Using blood plasma for monitoring organochlorine contaminants in juvenile white sturgeon, Acipenser transmontanus, from the lower Columbia River.

Organochlorine (OC) pesticide concentrations in blood plasma samples from 88 juvenile white sturgeon collected from the lower Columbia River were measured and compared to plasma sex steroid and OC tissue levels previously measured in corresponding fish. Significant squared correlation coefficients between summation operator DDT concentrations in sturgeon plasma and gonads and livers were 0.37 and 0.32, respectively. Significant negative correlations between plasma testosterone concentration and plasma Sigma DDT concentration in male fish (r(2)=0.26), plasma 17beta estradiol concentration and plasma Sigma DDT concentration in female fish (r(2)=0.38) and condition factor and plasma Sigma DDT concentration in all fish were found (r(2)=0.17). These results suggest that blood plasma may be a suitable nondestructive method for monitoring adult sturgeon population for persistent OC contaminants.

Mercury concentrations in gonad, liver, and muscle of white sturgeon Acipenser transmontanus in the lower Columbia River.

This study determined the partitioning of total mercury in liver, gonad, and cheek muscle of white sturgeon (Acipenser transmonatus) in the lower Columbia River. The relationship between tissue mercury concentrations and various physiologic parameters was assessed. White sturgeon were captured in commercial fisheries in the estuary and Bonneville, The Dalles, and John Day Reservoirs. Condition factor (CF), relative weight (Wr), and gonadosomatic index (GSI) were determined for each fish (n = 57). Gonadal tissue was examined histologically to determine sex and stage of maturity. Liver (n = 49), gonad (n = 49), and cheek muscle (n = 57) were analyzed for total mercury using cold-vapor atomic fluorescence spectrophotometry. Tissue protein concentrations were measured by ultraviolet-visible spectroscopy. Plasma was analyzed for testosterone (T), 11-ketotestosterone (KT), and 17ss-estradiol (E2) using radioimmunoassay. Mean tissue mercury concentrations were higher in muscle compared with liver and gonad at all sampling locations, except Bonneville Reservoir where mean liver mercury content was the highest tissue concentration observed in the study. Significant negative correlations between plasma androgens (T and KT) and muscle mercury content and plasma E2 and liver mercury content were found. A significant positive linear relationship between white sturgeon age and liver mercury concentrations was evident. Significant negative correlations between CF and relative weight and gonad and liver mercury content were found. In addition, immature male sturgeon with increased gonad mercury content had decreased GSIs. These results suggest that mercury, in the form of methylmercury, may have an effect on the reproductive potential of white sturgeon.

Plasma androgen correlation, EROD induction, reduced condition factor, and the occurrence of organochlorine pollutants in reproductively immature white sturgeon (Acipenser transmontanus) from the Columbia River, USA.

White sturgeon (Acipenser transmontanus) support an active fishery in the Columbia River, but there is poor reproductive success within the impounded sections. The poor reproductive success has been attributed to hydroelectric development; however, water pollution could be a significant factor. White sturgeon plasma, liver, and gonad samples were collected from four Columbia River locations and a California aquaculture facility. Total length and weight of the fish were measured, and plasma samples were analyzed for testosterone (T), 11-ketotestosterone (KT), 17 beta-estradiol (E2), and vitellogenin. Liver samples were analyzed for chlorinated pesticides and polychlorinated biphenyls, ethoxyresorufin-O-deethylase (EROD) activity and histopathology. Gonads were examined histologically to assess sexual maturity and characterize any lesions. Significant differences by location existed for p,p'-DDE, EROD activity, and condition factor. Plasma T was negatively correlated with p,p'-DDE in males and females, and plasma KT was negatively correlated in males. These data indicate that pollutants could be adversely affecting white sturgeon in the Columbia River basin.

Effects of stress on the reproductive performance of rainbow trout (Oncorhynchus mykiss).

