Female rainbow trout urine contains a pheromone which causes a rapid rise in plasma 17,20β-dihydroxy-4-pregnen-3-one levels and milt amounts in males

Previous studies have shown that spermiating male rainbow trout respond to the presence of female urine in the water with significant increases in plasma levels of gonadotrophin II, 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and testosterone. The present results show that males only need a single brief exposure to female urine in order to respond; levels of 17,20β-P rise significantly within 1 h of exposure, and peak between 3 and 4 h. Also, milt amounts increase significantly following exposure of males to female urine. Levels of 17,20β-P are also related positively to the amount of female urine to which the males are exposed. Furthermore, when live females are placed, out of physical and visual contact, in the same tank as males, levels of 17,20β-P rise in the same way as in males which are exposed to female urine. However, if females are fitted with urinary catheters (which drain the urine outside the tank), males respond more slowly. These results indicate that urine is the main source of the male 'priming' pheromone.     

Lack of species-specific primer effects of odours from female Atlantic salmon, Salmo salar, and brown trout, Salmo trutta

We exposed, in two successive spawning seasons, individually placed precocious male Atlantic salmon ( Salmo salar ) and brown trout ( Salmo trutta ) parr to odour stimuli (ovarian fluid and urine mix) from ovulated conspecific or heterospecific anadromous females. Atlantic salmon parr had significantly higher plasma concentrations of the hormones 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P), 11-ketotestosterone (11-KT) and testosterone (T) after exposure to odours from conspecific females or from brown trout females compared to parr exposed to a control solution (0.9% NaCl). We did not observe any significant differences between the hormone levels in salmon parr exposed to the two female odours. The salmon parr exposed to conspecific odours had significantly higher volumes of strippable milt compared to the controls, but we did not find any significant differences when comparing the effect of the two female odours. Brown trout parr had significantly higher plasma 17,20β-P levels following exposure to heterospecific female odours compared to control males, but there was no significant difference between males exposed to the different female odours. We did not observe any significant differences in plasma levels of T and 11-KT and in milt volumes between exposed and control trout. Taken together, the results from both tested species indicate that the potency of heterospecific stimuli in stimulating increased plasma sex steroid hormone levels in male parr was as strong as stimuli from conspecific females. The results are discussed in connection to observed hybridisation between the two sympatric species.     

Plasma levels of gonadotropin and 17α,20β-dihydroxy-4-pregnen-3-one in relation to spawning behaviour of rainbow trout, Oncorhynchus mykiss(Walbaum)

Manipulation of the opportunity to spawn was used to investigate the relationship between endocrine events, egg viability and spawning behaviour in female rainbow trout. Females were prevented from spawning by isolating them from males and gravel for up to 21 days after ovula- tion. Blood samples were taken before pairing with a male, at the onset of nesting activity, and at the completion of spawning. Plasma hormone levels of gonadotropin (GtH) and 17α,20β-dihydroxy-4-pregnen-3-one (17,2OP) were measured by specific radioimmunoassays. There were no qualitative or quantitative differences in the spawning behaviour of females paired on the day of ovulation or 7. 14, or 21 days after ovulation. There was a general decrease in the viability of eggs with increasing retention times. In females paired on the day of ovulation, or after 7 or 14 days, GtH levels increased with the onset of nesting behaviour and declined as fish reached the post-spawning condition. By day 21, GtH levels before pairing were significantly higher than prepairing levels in the other three treatment groups, and did not increase at the onset of nesting, or decrease in post-spawning fish. Plasma 17,20P remained high in prepairing and nesting samples of all four groups and declined to low levels in fish in post-spawning condition. In females paired on the day of ovulation there was a significant increase in 17,20P from the prepairing to the nesting stage. These results suggest that 17,20P plays a key role in the synchronization of behavioural and maturational events at the time of spawning.     

Urine of reproductively mature female rainbow trout, Oncorhynchus mykiss (Walbaum), contains a priming pheromone which enhances plasma levels of sex steroids and gonadotrophin II in males

Urine of reproductively mature female rainbow trout was shown to contain a priming pheromone which raised the levels of 17 a ,20β-dihydroxy-4-pregnen-3-one (17,20β-P), testosterone and gonadotrophin II in the blood plasma of reproductively mature male rainbow trout. Milt volumes, however, were unaffected.
Because it had been established in a previous study that the sulphated form of 17,20β-P is abundant in the urine of spawning rainbow trout, synthetic 17 a ,20β-dihydroxy-4-pregnen-3-one 20-sulphate was also tested for pheromonal activity. It was found to have only a smalt and inconsistent priming effect on steroid levels and did not alter the orientation or spawning activity of males.
It was also shown that, in the majority of experiments, there was a significant drop, over the course of 24 h, in the levels of 17,20β-P and testosterone in the control groups of males.     

