Out-of-season production of 17,20ß-dihydroxypregn-4-en-3-one in the roach Rutilus rutilus.

In this study, although the highest production of two physiologically significant progestins in teleosts [17,20ß-dihydroxypregn-4-en-3-one (17,20ß-P) and 17,20ß,21-trihydroxypregn-4-en-3-one (17,20ß,21-P)] was observed in the period just prior to spawning in both male and female roach Rutilus rutilus, there was also a substantial production (mean levels of 5-10 ng ml(-1) in blood; and a rate of release of 5-20 ng fish(-1) h(-1) into the water) in males and females in the late summer and early autumn (at least 7 months prior to spawning). During this period, the ovaries were increasing rapidly in size and histological sections were dominated by oocytes in the secondary growth phase [i.e. incorporation of vitellogenin (VTG)]. At the same time, the testes were also increasing rapidly in size and histological sections were dominated by cysts containing mainly spermatogonia type B. Measurements were also made of 11-ketotestosterone (11-KT) in males and 17ß-oestradiol and VTG in females. The 3 months with the highest production of 11-KT coincided with the period that spermatozoa were present in the testes. In females, the first sign of a rise in 17ß-oestradiol concentrations coincided with the time of the first appearance of yolk globules in the oocytes (in August). The role of the progestins during the late summer and autumn has not been established.

Variation in the reproductive potential of Schistocephalus infected male sticklebacks is associated with 11-ketotestosterone titre.

Parasites can impact host reproduction by interfering with host endocrine systems, but the adaptive nature of such effects is disputed. Schistocephalus solidus plerocercoids are parasites of three-spined sticklebacks Gasterosteus aculeatus that are often associated with impaired host reproduction. Here, we relate reproductive behavior and physiology to levels of the androgen 11-ketotestosterone (11KT) in naturally infected and non-infected male sticklebacks from two UK populations. In one population infected males harbored heavy infections and showed uniformly reduced 11KT titres and kidney spiggin (nesting glue protein) content compared to non-infected fish. However in a second population infection levels were more variable and males with smaller infections recorded 11KT and spiggin titres that overlapped those of non-infected fish; among infected males from this population 11KT and kidney spiggin also both correlated negatively with infection severity. Male reproductive behavior correlated closely with 11KT titre in both populations, and infected males with high 11KT levels exhibited normal reproductive behavior. Our results suggest that Schistocephalus infection per se does not block reproductive development in male sticklebacks, and that some male fish may have the ability to breed whilst infected. Our results are not consistent with the hypothesis that Schistocephalus adaptively castrates male hosts via endocrine disruption; rather they support the hypothesis that reproductive disruption is a side effect of the energetic costs of infection.

The role of the maturation-inducing steroid, 17,20beta-dihydroxypregn-4-en-3-one, in male fishes: a review.

The major progestin in teleosts is not progesterone, as in tetrapods, but 17,20beta-dihydroxypregn-4-en-3-one (17,20beta-P) or, in certain species, 17,20beta,21-trihydroxy-pregn-4-en-3-one (17,20beta,21-P). Several functions for 17,20beta-P and 17,20beta,21-P have been proposed (and in some cases proved). These include induction of oocyte final maturation and spermiation (milt production), enhancement of sperm motility (by alteration of the pH and fluidity of the seminal fluid) and acting as a pheromone in male cyprinids. Another important function, initiation of meiosis (the first step in both spermatogenesis and oogenesis), has only very recently been proposed. This is a process that takes place at puberty in all fishes and once a year in repeat spawners. The present review critically examines the evidence to support the proposed functions of 17,20beta-P in males, including listing of the evidence for the presence of 17,20beta-P in the blood plasma of male fishes and discussion of why, in many species, it appears to be absent (or present at low and, in some cases, unvarying concentrations); consideration of the evidence, obtained mainly from in vitro studies, for this steroid being predominantly produced by the testis, for its production being under the control of luteinizing hormone (gonadotrophin II) and, at least in salmonids, for two cell types (Leydig cells and sperm cells) being involved in its synthesis; discussion of the factors involved in the regulation of the switch from androgen to 17,20beta-P production that seems to occur in many species just at the time of spermiation; discussion of the effects of in vivo injection and application of 17,20beta-P (and closely related compounds) in males; a listing of previously published evidence that supports the proposed new function of 17,20beta-P as an initiator of meiosis; finally, discussion of the evidence for environmental endocrine disruption by progestins in fishes.