We investigated the effects of stress over the final stages of sexual maturation on the reproductive performance of female rainbow trout, Oncorhynchus mykiss. Stress was administered over the period of early vitellogenesis (1.5 mo), late vitellogenesis-final maturation (1.5 mo), or during both periods (3 mo). Each stress treatment and control was triplicated, with eight females in each replicate (n = 24 fish per treatment). The eggs and progeny of each female were kept separate, and observations were made for 4 mo after transfer to rearing tanks. Fish that experienced stress during final maturation and those that were under stress during the whole experiment ovulated on average 2 wk earlier than the control group. In contrast, fish stressed during the period of early vitellogenesis ovulated at the same time as controls. Absolute fecundity and fertilization were not significantly affected in any treatment group, but significant differences in relative fecundity were found. Stress applied early in vitellogenesis resulted in smaller eggs and swim-up fry. No significant differences were found in juvenile weight 8 wk after hatching. Furthermore, we found no differences in survival of the progeny or resistance to the fish pathogen Vibrio anguillarum. Thus, mild acute stresses applied to rainbow trout females may affect certain reproductive performance parameters such as timing of ovulation and relative fecundity; however, the progeny of such stressed females perform as well as controls with regard to juvenile growth and disease resistance.

Endogenous steroid metabolism is indicated by fluctuations of endogenous steroid and steroid glucuronide levels in early development of the steelhead trout (Oncorhynchus mykiss).

Concentrations of endogenous steroids and their glucuronide conjugates fluctuated during early development in steelhead trout Oncorhynchus mykiss. Whole body content of sex steroids and steroid glucuronides of both bisexual and gynogenetic (all female) steelhead trout were quantified by radioimmunoassay. Concentrations of 17beta-estradiol (E2) and cortisol increased 2-4 days before hatch. Two days after hatch, 11-ketotestosterone (11KT) increased in concentrations in both gynogenetic and bisexual populations, and 11KT glucuronide concentrations increased in the gynogenetic population. Testosterone (T) and E2 concentrations were at their lowest at 39 days postfertilization (dpf) for T and 39 and 61 dpf for E2. Changes in levels of steroid glucuronides were not consistently parallel to free steroids through time. T-, E2-, and 17alpha, 20beta dihydroxyprogesterone glucuronides declined slower than their free forms. Based on fluctuating concentrations of all steroid glucuronides, both populations of fish demonstrated an ability to form glucuronide conjugates of all steroids at the embryonic stage. The changes in levels of both free steroids and their glucuronides during early development of the trout indicate that steroid metabolism is active during development. This study also implicates steroid metabolism as an integral part of embryonic and postembryonic development.

In vivo steroid metabolism in embryonic and newly hatched steelhead trout (Oncorhynchus mykiss).

Radioactive pregnenolone (P5), testosterone (T), or 17-beta-estradiol (E2) was microinjected into steelhead trout, Oncorhynchus mykiss, embryos and newly hatched yolk-sac fry (alevins) to detect in vivo metabolism. We also assayed the water used to incubate animals for 10 hr after microinjection to detect possible metabolite excretion. High pressure liquid chromatography and thin layer chromatography were used to separate and tentatively identify steroid metabolites. Metabolites of P5 were androstenedione (AN), E2, T, and glucuronides of E2 and T in embryos and AN, E2, progesterone, 17-alpha, 20-beta-dihydroxyprogesterone, and P5 glucuronide in alevins. E2 and its glucuronide were synthesized from precursor T in the embryos and alevins; however, the amounts of E2 and E2 glucuronide synthesized in the embryos were 10 and 3 magnitudes greater than those detected in alevins. Testosterone glucuronide was synthesized in similar amounts in both stages of development. Embryos did not synthesize free metabolites from E2 precursor, but E2 glucuronide was detected from E2 precursor. Estradiol in alevins was metabolized into unidentified free and glucuronide-conjugated steroids. Three unknown metabolites synthesized from P5 precursors and seven unknown substances produced in animals injected with testosterone or estradiol precursors were detected. Free metabolites were detected in the incubation water that held the animals (embryos and alevins) for 10 hr after microinjection with T or E2. Glucuronide metabolites were not excreted by embryos into the incubation water 10 hr after microinjections with any of the steroid precursors; however, alevins excreted glucuronides into the incubation water when supplied with precursor T. These results imply that endogenous steroid metabolism of maternally contributed steroids is active during early development.

