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.     

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.     

Dynamics of excretion of 17α,20β-dihydroxy-4-pregnen-3-one 20-sulphate, and of the glucuronides of testosterone and 17β-oestradiol, by urine of reproductively mature male and female rainbow trout (Oncorhynchus myklss)

Levels of sulphated 17α20β-dihydroxy-4-pregnen-3-one (17,20 β -P; the oocyte maturation inducing steroid) in blood plasmas of sexually mature male and female rainbow trout Oncorhynchus mykiss , were very low in comparison to those of the free steroid. However, relatively large amounts were found in urine of both sexes.
Catheters were inserted into the urinary bladders of unovulated and ovulated females and of ripe-running males, and the fish then placed in spawning channels. Three-hourly urine samples were collected between 09.00 and 18.00 hours and then a 15-h sample between 18.00 and 09.00 hours the next morning. Measurements were made of 17,20 β -P-sulphale, testosterone glucuronide (T-G) and 17 β -oestradiol glucuronide (E2-G). In females, the highest rates of excretion of E2-G, T-G and 17,20 β -P-sulphate were found in unovulated, ovulating and ovulated females, respectively. The rates of excretion of 17,20 β -P-sulphate, T-G and E2-G in ovulated females were unaffected by the presence of a male. id males, however, there was a sharp increase in the rate of excretion of 17,20 β -P-suiphate and T-G in fish which were paired with an ovutated (nesting) female. A similar increase was found in males injected with male trout pituitary extract.     

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.     

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.     

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.     

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).     

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.     

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.     

Stimulatory and inhibitory effects of testosterone on testes in Atlantic salmon male parr

Immature 1-year-old Atlantic salmon Salmo salar parr were implanted with Silastic capsules of different sizes filled with testosterone (T). Testosterone had both positive and negative effects on testicular weights, spermatogenesis and steroidogenesis. The positive effects: higher incidence of males with enlarged gonads, spermiation, and high plasma levels of 11-ketotestosterone (11-KT) and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P), were most pronounced in males treated with small T capsules. The negative effects: suppression of gonadal development and depressed plasma levels of 11-KT and 17,20β-P compared with mature controls, were most evident in fish treated with large T capsules.     

Urinary sex steroids during sexual development in female mice and in proximate novel males.

The experiments described here were designed to determine whether males' capacity to accelerate female pubertal development is reflected in females' urinary steroid levels in mice, and whether steroids in males' urine are influenced by exposure to developing females. In the first experiment, measures from urine collected daily from female mice aged 31-59 days showed a gradual rise in 17beta-estradiol levels and a distinct linear rise in progesterone levels. In a second experiment, daily steroids were measured in females aged 30-42 days while they were either housed alone or underneath two novel outbred males. Females exposed to males showed accelerated development at day 43 in uterine weight, and to a lesser extent in ovarian and whole-body weights. Average steroid levels did not significantly differ between conditions, but intra-individual variance in estradiol measures was greater in male-exposed than in isolated females. Creatinine levels were higher in isolated females. Males exposed to developing females excreted higher levels of estradiol in their urine compared to isolated males. These data suggest that excreted steroids can reflect general pubertal development, but may not fully reflect substantial morphological impacts of exposure to novel males. Elevations of estrogen levels in males exposed to developing females could help to account for precocious puberty in such females.     

Isolation increases milt production in goldfish

Milt volume in goldfish is increased by female steroid and prostaglandin pheromones, by exposure to males with elevated gonadotropin levels, and by isolation from conspecifies. This study examined various aspects of the isolation effect on milt volume and serum gonadotropin II (GTH II). The latency of isolation-induced milt increase in this study (12-24 hr) was longer than the latencies to pheromone-induced milt increase in previous work (0.5-6.0 hr), was not affected by the time of day at which males were isolated, persisted for at least 72 hr in isolated males, and was terminated within 24 hr in males that were returned to groups. Isolated males maintained high milt production when separated from tank mates by a perforated barrier or when exposed to visual and odor cues from males in other tanks, suggesting that the unknown conspecific cues that maintain low milt production in groups operate at close range. Isolation appears to increase milt through a mechanism different from that mediating response to female pheromones because: (1) unlike female pheromones, which consistently increase serum GTH II, no isolation or regrouping treatment in this study affected male GTH II concentration; and (2) the effects of isolation and of exposure to the female pheromone 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) are additive. Finally, males that were previously isolated or exposed to 17,20 beta-P increased milt (but not GTH II) in grouped males, suggesting that the effects of isolation and pheromone exposure can indirectly stimulate male conspecifics. Although the biological function of the isolation effect is not clear, we propose that it illustrates the effect of removal from inhibitory cues normally received from male conspecifies.     