Synthesis of 17,20beta,21-trihydroxypregn-4-en-3-one by ovaries of reproductively mature Atlantic cod Gadus morhua.

Atlantic cod Gadus morhua ovaries were incubated in vitro with tritiated 17-hydroxypregn-4-ene-3,20-dione (17-P) to determine whether 17,20beta-dihydroxypregn-4-en-3-one (17,20beta-P) or 17,20beta, 21-trihydroxypregn-4-en-3-one (17,20beta,21-P), or both, were more likely to be the steroid responsible for inducing oocyte final maturation (i.e. resumption of meiosis). Only 17,20beta,21-P was produced, in addition to 11-deoxycortisol (17,21-P), which is intermediate between 17-P and 17,20beta,21-P. Also, the 5beta-reduced forms of 17-P, 17,21-P and 17,20beta,21-P were all found. Some sulphation of 21-hydroxylated steroids was demonstrated. The ability of female G. morhua to make 17,20beta,21-P but not 17,20beta-P was confirmed by radioimmunoassay of plasma samples from spawning fish. Although small amounts of 17,20beta-P immunoreactivity were detected in a few plasma samples, this was shown, by thin-layer chromatography, to be mostly due to cross-reaction with other unidentified compounds. The evidence strongly suggests that 17,20beta,21-P is more likely than 17,20beta-P to be the maturation-inducing steroid in G. morhua.

Further refinement of the non-invasive procedure for measuring steroid production in the male three-spined stickleback Gasterosteus aculeatus.

Measurement of steroids that are released into the water via the gills has previously been shown to be an effective way of studying the reproductive endocrinology of the male three-spined stickleback Gasterosteus aculeatus without having to kill the fish. In the present paper, a previous observation on the existence of a compound other than 11-ketotestosterone (11-KT) in water, which cross-reacted in the 11-KT radioimmunoassay was repeated. The amounts of this compound, however, were not sufficient to warrant a separation step prior to carrying out assay. The lack of association between androstenedione levels in water and those in plasma was also confirmed. For the first time, the amounts of testosterone released into the water were shown to be positively correlated with the amounts in plasma, the sampling procedure (placing the fish for 30 min in 50 ml water) had no effect on the rate of release of cortisol but caused a rapid drop in the rate of release of 11-KT (which means that the fish should not be sampled twice in short succession), physical interaction between two nesting males (which was accompanied by aggression) significantly increased the rate of release of 11-KT, androstenedione and testosterone (but not of cortisol) and the rate of release of 11-KT was at its maximum between 2 and 4 h after exposure.

Ethoxyresorufin-O-deethylase (EROD) and vitellogenin (VTG) in flounder (Platichthys flesus): system interaction, crosstalk and implications for monitoring.

The extent to which biological systems interact in fish from multi-contaminant areas needs to be understood for full interpretation of monitoring data. This study investigates the interaction between two biomarkers, ethoxyresorufin-O-deethylase (EROD) activity and plasma vitellogenin (VTG) in the European flounder (Platichthys flesus). Flounder were exposed to several waterborne EROD inducers and estrogenic chemicals on their own and in binary combinations. Each experimental exposure was for 10 days. The estrogenic chemicals suppressed PAH-mediated EROD induction. Ethynylestradiol (EE2) and nonylphenol (NP) had threshold concentrations of EROD inhibition similar to those at which they induced VTG production. Estradiol (E2), however, showed an ability to suppress EROD at a concentration much lower than that at which VTG was induced. This established that, although EE2 is a more potent VTG inducer than E2, it is less potent in its ability to inhibit EROD activity. The PAH, dibenz[a,h]anthracene (DbA), showed no effect on the VTG induction caused by EE2 and E2. A small effect was noted with NP at threshold concentrations for VTG induction. Archived data on flounder hepatic EROD activity collected during estuarine monitoring were reassessed in light of the project findings. It is hypothesised that published EROD monitoring data may be an underestimation of effects if it is assumed that estrogen-mediated MFO suppression is occurring in wild populations. A greater understanding of system interaction and other factors, including genetics, that influence biomarker response to contaminants would be required to interpret biomarker monitoring data.

