Steroidal Pheromone Research Articles

Methyl-Testosterone Induces Male-Typical Ventilatory Behavior in Response to Putative Steroidal Pheromones in Female Round Gobies ( Neogobius melanostomus)

Male round gobies ( Neogobius melanostomus) transiently increase their rate of ventilation when exposed to nanomolar concentrations of three putative steroidal pheromones (etiocholanolone, ETIO; estrone, E1; estradiol-glucuronide, E2-3g), whereas females exhibit this response only to ETIO. In this study we implanted females with Silastic capsules that were empty (Blank) or filled with methyl-testosterone (MT) to determine whether androgen induces a male-typical pattern of ventilatory response. As with untreated females in our previous studies, Blank females increased ventilation only in response to ETIO. In contrast, MT females tested 2–3 weeks postimplant responded not only to ETIO, but also to E1 and E2-3g. These results provide the first demonstration of an androgen inducing a male-typical behavioral response to a steroidal pheromone in adult female fish. Together with our previous findings of sexually isomorphic sensory responses to steroid odorants, the present results also suggest that a central action of androgen is responsible for sexually dimorphic behavioral responses to putative pheromonal steroids in Neogobius.

Water temperature and pH influence olfactory sensitivity to pre-ovulatory and post-ovulatory ovarian pheromones in male Barilius bendelisis.

The attractive response and sexual activity elicited by pre-ovulatory steroid sulphate and post-ovulatory 15K-PGF pheromones are greater in wild caught tubercular males and immature males which express breeding tubercles on the snout (at 12-13 days post androgen implant) than in non-tubercular and non-androgen implanted males of freshwater fish Barilius bendelisis. This shows that circulatory androgens exert an activational effect on olfactory receptors of male fish. Wild caught tubercular males and androgen implanted juvenile males exhibit a high responsiveness to steroid sulphate at the water temperature and pH which fish experience during the pre-spawning phase. The male's sensitivity to 15K-PGF is almost equally high at the water temperature and pH which they experience in wild during the both pre-spawning and spawning periods. This suggests that the differential olfactory sensitivity to the two classes of pheromones in androgen implanted males is due to the varied temperature and pH of water, and that during the breeding season the male's olfactory sensitivity to PGF pheromone is more widespread than to the steroidal pheromone. An increased and decreased olfactory sensitivity in mature males to sex pheromones and L-alanine respectively during the breeding phase is in agreement with the hypothesis that pheromonal stimuli dominate over feeding stimuli to promote spawning success.

Bile Acid secreted by male sea lamprey that acts as a sex pheromone.

We show that reproductively mature male sea lampreys release a bile acid that acts as a potent sex pheromone, inducing preference and searching behavior in ovulated female lampreys. The secreted bile acid 7alpha,12alpha,24-trihydroxy-5alpha-cholan-3-one 24-sulfate was released in much higher amounts relative to known vertebrate steroid pheromones and may be secreted through the gills. Hence, the male of this fish species signals both its reproductive status and location to females by secreting a pheromone that can act over long distances.

Milt production in goldfish: regulation by multiple social stimuli

Previous studies in goldfish ( Carassius auratus) demonstrate that milt (sperm and seminal fluid) volume is increased both by a preovulatory steroidal pheromone, 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P), that acts by increasing serum concentrations of gonadotropin II (GTH-II), and by a post-ovulatory prostaglandin (PG) pheromone, that acts by stimulating sexual interactions. Here, we show that male goldfish also increase milt volume when isolated for 24 h, or when placed with another male injected with human chorionic gonadotropin (hCG) for 12 h. In contrast to the milt increase induced by pheromonal 17,20β-P, the milt increase following isolation or exposure to hCG-injected males was not associated with increased serum GTH-II. Serum growth hormone also was unaffected, although serum testosterone increased in isolated males. The absence of GTH-II increase following isolation or exposure to hCG-injected males and the long latency time for these two types of responses, suggests these effects are mediated by a novel mechanism as yet undescribed. The present findings suggest that sperm competition has selected for complex mechanisms regulating milt production and fertility in goldfish: males regulate milt production not only in response to stimulatory pheromonal cues from ovulatory females, but also in response to unknown stimulatory and inhibitory cues from male competitors.

Synthetic musks in environmental samples: indicator compounds with relevant properties for environmental monitoring.