Serum levels of testosterone, 11‐ketotestosterone and oestradiol‐17β in three species of sturgeon during gonadal development and final maturation induced by hormonal treatment

Changes in serum concentrations of two androgens, testosterone (T) and 11‐ketotestosterone (11KT), and oestradiol‐17β(E2) in male and female giant sturgeon Huso huso , Russian sturgeon Acipenser gueldenstaedtii and stellate sturgeon Acipenser stellatus were studied at different stages of gonadal maturity and after final maturation induced by hormonal treatment. Both male and female fish displayed a distinct increase in serum steroid concentrations during gonadal development. 11KT levels were significantly higher in males than females, with a positive correlation detected between 11KT and T concentrations. In maturing males and females, higher values of both 11KT and T were observed in stellate sturgeon compared to giant and Russian sturgeons. Vitellogenesis and high E2 levels were correlated in maturing sturgeon females.     

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

The major progestin in teleosts is not progesterone, as in tetrapods, but 17,20β-dihydroxypregn-4-en-3-one (17,20β-P) or, in certain species, 17,20β,21-trihydroxy-pregn-4-en-3-one (17,20β,21-P). Several functions for 17,20β-P and 17,20β,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 17,20β-P in males, including listing of the evidence for the presence of 17,20β-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 17,20β-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 17,20β-P (and closely related compounds) in males; a listing of previously published evidence that supports the proposed new function of 17,20β-P as an initiator of meiosis; finally, discussion of the evidence for environmental endocrine disruption by progestins in fishes.     

17α,20β,21-Trihydroxy-4-pregnen-3-one: the probable maturation-inducing steroid of the Lusitanian toadfish

17,20β,21-Trihydroxy-4-pregnen-3-one (17,20β,21-P) was identified as the major metabolite of incubations of Lusitanian toadfish Halobatrachus didactylus ovarian follicles with [³H]-17hydroxyprogesterone. The potency of several steroids in inducing germinal vesicle breakdown of follicle-enclosed oocytes of Lusitanian toadfish was systematically examined by using an in vitro germinal vesicle breakdown (GVBD) bioassay. 17,20β-Dihydroxy-4-pregnen-3-one (17,20β-P) and 17,20β,21-P, two confirmed maturation-inducing steroids (MIS) in teleosts, were the most potent in inducing GVBD with ED₅₀s ranging between 9 and 271 nM. Structure-activity relationships followed similar patterns to what has been observed in similar bioassays, i.e. a vital requirement for 17- and 20β-hydroxyl groups in C21 steroids and a reduction in activity of 14 and 5–6%, respectively, for 5-pregnene and 5β-pregnanes compared to 4-pregnenes. Corticosteroids, testosterone and 17β-oestradiol were ineffective. Folliculated oocytes stimulated by pituitary homogenate produced 17,20β,21-P from endogenous substrates in amounts one order of magnitude higher than 17,20β-P. These results strongly support the hypothesis that 17,20β,21-P is the likely MIS in this species.     

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.     

Plasma concentrations of ovarian steroids in relation to oocyte final maturation and ovulation in female plaice sampled at sea

Blood plasma concentrations of free 17 β -oestradiol, free testosterone and glucuronidated testosterone were strongly positively related to the percentage of vitellogenic oocytes remaining in the ovaries of plaice Pleuronectes platessa caught at sea–being at their highest in pre-spawning (stage IV) females (i.e. those in which the oocytes were close to fully grown, but had not yet entered the stage of final maturation). In contrast, the concentrations of free and sulphated 17,20 β -P, 3α aL ,17, 20 β -P-5 β , and 3 α ,17,21-P-5 β were at their lowest in stage IV females. Free 17,20 β -P (the putative maturation-inducing steroid) became only slightly elevated (less than twofold) during spawning (i.e. in stage V and VI females with hydrated and/or ovulated eggs). Sulphated 17,20 β -P and 3 α ,17,21-P-5 β became slightly more elevated (three- to fourfold). However, sulphated 3 α , 17,20 β -P-5 β concentrations increased 30-fold and were at their highest in fish in which only 40% of vitellogenic oocytes remained in the ovaries. Sulphated 17,20 β -P, 3 α , 17, 20 β -P-5 β and 3 α ,17,21-P-5 β concentrations were significantly positively related to hyaline oocyte batch size; and sulphated 17,20 β -P and sulphated 3 α , 17,20 β -P-5 β were significantly negatively related to the degree of hydration of the hyaline oocytes. None of the steroid concentrations, however, was related to the time of capture. More ovulated females were found in the afternoon than at any other time of the day.     