Differential sensitivity of flounder (Platichthys flesus) in response to oestrogenic chemical exposure: an issue for design and interpretation of monitoring and research programmes.

This study was conducted as an initial investigation of 'differential response' in one of the main sentinel organisms used for monitoring programmes in United Kingdom estuaries, the flounder Platichthys flesus. It has been hypothesised that monitoring using species with a wide geographical spread and limited migration, such as flounder, might result in the comparison of different genetic stocks and certainly of populations with differing early life stage contaminant exposure histories. Furthermore, it is probable that these pre-exposure and genetic differences could manifest themselves in an ability to respond differently to contaminant exposure, so-called 'differential response'. It is important that the extent and nature of this response is understood, if we want to be able to fully interpret the monitoring data from such programmes. During this study, flounder were collected from four separate sources; wild caught fish from the estuaries of the Rivers Alde, Mersey and Tyne, and farmed flounder from Port Erin Farm, Isle of Man. Under controlled laboratory conditions, groups of fish from each source were exposed to water-borne concentrations of the synthetic oestrogen ethynylestradiol (EE2) at a nominal concentration of 50 ng/l. Plasma was taken from each male fish after 6 and 10 days exposure and analysed for the presence of vitellogenin (VTG) using an ELISA technique. Significant levels of VTG induction were evident in fish from all sources after both 6 and 10 days exposure. Flounder from the Mersey were the only fish with significantly elevated initial background levels of VTG (day 0) and this appeared to be reflected in that these specimens showed the highest induction response after day 6. However, after day 10, fish from all other sites had a slightly higher mean VTG than those from the Mersey which showed significantly (p < 0.05) lower mean plasma VTG. It is suggested that other differential responses may have been masked by the use of a high dose of EE2 which produced maximum induction in nearly all fish. The findings of the study are discussed in terms of implications for further research into the differential response issue and how the initial plasma VTG figures contribute to a time-series from the Mersey, Tyne and Alde estuaries.

Development and validation of a radioimmunoassay for peptides related to beta-melanocyte-stimulating hormone in human plasma: the lipotropins.

A radioimmunoassay is described for the measurement of human "beta-melanocyte-stimulating hormone" ("betah-MSH"). Two antisera have been used, one of which cross-reacts with synthetic betah-MSH as well as with the two larger pituitary peptides betah- and gammah-lipotropin (betah- and gammah-LPH) and the other mainly with betah-MSH and gammah-LPH. The sensitivity and reliability of the assay have been improved by employing a simple plasma extraction procedure, and the shelf-life of the iodinated betah-MSH tracer has been increased more than five-fold by storage in a concentrated human serum albumin solution. Using a 5 ml plasma sample the detection limit is 6 pg/ml. The mean resting "betah-MSH" level in normal subjects is 21 pg/ml (range 13-38 pg/ml) at 9 AM and 12 pg/ml (range 6-20 pg/ml) at 9 PM. Levels are considerably elevated (51-12,000 pg/ml) in patients with Addison's disease. Nelson's syndrome, Cushing's disease and the "ectopic" ACTH syndrome. After administration of insulin or pyrogen, the concentration of plasma "betah-MSH" increases in parallel with that of ACTH and they are approximately equivalent on a molar basis. The stability of purified betah- and gammah-LPH and endogenous "betah-MSH" when incubated in vitro in fresh blood or plasma are similar, in contrast to the less stable peptide synthetic betah-MSH. It is suggested that "betah-MSH" immunoreactivity in human plasma is due to betah- and gammah-LPH rather than betah-MSH.

17 alpha,20 beta-dihydroxy-4-pregnen-3-one 20-sulphate is a potent odorant in precocious male Atlantic salmon (Salmo salar L.) parr which have been pre-exposed to the urine of ovulated females.