Synthetic musks (nitro and polycyclic musks) are a group of chemicals offering a wide range of important properties for environmental monitoring programs. They are produced as odorous chemicals and added to a wide variety of perfumes, toiletry products and other household products. As such, they are directly applied in cosmetic products or in washed textiles to the human body in considerable concentrations and accumulate owing to dermal resorption. In addition, synthetic musks also enter the environment via waste water treatment. Several polycyclic musks are chiral. By using chiral gas chromatographic methods, it is possible to determine the enantiomeric ratio and assess their bioavailability. Although an comprehensive quality assurance program must be followed during the analysis of synthetic musks in environmental samples, the determination of these compounds is not very demanding and can be carried out by a standard analytical laboratory specialising in trace analysis of organic pollutants. Owing to the pheromone-like behavior of some synthetic musks, the induction of receptors in olfactory systems should be investigated. For HHCB (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta[g]-2-benzopyran, e.g., Galaxolide), three-dimensional structural similarities with androstenone (5 alpha-androst-16-en-3-one), a mammalian steroid pheromone, were found, which support the hypothesis of HHCB as an artificial pheromone. Owing to their environmental abundance, their relation to human activities and their potential for pheromone-like environmental behavior, synthetic musks are especially valuable as future indicator chemicals for environmental monitoring.

The steroid pheromone 4-pregnen-17,20ss-diol-3-one increases fertility and paternity in goldfish.

Previous studies in goldfish (Carassius auratus) showed that the oocyte maturation-inducing steroid 4-pregnen-17,20ss-diol-3-one (17,20ssP) functions after release as a pheromone that increases male serum gonadotropin II (GtH II) concentration, milt (sperm and seminal fluid) volume and sexual activity, effects hypothesized to increase male reproductive success in the sperm competition of multi-male spawnings. The present study tested this hypothesis by determining whether overnight exposure to 17,20ssP increases fertility. In pair spawnings, 17,20ssP-exposed males fertilized a greater percentage of eggs than did control males, apparently because 17,20ssP-exposed males had more releasable sperm at the onset of spawning. Microsatellite DNA paternity analysis showed that 17,20ssP-exposed males also fertilized more eggs in competitive spawnings involving one control male and one 17,20ssP-exposed male. This effect of 17,20ssP on competitive fertility could be due to demonstrated increases in spawning activity, milt volume, duration of sperm motility and proportion of motile sperm. However, it appears that a change in sperm quality is a major component of the pheromonal effect because, in competitive in vitro fertilizations, sperm from 17,20ssP-exposed males fertilized more eggs than did sperm from control males. The results indicate that the response to pheromonal 17,20ssP is a major determinant of reproductive success in male goldfish.

A Steroidal Pheromone and Spawning Stimuli Act via Different Neuroendocrine Mechanisms to Increase Gonadotropin and Milt Volume in Male GoldfishCarassius auratus

In goldfish (Carassius auratus), pheromonal 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and spawning stimuli (interaction with a sexually active female releasing prostaglandin pheromone) both increase gonadotropin-II (GtH-II) and milt volume. In the goldfish pituitary, GtH-II release is stimulated by gonadotropin-releasing hormone (GnRH) and inhibited by dopamine (DA). In this study, we investigated the possibility that 17,20β-P and spawning stimuli act via separate neuroendocrine mechanisms by determining whether their effects on GtH-II could be selectively disrupted by injection of DA type-2 receptor (D-2) agonists (bromocryptine and LY171555) or a goldfish GnRH antagonist, [Ac-Δ3-Pro1, 4FD-Phe2,d-Trp3,6]-mGnRH (analog E). D-2 agonists blocked 17,20β-P-induced increases in GtH-II and milt volume but did not affect spawning-induced responses. GnRH antagonist blocked 17,20β-P-induced increases in GtH-II and milt volume, and spawning-induced GtH-II increase, but did not affect spawning-induced increase in milt volume. These results suggest that (1) pheromonal 17,20β-P and spawning stimuli increase GtH-II increase via distinct neuroendocrine mechanisms, (2) the effect of pheromonal 17,20β-P on increasing milt volume is GtH-II-dependent, and (3) the effect of spawning stimuli on increasing milt volume is GtH-II-independent.

