Plasma levels of C18-, C19- and C21-steroids in captive and feral female sea bass

Morphometric analysis of the gonads of sea bass Dicentrarchus labrax revealed that captive fish matured 1 month later than feral fish, but levels of gonadal steroids were identical in both groups at the same stage of sexual development. 17β-oestradiol (E₂) (up to 3 ng ml-1) and testosterone (T) (up to 4 ng ml-1) were highest during the gametogenetic period while 17,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P) (free and sulphated) were maximal during the spawning period. Free 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) was very low and did not change (c. 0·5 ng ml⁻¹) while 17,20β-P-sulphate increased during the spawning period in both groups (up to 2 ng ml⁻¹). In contrast cortisol levels were higher in captive fish and increased during the spawning period (up to 100 ng ml⁻¹). These results suggest that captivity delays vitellogenesis and spawning in sea bass without affecting the final levels of the gonadal steroids and further indicates a role for cortisol in the latter period. The increased levels during the spawning period suggests a pheromonal role for 17,20β-P-sulphate and 17,20β,21-P-conjugates and the involvement of 17,20β,21-P in final ooccyte maturation.     

Luteinizing hormone and sexual steroid plasma levels after treatment of European sea bass with sustained-release delivery systems for gonadotropin-releasing hormone analogue

Spermiating male European sea bass Dicentrarchus labrax were treated with gonadotropin-releasing hormone agonists (GnRHa), either a GnRHa injection (IN; 25 μg kg⁻¹ body mass) or one of three types of controlled-release GnRHa-delivery systems: fast release implants (EVAc; 1OO μg kg⁻¹), slow release implants (EVSL; lOO μg kg⁻¹) and slow release microspheres (MC; 50 μg kg⁻¹). Luteinizing hormone (LH) release was highly stimulated by all GnRHa treatments, with elevated plasma levels lasting for 2 days in injected fish (IN) and 2, 4 and 6 weeks in controlled-release-treated fish (EVAc, MC and EVSL, respectively), correlating with a 1, 3, 5 and 5 week period of stimulation of milt production, respectively. Plasma levels of the androgens testosterone (T) and 11-ketotestosterone (11-KT), were not significantly affected by the GnRHa treatments. Plasma T was high at early spermiation and declined sharply near the end of this period. Plasma 11-KT levels declined continuously throughout the experiment. Levels of 17,20 β -dihydroxy-4-pregnen-3-one (17,20 β -P), a proposed maturation-inducing steroid (MIS) in European sea bass, fluctuated around 0.2–1 ng ml⁻¹ and were not greatly affected by the treatments. These results indicated a close correlation between sustained stimulation of LH release, achieved by GnRHa-delivery systems, and long-term enhancement of milt production. They also show an absence of changes in the common sex steroids, associated with elevated LH and enhanced spermiation.     

Stress-induced changes in concentrations of plasma sex steroids in black bream

Cortisol levels of black bream Acanthopagrus butcheri at capture did not change with time of day, gonadal stage or season and were 1·9±0·2 and 2·8±0·4 ng ml⁻¹ for male and female fish, respectively. Confinement resulted in significantly elevated cortisol levels at all time periods; however, levels after 24 h of confinement were significantly lower than peak cortisol levels (15 min for males and 1 h for females). Confinement stress resulted in reduced levels of 17β-oestradiol (E2) and testosterone (T) within 1 h in sexually mature females. In mature males, suppression of T and 11-ketotestosterone (11KT) occurred after 30 min and 6 h of confinement, respectively. The relationship between confinement stress and levels of 17,20β-dihydroxy-4-pregnen-3-one (17,20β P) was more complex, with levels in males being elevated after 15 min and 24 h and suppressed after 6 h of confinement. In contrast, 17, 20β P levels in females were elevated after 1 h of confinement. In regressed females, plasma E₂ and T concentrations were low at capture and were not affected by confinement stress whereas plasma 17, 20β P was elevated within 1 h. This study indicates that stress exerts a rapid inhibitory effect on gonadal steroidogenesis.     