Electrophysiological recordings from the olfactory epithelium have shown that 17 alpha,20 beta-dihydroxy-4-pregnen-3-one 20-sulphate (17,20 beta-P-sulphate; a conjugate of the oocyte-maturation-inducing steroid in teleosts) is a potent odorant in precocious male Atlantic salmon (Salmo salar L.) parr. However, the olfactory epithelium of these fish only appeared to be responsive to the steroid after stimulation with the urine of ovulated female Atlantic salmon. Immature fish did not respond at any time. Stimulation with urine from immature and precocious male Atlantic salmon parr did not make the olfactory epithelium of precocious male salmon parr responsive to the steroid. 17,20 beta-P-sulphate was found in the urines of ovulated females, precocious male parr and mature male Atlantic salmon. The findings are discussed in relation to the possible role of 17,20 beta-P-sulphate in the physiology of Atlantic salmon.

Conjugates of ovarian steroids, including 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (maturation-inducing steroid), accumulate in the urine of a marine teleost (plaice; Pleuronectes platessa).

Free and conjugated 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P), 17 alpha,21-dihydroxy-4-pregnene-3,20-dione (11-deoxycortisol) and 3 alpha,17 alpha,21-trihydroxy-5 beta-pregnan-20-one (3 alpha,17,21-P-5 beta) were measured by radioimmunoassay in matching blood plasma and urine samples from plaice (Pleuronectes platessa) females at several ovarian maturity stages: post-vitellogenesis (IV), final oocyte maturation (V), and ovulation (VI). Free steroids were generally low in all samples. Conjugated steroids were up to 2 orders of magnitude higher in urine than in plasma samples. Conjugated 17,20 beta-P was higher in stage V than in stage IV or VI females. Conjugated 11-deoxycortisol was higher in stage IV and V females. Conjugated 3 alpha,17,21-P-5 beta was higher in stage V and VI females. These results support earlier studies which indicated that 17,20 beta-P was the most likely maturation-inducing steroid (MIS) in plaice, and that the urine might be a vehicle for steroid pheromones synthesized by the gonads.

Incorporation of 14C-labelled amino acids into corticotrophin-like intermediate lobe peptide and alpha-melanocyte-stimulating hormone by the rat pituitary neurointermediate lobe in vitro, and the identification of four new pars intermedia peptides.

At least seven radioactive peptides, which fractionated on Biogel P6, were found in rat neurointermediate lobes after incubation for 6 h with [14C]proline. Only three of these could be tentatively identified; one as alpha-melanocyte-stimulating hormone (alpha-MSH) and two as forms of corticotrophin-like intermediate lobe peptide (CLIP). One other cross-reacted partially with a beta-malanocyte-stimulating hormone (beta-MSH) antiserum, was acidically charged and eluted on Biogel P6 in roughly the same position as ACTH. The other three peptides showed no resemblance to alpha-MSH, CLIP, beta-MSH or ACTH.

Light inactivation of metronidazole sensitivity discs.

We found that daylight inactivated metronidazole sensitivity discs. They should therefore be stored in light-proof containers.

ACTH release during passive avoidance behavior.

ACTH was measured by radioimmunoassay during learning and retention of a passive avoidance response. Electric footshock, used during the learning trial, appeared to be a weaker stimulus for ACTH release than was the retention test. Moreover high latency scores during retention were associated with high plasma ACTH levels, whereas shorter latencies were associated with lower levels. The results indicate that psychological mechanisms organizing behavioral coping are important in the response of the pituitary-adrenocortical system to stimuli which are related to a previous adversive experience.

The extent of oestrogenic contamination in the UK estuarine and marine environments--further surveys of flounder.