Two mechanisms for increasing milt volume in male goldfish,Carassius auratus

Milt volume in goldfish (Carassius auratus) is known to increase in response to both spawning interactions with prostaglandin F2α-injected females (spawning stimuli) and a steroidal pheromone, 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P). In this study, we sought evidence that spawning stimuli and 17,20β-P increase milt volume via different mechanisms, by determining latency of the milt response, and by comparing the effects of water temperature, hypophysectomy, and human chorionic gonadotropin (hCG) injection on milt response. At both 21 and 11°C, spawning stimuli increased milt volume within 2 hr, whereas 17,20β-P exposure or hCG injection did not increase milt volume until 4-6 hr at 21°C and 12-24 hr at 11°C. Hypophysectomy did not block spawning-induced milt increase but did block 17,20β-P-induced milt increase, consistent with earlier studies showing that pheromonal 17,20β-P acts by increasing blood gonadotropin-II (GtH-II) level. Furthermore, in males injected with hCG to swamp the effect of 17,20β-P-induced GtH-II increase, milt increase was observed in response to spawning stimuli but not to 17,20β-P exposure. These results indicate that distinct mechanisms mediate the effects of spawning stimuli and 17,20β-P on milt volume in goldfish. 17,20β-P evidently acts by releasing pituitary GtH-II, whereas the mechanism mediating the effect of spawning stimuli is unknown. The role of these two mechanisms in made reproductive function is discussed. © 1996 Wiley-Liss, Inc.

Milt production in common carp (Cyprinus carpio): stimulation by a goldfish steroid pheromone

This study examined the effect of a putative steroidal pheromone, 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) on milt volume and serum gonadotropin-II in precocious male common carp (40–285 g). 17,20β-P significantly increased both milt volume (14–18 h post-exposure) and GtH level (90 min post-exposure) at both doses tested (5 × 10−11 and 5 × 10−10 M in aquarium water), whereas a conjugated form of 17,20β-P (17,20β-P-sulphate) was ineffective at the only dose tested (5 × 10−11 M). Increases in milt, packed sperm volume, and GtH following exposure 5 × 10−10 M 17,20β-P also were compared to those induced by injection of Ovaprim (Syndel; mixture of salmon GnRH analogue and dopamine receptor antagonist) at two doses (0.1 and 0.5 ml/kg body weight). Increases in milt and GtH induced by the high Ovaprim dose were significantly greater than those induced by 5 × 10−10 M 17,20β-P. Increases in milt, and packed sperm volume, and GtH induced by the lower Ovaprim dose were not significantly larger than those induced by 17,20β-P. The results suggest: (1) in common carp, 17,20β-P has a pheromonal role in milt production similar to that demonstrated in goldfish; (2) waterborne 17,20β-P might be a practical non-invasive technique for enhancing milt production in cultured common carp.

Acute olfactory sensitivity of wild common carp, Cyprinus carpio, to goldfish hormonal sex pheromones is influenced by gonadal maturity

To determine whether wild common carp (Cyprinus carpio) employ as pheromones the same gonadal hormones as goldfish, and whether their olfactory sensitivity is influenced by gonadal condition, the olfactory responsiveness of Mississippi River carp to gonadal steroids was measured by electro-olfactogram recording during two spawning seasons. Thirty-three gonadal steroids and two prostaglandins were tested. Of the steroids, only 17α,20β-dihydroxy-4-pregnen-3-one (17α,20βP), 17α,20βP-sulfate (17α,20βP-S), and androstenedione (A) were specifically detected with high sensitivity. 17α,20βP was the most potent compound, with a threshold concentration of 10−13 M, while 17α,20βP-S, and A had thresholds of approximately 10−10 M. This pattern of sensitivity matches that of the goldfish, which strongly suggests that these species share steroidal pheromone systems. The olfactory sensitivities and specificities of adult male and female carp to steroids were similar to each other and did not vary with season. However, juvenile fish were less sensitive than adults to 17α,20βP, 17α,20βP-S, prostaglandin F2α, and 15-ketoprostaglandin F2α, and more sensitive to both A and L-serine: the sensitivity of the carp olfactory epithelium to putative pheromones was apparently influenced by gonadal maturity and likely also by hormonal condition.

Binding of the steroidal pheromone 17α,20β-dihydroxy-4-pregnen-3-one to goldfish (Carassius auratus) olfactory epithelium membrane preparations

Binding of the steroidal pheromone 17α,20β-dihydroxy-4-pregnen-3-one to goldfish (<i>Carassius auratus</i>) olfactory epithelium membrane preparations

Extreme olfactory specificity of male goldfish to the preovulatory steroidal pheromone 17?,20?-dihydroxy-4-pregnen-3-one