Plasma and ovarian thyroxine levels in relation to sexual maturation and gestation in female Sebastes inermis

Plasma T₄ (L-thyroxine) concentrations in the viviparous rockfish Sebastes inermis showed a peak (119·3±27·8 ng ml⁻¹) during the development of embryos. Ovarian T₄ concentrations increased during vitellogenesis and final maturation and decreased during embryogenesis.However, total T₄ content within the ovary increased continuously up to the larval stages. Plasma oestradiol-17β, (E₂) concentrations peaked at the final maturation stage and then recovered to the level seen during the non-reproductive periods. Plasma testosterone (T) concentrations showed a peak (5·33±1·08 ng ml⁻¹) at the embryo stage and high levels were maintained throughout the gestation period. Plasma T₄ concentrations during the embryo stage and ovarian T₄ content during vitellogenesis were much higher than the levels reported in oviparous fishes. These data suggest that in viviparous rockfish, T₄ may be required for sustaining gestation or embryo development within the maternal body. In addition, the high content of total T₄ in the ovary implies that there probably does exist a maternal-embryo relationship during gestation as well as during vitellogenesis.     

The role of F-series prostaglandins as reproductive priming pheromones in the brown trout

Electrophysiological studies demonstrated that the olfactory epithelium of mature male brown trout Salmo trutta parr was acutely sensitive to F-series prostaglandins (PGFs) PGF_1α and PGF_2α, with detection threshold concentrations of 10⁻¹¹ M. The olfactory epithelium was also sensitive to the PGF metabolite 15-ketoPGF_2α (threshold 10⁻⁸ m), but did not detect a further metabolite, 13,14,-dihydro-15-ketoPGF_2α Immature brown trout did not detect any of the prostaglandins tested. Exposure of mature male brown trout parr to waterborne PGF_1α and PGF_2α (concentration 10⁻⁸ m), resulted in significant increases in levels of expressible milt and the plasma concentrations of 17,20β-dihydroxy-4-pregnen-3-one, testosterone and 11-ketotestosterone. The olfactory epithelium of both immature and mature male brown trout parr was sensitive to the urine and ovarian fluid from ovulated female brown trout. Exposure of mature male brown trout parr to ovarian fluid resulted in an increase in the levels of plasma 17,20β-dihydroxy-4-pregnen-3-one whilst exposure to urine increased the levels of expressible milt. In addition, PGF_2α was found to be present within both the urine and ovarian fluid of mature female brown trout. It is suggested that the F-series prostaglandins have a role as priming pheromones in male brown trout.     

Evaluation of biochemical methods for the non‐destructive identification of sex in upstream migrating salmon and sea trout

Female‐specific markers of reproductive activity [plasma 17β‐oestradiol (E2), vitellogenin (VTG) and alkali‐labile phosphoprotein phosphorous (ALP)] were measured over 12 months in a captive population of brown trout Salmo trutta . During the early months of the reproductive season (February to May) and using the concentration of plasma E2 or plasma ALP as a marker for females the proportion of fish in which sex was misidentified was high (15–50%). The misidentification rate was considerably lower (1–8%) using plasma VTG. Preliminary evaluation of a commercial immunochromatographic VTG test system as a screen for the presence or absence of VTG in plasma from brown trout provided results that were consistent with those obtained from direct measurement of plasma VTG levels by enzyme‐linked immunosorbent assay (ELISA). These preliminary conclusions were verified by sampling upstream‐migrating anadromous brown trout, sea trout, and Atlantic salmon Salmo salar trapped over a 6 month period. Plasma E2 levels did not satisfactorily discriminate between male and female sea trout and Atlantic salmon. Plasma VTG levels in both species, however, were bimodally distributed and it was assumed that this divergence corresponded to male (plasma VTG levels <10 μg ml⁻¹) and female (plasma VTG levels >800 μg ml⁻¹) fishes. Plasma ALP provided a more accurate indication of sex in the wild Atlantic salmon and sea trout than was suggested by the pilot study on captive brown trout. The commercial immunochromatographic VTG test system provided results that were wholly consistent with the data obtained from the trapped fishes by direct measurement of plasma VTG.     