Characterization of a glucocorticoid receptor in the brains of chinook salmon, Oncorhynchus tshawytscha.

The effects of chronic stress on the brains of salmon may have important implications in light of the extremely high glucocorticoid levels experienced by migrating and spawning adults. The identification and the characterization of glucocorticoid receptors in salmon brains are the first steps in elucidating the effects of stress and high glucocorticoid levels on the brain. We have identified high-affinity, low capacity glucocorticoid receptors in chinook salmon (Oncorhynchus tshawytscha) brain cytosol and report the binding characteristics for the synthetic glucocorticoid triamcinolone acetonide (TA) and the naturally occurring salmonid glucocorticoid, cortisol. The binding characteristics for TA (Kd = 0.85 +/- 0.13 nM, B(max) = 22.4 +/- 2.97 fmol/mg protein, n = 7) and cortisol (Kd = 4.54 +/- 0.06 nM, B(max) = 25.40 +/- 2.20 fmol/mg protein, n = 2) demonstrated high-affinity, low capacity, and specificity for glucocorticoids. In competitive binding assays, TA, cortisol, and dexamethasone (a synthetic glucocorticoid) displaced [3H]TA most effectively, followed by RU38486. Corticosterone and RU28362 were weaker competitors. Cortisone was not a strong competitor nor were the sex steroids. Specific DNA binding was detected in DNA-cellulose chromatography assays. Receptors in nuclear extracts were not detected. These binding characteristics are consistent with published data on glucocorticoid receptors in other salmonid tissues.

Characterization of an androgen receptor in salmonid lymphocytes: possible link to androgen-induced immunosuppression.

Cytosol of rainbow trout (Oncorhynchus mykiss) leukocytes demonstrated specific and saturable binding of [3H]testosterone (Kd = 0.99 +/- 0.17 nM and Bmax = 30.4 +/- 4.9 fmol/mg protein; based on a total of 6 determinations from three different cytosolic pools). Specific binding of [3H]testosterone was high in leukocytes and other tissues with known androgen binding affinity (plasma, skin, and liver) and low in other tissues (heart, muscle, and red blood cells). Specific binding of [3H]testosterone was displaced by testosterone and dihydrotestosterone. Androstenedione displaced 50% of specifically bound [3H]testosterone between 10- and 100-fold excess, while 17-alpha-methltestosterone, 11-ketotestosterone, and progesterone displaced 50% of specifically bound [3H]testosterone between 100- and 500-fold excess. Cortisol, 17 beta-estradiol, 17 alpha,20 beta-dihydroxyprogesterone, the synthetic androgen mibolerone, and the synthetic estrogen ethenylestradiol did not displace [3H]testosterone binding, even at 500-fold excess. Treatment of cytosol with proteolytic enzyme significantly reduced the specific binding of [3H]testosterone. HPLC analysis determined that [3H]testosterone was not metabolized during assay incubation with cytosol. These data strongly suggest that androgen receptors exists in salmonid leukocytes and support the hypothesis that these receptors play a role in androgen induced immunosuppression.

Binding characteristics of an androgen receptor in the ovaries of coho salmon, Oncorhynchus kisutch.

