Ian Mayer

Male goldfish reproductive behaviour and physiology are severely affected by exogenous exposure to 17β-estradiol

Mature male goldfish were exposed to different concentrations of the natural hormone 17beta-estradiol (E2). Two methods of exposure were employed, via ingestion at 0, 1, 10 and 100 microg/g food and via the water at 0, 1 and 10 microg/l. The fish were exposed for 24-28 days during the spawning period. The males were then paired with an artificially induced, spawning female and their sexual behaviour was observed during a 15 min period. The physiological status of the fish was also examined with respect to GSI, presence of milt and spawning tubercles and the blood plasma concentration of E2. Despite the relatively short exposure period, exposure to physiological levels of E2 was shown to severely affect the male goldfish reproductive behaviour and physiology. In conclusion, the results from this study and the ability to interpret the effects on this well-studied species, show that the effects of E2, and possibly other estrogenic EDCs, have severe effects at several vital levels of male goldfish reproduction. The results also suggests that the hormone E2 can act as an endocrine disrupting chemical (EDC) in the environment.

Molecular biomarkers of endocrine disruption in small model fish

A wide range of environmental contaminants can interfere with hormonal regulation in vertebrates. These endocrine disrupting chemicals (EDCs) are of high relevance for human and wildlife health, since endocrine signalling controls many essential physiological processes which impact on the individuals health, such as growth and development, stress response, and ultimately reproduction and population development. Small fish represent a cost-effective model for testing potential EDCs allowing the possibility to integrate from molecular to phenotypic and functional effects. We have comprehensively reviewed exposure-effect data from four different small model fish: zebrafish, medaka, fathead minnow, and the three-spined stickleback. The majority of available data refer to EDCs interfering with reproductive hormones. However, we have also included interactions with other hormone systems, particularly the thyroid hormones. We demonstrate that the available data clearly indicates the predictive potential of molecular biomarkers, supporting the development and regulatory application of simple molecular-based screening assays using small model fish for EDC testing.

Melatonin in non-mammalian vertebrates: Physiological role in reproduction?

Abstract Although the pineal organ has been clearly implicated in the photoperiodic control of scasonal reproduction in several non-mammalians, the possible physiological role for melatonin, the major pineal hormone, is far from clear. In many studies on birds, reptiles, amphibians, fishes and even some invertebrates, it has been reported that melatonin administration can influence (usually suppress) reproductive parameters. However, in the vast majority of these studies, melatonin has been administered in an unphysiological manner, usually by injection, and/or dose. It is notable that in most studies where melatonin has been administered either using continuous-release implants or by treatments attempting to mimick the natural diurnal melatonin rhythm, no or limited effects on reproduction were observed. In conclusion, the data available to date do not support the concept that melatonin plays an important physiological role in the photoperiodic control of reproduction in non-mammalians.

The potential of the three-spined stickleback (Gasterosteus aculeatus L.) as a combined biomarker for oestrogens and androgens in European waters

The majority of endocrine disruption studies in Europe have been on non-indigenous species (some of them tropical!)--and none of which has traits that make them suitable for the detection of androgenic compounds. To overcome these problems, we have been developing the stickleback as a model biomarker for testing the effect of endocrine disrupters in European waters. Its advantages are: it is the only fish with a quantifiable in vivo androgen and anti-androgen endpoint (the production of the glue protein, spiggin, by the kidney); it is the only fish in which it will be possible to simultaneously test oestrogenic and androgenic properties of compound; it has a genetic sex marker; it is found in all EU countries; it survives and breeds in both seawater and freshwater; it is extremely robust and can be readily deployed in situ; it displays a variety of pronounced reproductive behaviours; it has a simple and short life cycle, low fecundity and high egg/fry survival rates.