Antibacterial activity of anacardic acid and totarol, alone and in combination with methicillin, against methicillinresistant Staphylococcus aureus

The inhibitory and bactericidal activities of anacardic acid and totarol, alone and in combination with methicillin, were investigated against methicillin-resistant Staphylococcus aureus (MRSA). The growth of two MRSA strains was inhibited by 6·25 μg ml^-1 of anacardic acid and 0·78 μg ml^-1 of totarol. The time-kill curve study showed that these two compounds were bactericidal against MRSA. Anacardic acid killed MRSA cells more rapidly than totarol, and no viable cells were detected after being exposed to 6·25 μg ml^-1 of anacardic acid for 6 h. Anacardic acid showed bactericidal activity against MRSA at any stage of growth, and also even when cell division was inhibited by chloramphenicol. In the combination studies, the minimal inhibitory concentration (MIC) of methicillin was lowered from 800 to 1·56 μg ml^-1 for MRSA ATCC 33591, and from 800 to 6·25 μg ml^-1 for MRSA ATCC 33592, by combining with 1/2 X MIC of anacardic acid. The time-kill curves demonstrated synergistic bactericidal activities for these combinations.     

Pheromone of male blue gourami and its effect on vitellogcnesis, steroidogenesis and gonadotropin cells in pituitary of the female

Female Trichogaster trichopterus were exposed to aquarium water in which males had built nests. Gonadotropin cells in the pituitary gland, and exovitellogenesis and steroidogenesis in the ovary were studied. In females in which the percentage of oocytes in vitellogenesis (%V) was low initially, it rose significantly in comparison with an unexposed control group. In females in which the %V was higher initially, it increased further, and in addition a significant percentage of oocytes reached maturation. Thin layer chromatography, using the precursors ³H-pregnenolone and ¹⁴progesterone, revealed high yields of the steroids 17β-estradiol (E₂), 17α,20β-dihydroxy-4-pregnen-3-one (17,20-P), 5β-pregane,3α,17α,20β-triol (5β-P-triol) and 11-ketotestosterone (11KT) in both experimental groups. Significant differences were found in E₂, 17,20-P and 5β-P-triol between the test and control groups. The immunoresponse of GtH-producing cells in the pituitary of the females maintained in nest water was lower than in the control group, suggesting that the GtH was secreted from the cells, which would explain the vitellogenic and steroidogenic changes found in the ovary.     

Seasonal variations in plasma concentrations of 11-ketotestosterone and testosterone in male rainbow trout, Salmo gairdnerii Richardson

Measurements were made of plasma 11-ketotestosterone and testosterone in control and sex-reversed male rainbow trout, before and during their first spawning season (winter 1978/1979). Plasma concentrations of both androgens (in both groups) were 2–3 ng ml^-1 in January 1978 (apart from some precocious spawners), rose slowly to 9–11 ng ml^-1 in April and July and then increased rapidly to 100–150 ng ml^-1 in November. From this time, testosterone levels declined but those of 11-ketotestosterone continued to rise to a peak of 260 ng ml^-1 in February 1979. No significant differences in hormone levels were found between control male and masculinized female fish.     