Androgen receptors were identified in the cytosol from ovaries of juvenile coho salmon, Oncorhynchus kisutch. The binding for the synthetic androgen mibolerone was specific and saturable (Kd = 0.32 +/- 0.02 nM; Bmax = 15.31 +/- 4.31 fmol/mg protein). Bound [3H]mibolerone was much higher in ovarian cytosol than in cytosolic extracts from heart, liver, and muscle. [3H]mibolerone specific binding was 50% lower in the plasma than in the ovarian cytosolic extracts. [3H]mibolerone binding was displaced most effectively by those 17 alpha-methylated synthetic androgens (mibolerone, methyltestosterone, methylandrostanolone) that can induce functional masculinization in fish. The naturally occurring androgens 11-ketotestosterone and 5 alpha-dihydrotestosterone both displaced [3H]mibolerone binding, but they were 10- to 100-fold less effective than the 17 alpha-methylated androgens. Testosterone, 11 beta-hydroxyandrostenedione, estradiol, progesterone, and 17 alpha,20 beta-dihydroxyprogesterone were not potent competitors. [3H]mibolerone specific binding was reduced after preincubation with trypsin. About 25% of the binding in the cytosolic extract had DNA binding affinity under experimental conditions. The characteristics of this androgen binding site are consistent with a model of receptor-mediated steroid-induced sex inversion.

In vitro steroid secretion during early development of mono-sex rainbow trout: sex differences, onset of pituitary control, and effects of dietary steroid treatment.

Sex differentiation in many teleost species can be controlled by treatment with steroids. To investigate the development of steroidogenesis during both natural and controlled sex differentiation, the production of androstenedione, testosterone, and estradiol were determined in tissues from populations of all-female and all-male rainbow trout (Oncorhynchus mykiss). At various times from hatching through gonadal sex differentiation, explants of steroidogenic tissues were incubated in vitro alone or in the presence of partly purified salmon gonadotropin and the resulting media were assayed for steroids. Androstenedione and testosterone were produced at higher levels in media from testes than from ovaries within 2 weeks of the onset of feeding (before any dramatic gonadal differentiation). Gonadal estradiol secretion was nondetectable until about 1 month after the onset of feeding when females produced more than males. Gonadotropin stimulated gonadal steroid production only after differentiation, but stimulated anterior kidney (interrenal) production of androstenedione much earlier in development. Dietary treatment of rainbow trout with either estradiol or 17 alpha-methyltestosterone (MT) inhibited in vitro gonadal steroid production and this effect persisted in MT-fed fish even after withdrawal of dietary steroids.

Evidence for ultra-short-loop feedback in ACTH-induced interrenal steroidogenesis in coho salmon: acute self-suppression of cortisol secretion in vitro.

Interrenal tissues from coho salmon (Oncorhynchus kisutch) were incubated in a defined medium under blood-gas atmosphere at 17 degrees. Rates of cortisol secretion by tissues incubated in media containing 50 mU/ml porcine-ACTH were initially much greater than those of resting tissues in hormone-free media, but after 3 to 6 hr returned to resting rates. The time course of cortisol accumulation in ACTH-containing media was the same when tissues were incubated in different volumes; the final concentrations of cortisol in these incubations were similar to each other and resembled peak in vivo concentrations in juvenile coho subjected to acute stress. Cortisol secretion rates of tissues sequentially transferred to fresh ACTH-containing media every 6 hr did not return to resting levels but remained elevated for at least 24 hr. Cortisol secretion in response to ACTH was attenuated or completely abolished in tissues incubated in media containing exogenous cortisol; this effect was reversible and dose-dependent. Our results suggest that in coho salmon, cortisol may exert ultra-short-loop negative feedback directly at the level of the interrenal gland to effect self-suppression.

Sex steroid profiles of coho salmon (Oncorhynchus kisutch) during early development and sexual differentiation.