Seasonal changes in and effect of castration/androgen replacement on the plasma levels of five androgens in the male three-spined stickleback, Gasterosteus aculeatus L

The seasonal changes in plasma levels of the androgens 11-ketotestosterone (OT), testosterone (T), 11 beta-hydroxytestosterone (OHT), 11-ketoandrostenedione (OA), and 11 beta-hydroxyandrostenedione (OHA) were measured in the male three-spined stickleback (Gasterosteus aculeatus L). OT was the dominant plasma androgen in the breeding season in summer and was the only androgen that peaked during this period. The levels of OT correlated closely with the development of male secondary sexual characters and reproductive behavior. T and OHT were low in all seasons, whereas OHA and OA displayed the highest levels in early winter. During the postbreeding period, the time of active spermatogenesis, all measured steroids were low. Castration resulted in an almost complete loss of plasma OT and reduced T, whereas OHT, OHA, and OA were not reliably influenced. Androstenedione implants in castrated fish increased plasma T and OA implants increased plasma OT, suggesting a nontesticular site of conversion.

Androgen and behavior in the male three-spined stickleback, Gasterosteus aculeatus I.--changes in 11-ketotestosterone levels during the nesting cycle.

Circulating 11-ketotestosterone (11KT) levels are higher during the courtship phase than during the later parental phase in a number of male teleosts. The present study describes the temporal changes in 11KT levels and their relationships to changes in courtship behavior, after different number of spawnings, over the nesting cycle of the male three-spined stickleback, Gasterosteus aculeatus, a small teleost showing pronounced paternal behavior. Plasma 11KT levels, measured by radioimmunoassay, were approximately 34 times higher during the initial courtship phase than at the end of the following parental phase in spawned males. In addition, males that spawned with three or more females on a single day showed an earlier decline in 11KT levels and in courtship behavior compared to males that were only allowed to spawn with a single female. Plasma 11KT levels remained high in males not allowed to spawn.

Male–male competition and female choice in brown trout

In some salmonid species, the females have been assumed to choose their mates on the size of the males adipose fin. This hypothesis was tested in a stream water aquarium, in which 19 brown trout, Salmo trutta, females were allowed to choose between two males of the same body size but with different adipose fin sizes. The two males were separated from each other in cages. After the female had started to prepare her nest close to one of them, the males were released and allowed to fight each other for the opportunity to spawn. Out of 19 females, 14 prepared a nest closest to the male with the larger adipose fin. However, only six of the 14 females spawned with this male. Males that spawned were more dominant (i.e. were more likely to win fights). When the female spawned with the male she chose, he was less aggressive towards her than when she spawned with the other male. There were no significant differences in the plasma levels of testosterone (T) and 11-ketotestosterone (11-KT) between the chosen males and those not chosen. However, the dominant males had significantly higher plasma levels of T and 11-KT both before and after the experiment. The results support the view that female brown trout exhibit mate choice, but their choice is overruled by male-male competition. Copyright 1999 The Association for the Study of Animal Behaviour.

Serotonin, but not melatonin, plays a role in shaping dominant-subordinate relationships and aggression in rainbow trout

The aim of this study was to clarify to what extent the effects of elevated dietary L-tryptophan (Trp) on aggressive behavior and stress responsiveness in rainbow trout are mediated by circulating melatonin and central serotonin (5-HT), respectively. Isolated rainbow trout were paired for 1h a day for 7 days in order to create fish with experience of being dominant and subordinate. Following this week, the fish were tested for aggressive behavior using a resident-intruder test after which they were subjected to one of four treatments: (1) tryptophan, (2) the selective serotonin reuptake inhibitor (SSRI) citalopram, (3) melatonin, and (4) no treatment (controls). After 7 days of treatment, the fish were subjected to a second resident-intruder test. Trp-supplemented feed resulted in a suppression of aggressive behavior in fish with experience of being dominant. Moreover, fish fed Trp-supplemented feed, regardless of social experience, also displayed lower plasma cortisol levels than controls. These effects of elevated dietary Trp were closely mimicked by citalopram treatment, whereas exogenous melatonin had no effect on either aggressive behavior or plasma cortisol. Thus, the effect of elevated dietary Trp on aggressive behavior and stress responses does not appear to be mediated by melatonin even though elevated dietary intake of Trp resulted in an increase in plasma melatonin concentrations.

