations differed significantly among sites and between samples. In the five CGA sites, the maximum atrazine concentrations measured during the study ranged from 1.2 to 9.3 microg/L. Although no atrazine was recorded as being applied in the catchment of the three NCGA sites, maximum concentrations from 0.39 to 0.84 microg/L were measured during the study, possibly as a result of atmospheric transport. Maximum measured concentrations of terbuthylazine ranged from 1.22 to 2.1 microg/L in the NCGA sites and from 1.04 to 4.1 microg/L in the CGA sites. The source of terbuthylazine in the NCGA sites may have been in use other than in corn. The triazine degradation products, deisopropylatrazine (DIA) and deethylatrazine (DEA) and diaminochlorotriazine (DACT) were also found in water from both the CGA and NCGA sites. Concentrations of DIA were > or = 1 microg/L throughout the season, while DEA concentrations were mostly <0.5 microg/L before planting but increased after planting and application of herbicides to concentrations >2 microg/L in some locations. Concentrations of DACT were highly variable (LOD to 8 microg/L) both before and after planting and application, suggesting that they resulted from historical use of triazines in the area. Other herbicides such as simazine and acetochlor were only detected infrequently and pesticides such as S-metolachlor, cypermethrin, monocrotophos, and terbuphos, known to be used in the CGA, were not detected in any of the samples. Because of dilution by higher than normal rainfall in the study period, these concentrations may not be predictive of those in years of normal rainfall.

The effects of pulp mill effluent on the sex steroid binding protein in white sucker (Catostomus commersoni) and longnose sucker (C catostomus).

The objective of this study was to characterize the effects of pulp mill effluent on the properties of the sex steroid binding protein (SBP) in the plasma of white sucker (Catostomus commersoni) and longnose sucker (C. catostomus). SBPs which specifically bind estradiol and testosterone with high affinity (k(D) approximately 3 nM) and low capacity (B(max) approximately 73-81 nM) were identified in both species. Subsequent studies determined if the properties of the SBP in white sucker exposed to bleached kraft mill effluent (BKME) at Terrace Bay, ON, and in longnose sucker exposed to BKME at Grande Prairie, AB. differed from appropriate reference fish. There were no effects of BKME exposure on the binding affinity (k(D)) of the SBP in either species, but there was a significant increase in the binding capacity (B(max)) of longnose sucker SBP exposed to BKME. The livers of nai;ve white sucker exposed to effluent at Terrace Bay or a bleached sulfite/groundwood mill in Edmundston, NB, rapidly accumulated compounds of differing hydrophobicity that bound to both the white sucker and goldfish (Carassius auratus) SBP. Conversely, there was reduced accumulation of SBP ligands in the bile of effluent-exposed fish. We have demonstrated that constituents present within pulp mill effluent bind to both the white sucker and goldfish SBP, and that native species residing downstream of pulp mill effluents may experience modifications in the properties of their SBP.

Milt production in goldfish: regulation by multiple social stimuli.

Previous studies in goldfish (Carassius auratus) demonstrate that milt (sperm and seminal fluid) volume is increased both by a preovulatory steroidal pheromone, 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P), that acts by increasing serum concentrations of gonadotropin II (GTH-II), and by a post-ovulatory prostaglandin (PG) pheromone, that acts by stimulating sexual interactions. Here, we show that male goldfish also increase milt volume when isolated for 24 h, or when placed with another male injected with human chorionic gonadotropin (hCG) for 12 h. In contrast to the milt increase induced by pheromonal 17,20 beta-P, the milt increase following isolation or exposure to hCG-injected males was not associated with increased serum GTH-II. Serum growth hormone also was unaffected, although serum testosterone increased in isolated males. The absence of GTH-II increase following isolation or exposure to hCG-injected males and the long latency time for these two types of responses, suggests these effects are mediated by a novel mechanism as yet undescribed. The present findings suggest that sperm competition has selected for complex mechanisms regulating milt production and fertility in goldfish: males regulate milt production not only in response to stimulatory pheromonal cues from ovulatory females, but also in response to unknown stimulatory and inhibitory cues from male competitors.

Development of a retinoic acid receptor-binding assay with rainbow trout tissue: characterization of retinoic acid binding, receptor tissue distribution, and developmental changes.