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.     

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.     

Large and persistent effect of a female steroid pheromone on ejaculate size in goldfish Carassius auratus

This study determined if ejaculate size in male goldfish Carassius auratus is increased by the female preovulatory steroid pheromone 4‐pregnen‐17,20β‐diol‐3‐one (17,20βP), which previously has been shown to affect male behaviour and to increase sperm motility and stripped sperm number, and also to increase paternity in competitive spawning and competitive in vitro fertilization. Experimental males were exposed overnight to 17,20βP whereas control males were not. The morning following exposure, each male was placed with a reproductively active female and, after one to 20 spawning acts, aquarium water was sampled to quantify released sperm. Although exposure to 17,20βP induced a five‐fold difference in the number of sperm that could be stripped, the median number of sperm in first ejaculates of pheromone‐exposed males was >60 sixty times that of control males, a pheromonal effect on ejaculate size that persisted for at least 20 spawning acts. The magnitude of the pheromone effect on ejaculate size indicates that it is a critical component of C. auratus sperm allocation, and that examining this effect in concert with other factors (e.g. presence of competitors, male and female size and frequency of spawning) will reveal the contribution of the preovulatory pheromone to male fitness in this promiscuous 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.     

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.     

Changes in reproductive parameters during the spawning migration of pink salmon, Oncorhynchus gorbuscha (Walbaum)

The hormones 17β-estradiol, 17α-hydroxy-20β-dihydroprogesterone(17α, 20β-P), 11-ketotestosterone, testosterone, gonadotropin and also vitellogenin, were determined during the spawning migration of wild pink salmon in the Fraser and Thompson Rivers in British Columbia. This stock of pink salmon takes approximately 2 weeks to migrate the 333 km upstream to the spawning grounds. Both sexes were at an advanced stage of sexual development when they entered fresh water. In females both the 17β-estradiol and vitellogenin levels fell precipitously during the migration, to be very low at spawning, whereas the 17α,20β-P level rose rapidly, to be highest at arrival on the spawning grounds. The gonadotropin level also rose rapidly during the migration, and was highest in spent fish. Testosterone was at a high level throughout, although this level decreased steadily during migration. In many respects similar endocrine changes were observed in the male. For example, in the case of androgen levels, both testosterone and 11-ketotestosterone fell steadily during migration but were still relatively high at spawning, whereas both gonadotropin and 17α, 20β-P levels rose markedly as migration progress. However, although the qualitative changes were often similar between the sexes, the levels of 17α, 20β-P, testosterone, and gonadotropin were considerably higher throughout in females than in males. It is concluded that this stock of pink salmon is at an advanced stage of sexual development when it enters fresh water. The endocrine changes observed during this study represent those controlling the final stages of reproduction, specifically final oocyte maturation and ovulation in females, and the final stages of spermatogenesis and spermiation in males.     

Effects of copper on olfactory‐mediated endocrine responses and reproductive behaviour in mature male brown trout Salmo trutta parr to conspecific females

In the present study, the effects of copper (CuSO₄) on the ability of mature male brown trout Salmo trutta parr to detect and react both physiologically and behaviourally to female pheromones were studied. The study was composed of two parts. In the first experiment, priming effects of the female pheromone prostaglandin F_2α (PGF_2α) were evaluated by determining the amount of milt produced and the blood plasma levels of 11‐ketotestosterone (11‐KT) and 17α,20β‐dihydroxy‐4‐pregnen‐3‐one (17,20β‐P) after the PGF_2α exposure. In the second experiment, male parr were placed in a large stream tank together with a group of adult males and ovulated females and their individual behaviours were recorded. In the priming experiment, the amount of expressible milt was significantly lower, less than half, in groups exposed during 4 days to 10 or 100 µg l^?1 copper compared with control parr only exposed to water. No significant differences were observed in plasma levels of 11‐KT and 17, 20β‐P. During the behavioural experiment, exposed parr spent less time with the female and had a lower number of courting events. Blood plasma levels of 11‐KT were, however, significantly higher in the group exposed to 100 µg l^?1 copper compared with the control group. Furthermore, the exposed group spent significantly less time swimming upstream than did the control group. The present study demonstrates that exposure to copper affects reproductive behaviours and endocrinology of S. trutta male parr.     

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.