Androgens and Behaviour in the Three-Spined Stickleback

In vitro incubations showed 11-ketoandrostenedione (11KA) to be the main androgen produced by the testes of the breeding stickleback. In breeding males the plasma levels of 11-ketotestosterone (11KT) are higher than those of other measured androgens, and display a clear peak in the breeding season. 11 KA can be converted extra-testicularily to 11KT. In breeding females the plasma levels of 11KT are lower than in breeding males, whereas the opposite is the case for testosterone. Castration leads to the disappearence of male reproductive behaviour and secondary sexual characters. 11-androgens are more effective than other androgens in stimulating secondary sexual characters, notably kidney hypertrophy. All aspects of male reproductive behaviour can be restored in castrated males with 11KA capsules. 11KA was more effective than other tested androgens in this respect. The effcctiveness of 11KA on nesting is not influenced by photoperiod.

Detection of the anti-androgenic effect of endocrine disrupting environmental contaminants using in vivo and in vitro assays in the three-spined stickleback

We have previously developed a novel in vitro assay that utilises cultures of primed female stickleback kidney cells for the screening of potential androgenic and anti-androgenic environmental contaminants. Stickleback kidney cells are natural targets for steroid hormones and are able to produce a protein, spiggin, in response to androgenic stimulation. We undertook a combined in vivo/in vitro study where we used the magnitude of spiggin production as an endpoint to test the anti-androgenic properties of the pharmaceutical androgen antagonist flutamide and three environmental contaminants: the organophosphate insecticide fenitrothion, the urea-based herbicide linuron and the fungicide vinclozolin. In vitro, kidney cells were exposed to a range of concentrations [from 10(-14) M (2.5 pg/L) up to 10(-6) M (280 microg/L)] of the test compounds alone for determining agonist activities, or together with 10(-8) M (3 microg/L) dihydrotestosterone (DHT) for determining antagonist activities. An in vivo flow-through aquarium-based study was carried out in parallel. Female sticklebacks were exposed to a range of concentrations of the same chemicals alone or in combination with DHT (5 microg/L) for 21 days. All of the compounds significantly inhibited DHT-induced spiggin production in a concentration-dependent manner in both the in vitro (FN > or = FL > or = LN > VZ) and in vivo (FN > FL > or = VZ > LN) assays. Fenitrothion and flutamide inhibited spiggin production in vitro at a concentration as low as 10(-12) M (P < 0.05), while linuron and vinclozolin inhibited DHT-induced spiggin production at concentrations of 10(-10) M (P < 0.05) and 10(-6) M (P < 0.001) respectively. Similarly, fenitrothion and flutamide were the most potent chemicals in vivo and significantly reduced DHT-induced spiggin production at a concentration of 10 microg/L and 25 microg/L respectively (P < 0.01). Both linuron and vinclozolin induced a significant decrease in DHT-induced spiggin production at a concentration of 100 microg/L when tested in vivo. In addition, kidney cell primary culture was used to test the (anti-)androgenic effects of the major environmental contaminants: oestradiol (E2), nonylphenol (NP) and bisphenol A (BPA) for the first time in teleosts. We observed that these compounds were able to significantly inhibit spiggin production at high doses (E2: 270 microg/L; NP: 2.2 microg/L; BPA: 2.3 microg/L). When tested in the absence of DHT, none of the compounds showed a significant agonistic activity in either in vivo or in vitro assays. Overall, our data further demonstrate that kidney cell primary culture is a reliable and a sensitive screening tool for the detection of (anti-)androgenic compounds. In addition, our study represents the first attempt to develop a combined in vivo/in vitro screening strategy for assessing the effects of (anti-)androgenic endocrine disrupters.