Retinoic acid (RA) regulates the transcription of various genes required for several essential functions in vertebrates through binding to two classes of nuclear receptors, the retinoic acid receptors (RAR) and retinoid X receptors (RXR). We investigated nuclear RA binding in tissues from rainbow trout using the radiolabeled all-trans and 9-cis isomers of RA. Specific binding (indicative of receptor binding) of both all-trans- and 9-cis-RA was found in all tissues tested, including the adult trout ovary, testis, gill, liver, kidney, blood, white muscle, and heart. The kinetics and absolute amount of RA binding were dependent on both the tissue and the isomer of RA used. All-trans-RA bound with high affinity (K(d) approximately 1.0-3.9 nM), and low capacity (B(max) approximately 75-484 fmol RA/mg protein), while 9-cis-RA bound with lower affinity (K(d) approximately 7-56 nM), but with a greater capacity (B(max) approximately 214-1076 fmol RA/mg protein). The B(max) results were used to estimate RAR and RXR levels and revealed that the gill possesses primarily RARs while the liver possesses primarily RXRs. The RAR-specific competitor TTNPB was able to effectively displace all-trans-[3H]RA in most tissues, and the RXR-specific competitor AGN 194204 was able to effectively displace 9-cis-[3H]RA. However, TTNPB and AGN 194204 could not displace all of the RA in the kidney and testis, suggesting the existence of another nuclear RA binding protein. Binding of all-trans- and 9-cis-RA was also found in developing trout embryos and fry. Kinetic analysis revealed that RAR levels predominated at the eyed-embryo stage, but decreased 87% by the swim-up fry stage, while RXR levels remained relatively constant over the same time period. These findings suggest that RA and its receptors may play a key role in early trout development. This study has provided a simple and rapid radioligand binding assay that can identify RAR and RXRs in trout tissues.

Stimulatory effects of selected PAHs on testosterone production in goldfish and rainbow trout and possible mechanisms of action.

This study investigates the effects of selected polycyclic aromatic hydrocarbons (PAHs) on testicular function in fish. In vitro incubations of goldfish testis tissue exposed to three selected PAHs [naphthalene, beta-naphthoflavone (betaNF), or retene] resulted in biphasic potentiation of gonadotropin (GtH)-stimulated testosterone (T) production, with no effect on basal levels. In vivo exposure of goldfish to betaNF also resulted in biphasic increases in plasma T levels. Experiments with rainbow trout revealed increased levels of in vitro T production when exposed to naphthalene, albeit only at higher concentrations than the responses observed in goldfish. Studies of possible sites of action at the level of the goldfish testis showed that betaNF exerted its most potent action when T production was initiated by a ligand such as GtH or prostaglandin E(2). Within the steroidogenic pathway, betaNF potentiation of GtH-stimulated T production may be partially mediated by increases in cAMP production, but appear to be independent of cytosolic calcium content. These studies demonstrate that PAHs may function as disruptors of reproductive endocrine function in teleosts through the unexpected effect of enhancing testicular steroidogenesis.

An interlaboratory study on the use of steroid hormones in examining endocrine disruption.

In recent years, there has been an increased use of the measurement of sex steroid hormone levels in the blood of animals exposed to chemicals as an indicator of reproductive impairment or an alteration in endocrine function. Although levels of hormones are often compared among animals and laboratories, there has been no study to examine the between-laboratory variability in actual steroid measurements. Therefore, we initiated a study with white sucker collected from a site receiving pulp mill effluent, previously documented as having reduced steroid levels, to address this issue. Samples of plasma and media from in vitro gonadal incubations were delivered to eight outside laboratories with the ability to measure steroid hormones. These laboratories ranged from well-established fish endocrine laboratories to wildlife toxicology laboratories, which have recently implemented the methods to measure steroid hormones. In this study, we have considered both the absolute measure of steroid content between laboratories as well as the ability to discriminate between reference and exposed populations as important criteria when evaluating the utility of these measures. Of the eight outside laboratories conducting the analyses, six detected identical site differences in circulating levels of testosterone and 17beta-estradiol to those documented by our Burlington laboratory (ON, Canada). However, the absolute value of the steroid hormones measured in the plasma varied significantly (plasma testosterone 0.6-23.1 ng/ml, 17beta-estradiol 77.6-1782.7 pg/ml) with coefficients of variation of 70.4% and 60.3% respectively. Similar results were demonstrated for the measurement of steroid hormones in media following in vitro gonadal incubation. Although there was a fair amount of variability in the absolute measure of steroid hormone levels, we would predict a far greater coherence of interlaboratory results through the sharing of reagents and the use of a common methodology between laboratories. These results are very promising, providing evidence for the inclusion of steroid hormones in monitoring endocrine disruption in wildlife species.