In 1996, The Centre for Environment, Fisheries and Aquaculture Science (CEFAS) initiated a project to establish whether oestrogenic materials are present in UK estuarine and marine waters at biologically significant concentrations, and to investigate some of the possible effects which these may have in flounder (Platichthys flesus). Early results are published elsewhere; this paper describes the results of a second wider survey of vitellogenin and reproductive abnormalities in UK flounder. Vitellogenin levels in male blood plasma in the period from spring to winter 1997 were found to be significantly elevated (in comparison with a clean reference site on the Alde estuary) in at least one sample from most of the 11 estuaries investigated. The exceptions were the Tamar and the Dee where all fish appeared entirely normal. In broad terms, the degree of oestrogenic contamination as measured by male vitellogenesis in the various estuaries was ranked in the following descending order: Tees > Mersey > Tyne > Wear = Humber = Clyde = Southampton Water = Thames > Dee = Tamar. VTG concentrations in Tees, Mersey and Tyne male fish were extremely high (> 100,000 ng/ml), and often exceeded those normally found in sexually mature females. At most locations, ovotestis conditions in male flounder were entirely absent but 9% of male Mersey fish and 7% of male Tyne fish contained ovotestis. In a few cases, eggs were fully developed with yolk granules. Most testes did not show gross morphological abnormalities related to oestrogenic exposure, although one testis from a Mersey fish appeared to be almost entirely composed of eggs. Abnormal sex ratios were not seen in any estuary. The paper concludes by outlining a new research programme which will be addressing the biological significance of these observations.

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.

A comparison of alprazolam with amitriptyline in the treatment of patients with neurotic or reactive depression. A report of a randomised, double blind study by a General Practitioner Working Party.

104 patients suffering from neurotic or reactive depression were treated with either alprazolam or amitriptyline in randomised, double-blind fashion. Seventeen patients were either lost to follow-up or withdrawn before week 2 (13 due to side effects and 1 because she was feeling better). A further 7 patients did not comply with the protocol, giving a total of 24 patients whose data were not considered suitable for inclusion in the analysis of therapeutic assessments. Evaluation of the 80 patients (40 in each group) who completed at least 2 weeks of the 4-week study demonstrate that both treatments produced a statistically significant response rate. There was a more rapid effect in those patients who received amitriptyline, but there was no significant difference in response between the treatment groups after 4 weeks treatment. Analysis of safety and side effect data on 101 patients (50 treated with alprazolam and 51 with amitriptyline) shows no statistically significant difference in the overall number of side effects experienced in each group, although 11 of those patients who received amitriptyline withdrew because of adverse reactions before completing the study compared to 6 in the alprazolam group. These results suggest that alprazolam may be a useful treatment for patients with neurotic or reactive depression not requiring hospitalisation.

Anti-androgens act jointly in suppressing spiggin concentrations in androgen-primed female three-spined sticklebacks - prediction of combined effects by concentration addition.

Increasing attention is being directed at the role played by anti-androgenic chemicals in endocrine disruption of wildlife within the aquatic environment. The co-occurrence of multiple contaminants with anti-androgenic activity highlights a need for the predictive assessment of combined effects, but information about anti-androgen mixture effects on wildlife is lacking. This study evaluated the suitability of the androgenised female stickleback screen (AFSS), in which inhibition of androgen-induced spiggin production provides a quantitative assessment of anti-androgenic activity, for predicting the effect of a four component mixture of anti-androgens. The anti-androgenic activity of four known anti-androgens (vinclozolin, fenitrothion, flutamide, linuron) was evaluated from individual concentration-response data and used to design a mixture containing each chemical at equipotent concentrations. Across a 100-fold concentration range, a concentration addition approach was used to predict the response of fish to the mixture. Two studies were conducted independently at each of two laboratories. By using a novel method to adjust for differences between nominal and measured concentrations, good agreement was obtained between the actual outcome of the mixture exposure and the predicted outcome. This demonstrated for the first time that androgen receptor antagonists act in concert in an additive fashion in fish and that existing mixture methodology is effective in predicting the outcome, based on concentration-response data for individual chemicals. The sensitivity range of the AFSS assay lies within the range of anti-androgenicity reported in rivers across many locations internationally. The approach taken in our study lays the foundations for understanding how androgen receptor antagonists work together in fish and is essential in informing risk assessment methods for complex anti-androgenic mixtures in the aquatic environment.