Sexual maturity in sea trout, Salmo trutta L., running up the River Calonne (Normandy, France) at the 'finnock' stage

Sexual maturation in sea trout ( Salmo trutto L.) running up the River Calonne (Normandy, France), at the finnock stage, is characterized. Autopsy of 118 finnock in summer showed that 87 ± 7% aged 2+ and 33 ± 15% aged 1 + were undergoing gametogenesis. In autumn all the fish were maturing. The analysis of plasma levels of 11-ketotestosterone, 17β-oestradiol, maturational gonadotropin and vitellogenin showed that only females in vitellogenesis can be well identified in summer (plasma vitellogenin greater than 0.30 mg ml^-1). The morphological recognition of females among 134 autumn finnock and the ELISA assay of plasma vitellogenin of 814 summer finnock, demonstrated that vitellogenic females represented 12 ± 2%0 of the 1 + individuals and 46 ± 2% of 2+ fish captured during the 1985 run. The question of a possible relationship between the salinity tolerance ofjuvenile trout migrating towards the sea in the spring and their age on first migration in the river is discussed.     

Reproductive biology and endocrine regulation of final oocyte maturation of captive white bass

The ovarian development, and plasma levels of gonadotropin II (GtH II) and sex-steroid hormones at the end of vitellogenesis were examined in captive white bass Morone chrysops. The changes in plasma hormone levels and oocyte morphology associated with gonadotropinreleasing hormone agonist (GnRHa)-induced final oocyte maturation (FOM) were studied. Although plasma 17β-oestradiol (E₂) and oocyte diameter increased, there were no changes in GtH II, testosterone (T), 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) or 17,20β,21-dihydroxy-4-pregnen-3-one (17,20β,21-P) in non-hormone-treated females, and no FOM was observed. Treatment with a sustained-release GnRHa delivery system (GnRHa implant) induced two FOM cycles separated by about 24 h, with the release of approximately equal numbers of eggs in each spawn. Plasma GtH II levels were elevated significantly throughout FOM, reaching a maximum of 9·07 ± 1·55 ng ml^?1 in ovulated fish. Both plasma E₂ and T increased soon after the GnRHa treatment, but E₂ declined in fish undergoing germinal vesicle (GV) migration. Plasma T increased further during FOM (7·55 ± 2·87 ng ml^?1), but declined precipitously at ovulation. A surge in plasma 17,20β-P and 17,20β,21-P (4·11 ± 0·97 ng ml^?1 and 3·10 ± 0·77 ng ml^?1, respectively) was observed in females undergoing GV breakdown (GVBD). Based on the involvement of different sex-steroid hormones, FOM was separated into two stages. Early FOM included lipid-droplet coalescence and GV migration, and was associated with elevations in plasma GtH II and T. Late FOM included GVBD and yolk-globule coalescence, and was associated with elevations in plasma GtH II, 17,20β-P and 17,20β,21-P. The results of this study point to the absence of a surge in plasma GtH II as the missing link in the reproductive axis responsible for the failure of captive white bass to undergo FOM at the end of vitellogenesis. Sustained elevation of plasma GtH II via treatment with a GnRHa implant induced two consecutive spawns with an overall egg production two- to eightfold higher than previously obtained from captive broodstocks, and similar to annual egg production Values reported for wild fish.     

Sublethal effects of the pesticide Diazinon on olfactory function in mature male Atlantic salmon parr

Diazinon, an organophosphate pesticide, had a sublethal effect on the olfactory system of mature male Atlantic salmon parr. The olfactory responses of the parr to prostaglandin F_2a (PGF_2a) were studied after exposure of the epithelium to different concentrations of Diazinon in water. Electrophysiological recordings from the epithelium indicated that the responses to this prostaglandin were significantly reduced at nominal concentrations as low as 1.0μg l⁻¹ and the threshold of detection was reduced 10-fold at 2.0 μg 1⁻¹ . Mature male salmon parr exposed for a period of 120 h to Diazinon (nominal concentrations 0.3, 0.8, 1.7, 2.7, 5.6, 13, 28 and 45 μg 1 ⁻¹) also had significantly reduced levels of the reproductive steroids, 17,20β-dihydroxy-4-pregnen-3-one, testosterone and gonadotrophin II in the blood plasma after priming with ovulated female salmon urine. Both prostaglandin F_2a and ovulated female urine are known to have important roles in synchronizing reproductive physiology and behaviour in salmonids as well as other fish species. The results are therefore discussed in relation to the possible sublethal effects of Diazinon on reproduction in the Atlantic salmon and possible effects on populations of salmonids.     