Seasonal changes in serum steroid hormones in a protandrous teleost, the sobaity (Sparidentex hasta Valenciennes)

Serum concentrations of 11-ketotestosterone, 11β-hydroxytestosterone, testosterone, testoster-one glucuronide, oestradiol and 17,20β -dihydroxy-4-pregnen-3-one (17,20β -P) were measured in the sobaity at monthly intervals through their second breeding season. Concentrations of the 11-oxygenated androgens in the males and of oestradiol in the females peaked during the spawning season in January-February, while maximum levels of testosterone were found in the summer when these steroids were low. Testosterone glucuronide showed two peaks, one in the post-spawning period as oestradiol and the 11-oxygenated androgens were falling and the other coincident with the summer peak of testosterone. 17,20β -P was detectable in only one male and one female fish in February. Serum concentrations of 11-oxygenated androgens are more reliable than those of oestradiol for determining the sex of sobaity, and may also be used as indicators of the occurrence of sex reversal. The seasonal pattern of serum steroids correlated well with the changes of sexual status of the gonads during regression and recrudescence observed histologically and suggests that oestradiol may be involved in the sex inversion of this species.     

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.     

Male priming pheromone is present in bile, as well as urine, of female rainbow trout

The bile of sexually mature female rainbow trout Oncorhynchus mykiss has pheromonal activity which causes a significant increase in concentrations of 17,20β-dihydroxy-4-pregnen-3-one in the plasma of males. Bile from male trout is inactive. The activity in the female bile binds to octadecylsilane and can be eluted with methanol. The synthetic bile acids, taurocholic acid and taurolithocholic acid, are inactive.     

Regulation by Androgen of Levels of the β Subunit of Nerve Growth Factor and Its mRNA in Selected Regions of the Mouse Brain

Abstract: Our previous studies showed that the concentration of the β subunit of nerve growth factor (β-NGF) in nervous tissues is higher in male than in female mice. To identify the brain regions that are affected by androgens, the amounts of β-NGF protein and its mRNAs were measured in male, female, and castrated male CD-1 mice and testicular feminization mice at 3–4 months of age. Among tissues examined, the hypophysis of males contained the highest average concentration of β-NGF protein. In most regions of the brain, individual levels were more variable in males than in females. However, after the castration, such variations in β-NGF levels disappeared. Average levels of β-NGF protein in males were higher in the cerebellum (eightfold higher), olfactory bulb (12-fold higher), hypothalamus (sixfold higher), and hypophysis (72-fold higher) than thope in corresponding regions of females. No significant differences were observed in levels of β-NGF protein in the hippocampus, cerebral cortex, striatum, septum, and brainstem. The castration of male mice caused a reduction in levels of β-NGF protein in the hypothalamus and hypophysis, but not in the cerebellum and olfactory bulb, to the femgle levels. The concentrations of β-NGF protein in testicular feminization mice were similar to those in female CD-1 mice in all regions. The concentrations of mRNA for β-NGF in the olfactory bulb and hypophysis from males were higher than those from females. By contrast, northern blots showed no remarkable differences in the amounts of brain-derived neurotrophic factor and neurotrophin-3 between the two sexes. Thus, in some regions of the brain, the production of β-NGF appears to be regulated by testosterone, but the regulatory mechanisms do not appear to be simple. Our present results indicate that the binding of testosterone to its receptor is an important step in the regulation of the level of β-NGF in these region.     

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.     

Release of sex steroids into the water by roach

Electro‐olfactogram (EOG) recordings of the olfactory epithelium of both male and female roach Rutilus rutilus demonstrated that both sexes were able to detect free and glucuronidated 17,20β‐dihydroxy‐4‐pregnen‐3‐one (17,20β‐P) with high sensitivity. Male, but not female, roach were also sensitive to androstenedione. Sexually mature female roach were shown to release free 17,20β‐P, glucuronidated 17,20β‐P and androstenedione into the water; for all three steroids, the rate of release was significantly enhanced by injection of carp pituitary extract (CPE). A series of trials was also carried out which showed that mature males, and to a lesser extent immature males and females, were able also to release free and glucuronidated 17,20β‐P, both before and after CPE treatment. Water extracts from containers that had held CPE‐treated mature male and female roach were examined for the presence of other steroids. This revealed that free and glucuronidated 17,20β‐P plus free and glucuronidated 17,20β,21‐trihydroxy‐4‐pregnen‐3‐one (17,20β, 21‐P) predominated in water extracts from both sexes. The free moieties of 17,20α‐dihydroxy‐4‐pregnen‐3‐one, 17‐hydroxyprogesterone and 11‐deoxycortisol were found at concentrations which were between four and 20 times lower than those of free 17,20β‐P. Androstenedione was found at concentrations which were 25‐fold lower than those of 17,20β‐P. Despite its apparent high rate of release by sexually mature male and female roach, free 17,20β,21‐P was found not to exhibit any EOG activity at the highest dose tested (10⁻⁷ M).     