Sex steroids were measured by radioimmunoassay in whole-body extracts of coho salmon, Oncorhynchus kisutch, during early development and sexual differentiation. Profiles were developed for fish from the time of fertilization until 87 days postfertilization (dpf) for six steroids: testosterone (T), 11-ketotestosterone (KT), androstenedione (A), progesterone (P4), 17 alpha-hydroxy-20 beta-dihydroprogesterone (DHP), and 17 beta-estradiol (E2). Ovarian fluid was also examined for steroid content. Steroid profiles of unfertilized eggs essentially paralleled those of ovarian fluid. In one experiment, steroids in developing embryos declined precipitously after fertilization until 30 dpf; at hatching, all steroids increased slightly and then declined during yolk sac absorption. Results from a second experiment basically supported those of the first except that only testosterone increased at the time of hatching. Bimodality was evident in the data on steroid levels for fish collected between 42 and 56 dpf and again after 87 dpf. The hormone levels generally decreased or remained constant after the onset of exogenous feeding. Histological analyses during the first experiment showed the presence of undifferentiated gonads between hatching and 70 dpf, but by 77 dpf ovarian development was evident. In the second experiment, in which fish were more frequently sampled for histological analysis, undifferentiated gonads were present from hatching to 59 dpf. Development of oogonia was observed between 66 and 73 dpf and by 75 dpf ovarian development could be easily discerned. The sex of fish sampled at 101 dpf was determined by examining gonadal morphology, and steroid levels of those fish were determined. A sexual dimorphism was apparent in levels of T, KT, and A, but not of DHP or E2. The dynamics of steroid content of developing coho salmon at hatch, coupled with their bimodal distributions during yolk sac absorption, may suggest a role of sex steroids in the process of sexual differentiation apparent later in development. Changes in whole-body steroid levels at hatch may also be indicative of the onset of sexual differentiation even though no signs of gondal differentiation were histologically discernible at that time.

Regulation of the interrenal of fishes: non-classical control mechanisms.

The regulation of the interrenal of teleostean fishes is reviewed from the perspective of non-classical control mechanisms and new evidence is presented suggesting gonadotropic control of the interrenal. Cortisol secretion by the interrenal, in addition to regulation by ACTH, appears to be mediated by other hormones. Physiologically relevant, direct control of interrenal function by hydromineral factors is unclear.In vitro experiments with interrenals of coho salmon (Oncorhynchus kisutch) indicate that salmon gonadotropin is extremely corticotropic and both ACTH and gonadotropin stimulate the secretion of large quantities of androstenedione from the interrenal.

Profiles of plasma sex steroids and gonadotropin in coho salmon, Oncorhynchus kisutch, during final maturation.

The plasma concentrations of estradiol-17 alpha (E2), 17 mu-hydroxy-20 beta-dihydroprogesterone (DHP), progesterone, 11-ketotestosterone (11-KT), testosterone (T), and gonadotropin (GtH) were measured in adult coho salmon (Oncorhynchus kisutch) concomitantly with advancing maturation of the gonads during the spawning season. Concentrations of E2 were higher in females with eggs showing central (premigrating) germinal vesicles, migrating germinal vesicles, or peripheral germinal vesicles than in fish in which the eggs had undergone germinal vesicle breakdown or ovulation. Conversely, plasma levels of DHP were low at central, migrating, and peripheral germinal vesicle stages and then increased dramatically at germinal vesicle breakdown and ovulation. Levels of 11-KT, T, and GtH generally increased with advancing maturity of the eggs. When time rather than degree of maturation was used as a variable, plasma E2 was high in females first returning from the ocean but then dropped off precipitously in fish sampled at the end of the run. Plasma DHP followed the opposite pattern, being low in fish sampled early in the run and high in fish sampled late in the run. No easily discernible patterns emerged from the profiles of the other steroids and GtH, although some significant variation in the concentrations occurred. Even though sampling was initiated at the time when the salmon first returned from the ocean, most of the hormones were not variable in males when viewed according to sampling date or predominant cell type in the testes, which indicates that the males were extremely close to final maturity during the sampling period. Plasma levels of DHP were higher in males that were producing milt than in any other group of males. Concentrations of 11-KT were higher than those of T in all males, but no patterns in the levels of either steroid emerged.