Reproductive sensitivity to elevated water temperatures in female Atlantic salmon is heightened at certain stages of vitellogenesis

In order to compare the effects on reproductive performance of short-term or prolonged exposure to elevated temperatures during vitellogenesis, female Atlantic salmon Salmo salar were held at a water temperature of 22° C for periods of 4 or 6 weeks during the austral summer and autumn. Plasma levels of 17β-oestradiol (E₂), testosterone (T) and vitellogenin (Vtg) were monitored and reproductive success was compared to that in groups of fish maintained at 14 or 22° C for 12 weeks from mid-January. Significant endocrine effects were observed within as few as 3 days of the commencement of exposure to 22° C, when plasma levels of E₂ ( c. 0·5 ng ml⁻¹) and Vtg ( c. 1·4 mg ml⁻¹) were approximately half those observed in fish maintained at 14° C ( c. 1·0 ng ml⁻¹ and 2·7 mg ml⁻¹ respectively). The fertility and survival to the eyed stage of ova from fish held at 14° C exceeded 85 and 70% respectively, whereas ova from fish held at 22° C for 6 or 12 weeks exhibited significantly reduced fertility (<70 and <45% respectively) and survival ( c. 40 and 13% respectively). In spite of significant endocrine effects at all stages, a 4 week exposure to 22° C only generated significant reductions in egg fertility (<65%) and survival ( c. 30%) when it occurred between mid-February and mid-March. Together, these data confirm that high temperature spikes can affect reproductive success as strongly as more prolonged exposures, and indicate that there is a critical period of reproductive sensitivity to elevated temperature in late February and early March in this stock of Atlantic salmon.     

Study of the sexual maturity of female bluefin tuna: purification and partial characterization of vitellogenin and its use in an enzyme-linked immunosorbent assay

Plasma steroid levels of female bluefin tuna (BFT) Thunnus thynnus rose from c. 1·5 ng ml⁻¹ during the quiescent period (March) to c. 7 ng ml⁻¹ during the ripening period (May). Testosterone (T) increased further to c. 8 ng ml⁻¹ during the pre-spawning period (June) while 17β-oestradiol (E₂) began to decrease. In the post-spawning period (August) steroid levels decreased to < 1 ng ml⁻¹. Vitellogenin (Vtg) plasma levels seemed to follow changes in E₂, showing an increase from the quiescent period to the ripening period of c. 18 mg ml⁻¹, decreasing slightly before spawning, and then decreasing after spawning. The Vtg content in plasma showed a good correlation both with the plasma levels of E₂ and T and with the percentage of vitellogenic oocytes at different periods of the reproductive cycle. Thus the ELISA could be taken as validated. Immunohistochemical staining of ovaries with anti BFT-Vtg serum demonstrated a high cross-reactivity with yolk proteins allowing the identification of vitellogenic oocytes.     