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.     

A study on the 3β- and 17β-hydroxysteroid dehydrogenase activities in the gonads of Monopterus albus (Pisces: Teleostei) at various sexual phases during natural sex reversal

Activities of 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD) in Monopterus gonads were studied at different sexual phases during natural sex reversal. Before sexual transformation, positive reactions for 3β-HSD in the follicular epithelium were found in the granulosa cells of some large, maturing follicles in some females during the breeding season. Weak reaction for this enzyme was also detected in some scattered interstitial cells found occasionally in some ovaries. At the intersexual and the male phases, intense 3β-HSD activities were demonstrated exclusively in the interstitial Leydig cells. No 17β-HSD activities were observable in the gonads at any stage of development. The reaction intensity of 3β-HSD in the interstitial cells exhibited a marked increase during the process of sex change from female to the intersexual and the male phases and there is a definite correlation with the density and nuclear size of these cells. It is concluded that in Monopterus , the granulosa cells in the ovary and the interstitial cells of the intersexual and male gonads are the major sites for the biosynthesis of oestrogens and androgens, respectively, and that the intensive development of interstitial tissue with increasing steroidogenic enzyme activities at the intersexual and male phases was directly related to the increase in androgen production in vitro reported previously. The occasional presence of some 3β-HSD positive interstitial cells in the ovary suggests that interstitial cell development might precede testicular lobule formation during natural sex reversal.     

Immunohistochemical Localization of G Protein β1, β2, β3, β4, β5, and γ3 Subunits in the Adult Rat Brain

Abstract: The regional distributions of the G protein β subunits (Gβ1–β5) and of the Gγ3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gβ and Gγ3 subunits were widely distributed throughout the brain, with most regions containing several Gβ subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gβ immunostaining. Negative immunostaining was observed in cortical layer I for Gβ1 and layer IV for Gβ4. The hippocampal dentate granular and CA1–CA3 pyramidal cells displayed little or no positive immunostaining for Gβ2 or Gβ4. No anti-Gβ4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gβ1 was absent from the cerebellar molecular layer, and Gβ2 was not detected in the Purkinje cells. No positive Gγ3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double-fluorescence immunostaining with anti-Gγ3 antibody and individual anti-Gβ1–β5 antibodies displayed regional selectivity with Gβ1 (cortical layers V–VI) and Gβ2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gβ1–β5 with Gγ3, specific dimeric associations in situ were observed within discrete brain regions.     

Impact of γ-hexachlorocyclohexane exposure on plasma gonadotropin levels and in vitro stimulation of gonadal steroid production by carp hypophyseal homogenate in Carassius auratus

Male and female Carassius auratus were exposed to safe (SC; 0.01 ppm) and sublethal (SL; 0·1 ppm) concentrations of an organochlorine pesticide γ-hexachlorocyclohexane (γ-HCH) for 4 weeks during the pre-spawning phase (June) of the annual reproductive cycle. Gonadosomatic index and gonadotropin levels were significantly lower after exposure to both γ-HCH concentrations than in control fish. After 4 weeks exposure, gonadal tissue from control, SC and SL exposed fish was incubated with carp hypophyseal homogenate (chh). The chh stimulated production of testosterone, 17,20β-dihydroxy-4-pregnen-3-one (17,20βP), 11-deoxycortisol, and 11-ketotesterone (11-KT) was estimated in the unconjugated (free) and conjugated (glucuronide) fractions by radioimmunoassay. In both sexes, testosterone production was greatly decreased in γ-HCH exposed fish compared to controls. 17,20βP production was low in all fish and was unaffected by γ-HCH. Free 11-deoxycortisol production by testicular fragments was higher in SL and SC compared with controls, while conjugated 11-deoxycortisol was increased only higher in the SL. In ovarian fragments from exposed fish, free 11-deoxycortisol decreased while glucuronide concentrations increased compared with controls. 11-KT production was significantly decreased in testicular fragments of exposed fish. The results indicate that γ-HCH inhibits gonadal recrudescence by decreasing both gonadotropin secretion and its potential for stimulation of steroidogenesis.     

Effects of testosterone and β‐glucan on immune functions in tench

A hormone manipulation was performed to examine the effects of testosterone on basal and β‐glucan‐induced immune functions in wild‐caught male and female tench Tinca tinca . Testosterone administration elevated testosterone concentration in plasma, but did not suppress lytic activity of plasma or the chemiluminescence response of blood or head kidney phagocytes in any of the three successive samples or in any of the treatment groups. Both testosterone and β‐glucan administrations had a negative effect on the relative mass of the spleen, and testosterone‐treated fish lost more mass than control fish. Males had a relatively larger spleen than females, but there were no gender differences in immune function. β‐glucan‐administration did not affect the plasma testosterone concentration.