Steroid metabolism by ovarian follicles of the sea bass Dicentrarchus labrax

Ovarian samples from fear sea bass, Dicentrarchus labrax, were collected for the in vitro incubations during the spawning period. Follicles with fully developed vitellogenic oocytes showing central germinal vesicle (stage I follicles) and follicles with oocytes showing initial germinal vesicle migration (stage II follicles) were treated with either (1) 20 μg sea bass hypophysis plus 50 ng 17-hydroxyprogesterone (17-P), (2) 20 μg hypophysis alone, (3) 50 ng 17-P alone and (4) media alone. Structure-activity experiments used stage II follicles treated with several dosages (0.1, 1.0 and 10.0 ng/ml) of either 17-P, 17,20β-P, or 17,20β,21-P. Free and conjugated (sulfates and glucuronides) levels of the established teleost oocyte maturation inducing steroids (MIS), i.e. 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and 17,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P) were measured in the incubation media by high performance liquid chromatography. Our results show that the synthesis of free and conjugated 17,20β-P is constant (0.1–0.2 ng/ml) in all incubates. In contrast, the synthesis of free and conjugated 17,20β,21-P is higher in incubates containing stage II follicles (up to 5 ng/ml) than in those having stage I follicles (up to 3 ng/ml; P<0.01). Structure-activity data reveal that 17-P is not effective at inducing in vitro germinal vesicle breakdown whereas both 17,20β-P and 17,20β,21-P are equally potent and highly effective. These results demonstrate that 17,20β-P and 17,20β,21-P are synthesized in vitro by follicles of sea bass and that sulfation is the main route for the metabolism of the C₂₁-steroids in riper follicles. The highest levels of 17,20β,21-P, found in incubates containing stage II follicles, points at 17,20β,21-P, rather than 17,20β-P, as the most probable MIS in sea bass, nonetheless, this hypothesis requires further confirmation.     

In vitro steroid production by isolated ovarian follicles of the striped trumpeter

Ovarian follicles from striped trumpeter Latris lineata were incubated in L15 medium alone, or medium supplemented with gonadotropin (GtH) preparations (human chorionic GtH, carp maturational GtH or partially purified salmon GtH), testosterone (T) or 17-hydroxyprogesterone (17P). Levels of oestrone (E₁), 17 β -oestradiol (E₂), T, and 17,20 β -dihydroxy-4-pregnen-3-one (17,20 β P) in the medium after incubation were measured by radioimmunoassay. Basal production of E₂ was high from previtellogenic follicles, whereas little T was produced. Both T and E₂ production increased in response to treatment with GtH or steroid precursors. Vitellogenic follicles showed basal production of both T and E₂, and T but not E₂ levels generally increased in response to hormone treatment. Preparations containing follicles nearing final maturation showed low basal production of E₂ but high production of T. Treatment with steroids resulted in little change in E₂ but often very large increases in T production, whereas GtH stimulated lesser increases. 17,20 β P production was detectable from incubations of maturing follicles from two out of five fish, and in those two incubations, increased in response to treatment with 17P. E₁ was not detectable in any incubations. The results indicate that there is a shift in steroidogenesis from E₂ to T production during oocyte development, and provide further evidence that steroid biosynthesis in non-salmonids is principally regulated by substrate availability.     

Comparative effects of long-term hypoxia on growth, feeding and oxygen consumption in juvenile turbot and European sea bass

When juvenile turbot Scophthalmus maximus and sea bass Dicentrarchus labrax were fed to satiation, growth and food intake were depressed under hypoxia (3·2±0·3 and 4·5±0·2 mg O₂ l^-1). However, no significant difference in growth was observed between fishes maintained in hypoxia and fed to satiation and fishes reared in normoxia (7·4±0·3 mg O₂ l^-1) and fed restricted rations (same food intake of fishes at 3·2 mg O₂ l^-1). Routine oxygen consumption of fishes fed to satiation was higher in normoxia than in hypoxia due to the decrease in food intake in the latter. Of the physiological parameters measured, no significant changes were observed in the two species maintained in hypoxia. This study confirms the significant interaction between environmental oxygen concentrations, feeding and growth in fishes. Decrease in food intake could be an indirect mechanism by which prolonged hypoxia reduces growth in turbot and sea bass, and may be a way to reduce energy and thus oxygen demand.     

Reproductive priming in mature male Atlantic salmon parr exposed to the sound of redd cutting

The sounds of a wild female Atlantic salmon cutting a redd were associated with significant increases in the levels of plasma 17,20β-dihydroxy-4-pregnen-3-one (17,20βP) and of expressible milt in mature male parr, comparable with levels of the steroid and milt produced in parr exposed to the priming pheromone, prostaglandin F_2a. Hence auditory cues may have a significant role in synchronising reproductive physiology in Atlantic salmon.