1. To determine the specificity of the goldfish (Carassius auratus) olfactory system to the reproductive pheromone 17α,20β-dihydroxy-4-pregnen-3-one (17α,20βP), and to determine if related sex steroids might also function as pheromones, electro-olfactogram (EOG) responses were recorded from mature male goldfish. 2. Of the 24 steroids tested, 17α,20βP was the most stimulatory. It had a detection threshold of 10−12M and at a concentration of 10−8M elicited an EOG response 3 times that elicited by 10−5M L-serine 3. 17α,20β,21-triol-4-pregnen-3-one, a metabolite of 17α,20βP, was the only other highly stimulatory steroid. Its threshold was 10−11M. 4. In cross-adaptation experiments EOG responses to all 21-carbon steroids were inhibited during adaptation to 17α,20βP: responses to this pheromone are transduced by a single receptor/transduction mechanism. 5. To verify the biological relevance of EOG recording whole animal responsiveness was determined by measuring blood gonadotropin. When goldfish were placed into homogeneous steroid solutions endocrine responsiveness strongly correlated with EOG recording. However, when steroids were added to aquaria containing fish, responses were less specific indicating that transient wisps of steroids trigger endocrine responses. 6. Although the extreme sensitivity and specificity of the goldfish olfactory system to 17α,20βP gives it the potential to serve as a highly specific cue, realization of this potential is probably determined by the dynamics of pheromone exposure.

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

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

Pheromone detection and olfactory pathways in the brain of the female African catfish, Clarias gariepinus

The olfactory tract of the African catfish, Clarias gariepinus, consists of two tracts, the medial and lateral olfactory tract. Ovulated female catfish are attracted by male steroidal pheromones. Attraction tests with catfish in which the medial and lateral olfactory tract have been selectively lesioned show that the effects of these pheromones are mediated by the medial olfactory tract. The central connections of the medial and lateral olfactory tract have been studied by retro- and anterograde transport techniques using horseradish peroxidase as a tracer. Upon entering the forebrain, the medial olfactory tract innervates the posterior pars ventralis and pars supracommissuralis of the area ventralis telencephali and the nucleus preopticus periventricularis, the nucleus preopticus and the nucleus recessus posterioris. Application of horseradish peroxidase to the olfactory epithelium shows that part of the innervation of the area ventralis telencephali and the nucleus preopticus periventricularis can be attributed to the nervus terminalis, which appears to be embedded in the medial olfactory tract. The lateral olfactory tract sends projections to the same brain areas but also innervates the nucleus habenularis and a large terminal field in the area dorsalis telencephali pars lateralis ventralis. Furthermore, the medial olfactory tract carries numerous axons from groups of perikarya localized in the area dorsalis telencephali. Contralateral connections have been observed in the olfactory bulb, telencephalon, diencephalon and mesencephalon. It is suggested that processes of the medial olfactory tract innervating the preoptic region may influence the gonadotropin-releasing hormone system and in doing so may lead to behavioral and physiological changes related to spawning.

Extreme olfactory sensitivity of mature and gonadally-regressed goldfish to a potent steroidal pheromone, 17?,20?-dihydroxy-4-pregnen-3-one

1. Electrical responses (electro-olfactogram; EOG) were obtained from the olfactory epithelium of goldfish exposed to a variety of sex steroids thought to function as pheromones, severall-amino acids, and a bile acid. Responses of male and female goldfish with both mature and regressed gonads were compared. 2. 17α,20β-dihydroxy-4-pregnen-3-one (17,20P) and progesterone were potent odorants. 17,20P had a detection threshold of 10−13M and at a concentration of 10−8M evoked an EOG response two to three times that of 10−5Ml-serine. Progesterone was less stimulatory than 17,20P. This finding corroborates recent endocrinological and behavioral studies (Stacey and Sorensen 1986; Dulka et al. 1987) which indicate that 17,20P is a potent pheromone in mature male goldfish. 3. Preliminary cross-adaptation experiments suggest that 17,20P is detected by receptor site(s) differing from those which detect bile acids andl-amino acids. 4. The goldfish olfactory epithelium did not respond to etiocholanolone glucuronide, 17β-estradiol glucuronide, testosterone glucuronide and 17β-estradiol, which have also been hypothesized to function as pheromones in fish. 5. The olfactory thresholds of goldfish tol-amino acids and taurocholic acid were similar to other species of fish: approximately 10−13M forl-serine and 10−9M forl-cysteine and taurocholic acid. 6. The olfactory sensitivities of mature male, mature female and gonadally-regressed fish were similar to all compounds tested suggesting that differences in olfactory receptor function are not responsible for the fact that only mature males exhibit behavioral and endocrine responses to pheromonal 17,20P.

Analysis of the steroid receptor of Achlya ambisexualis.

We have previously reported the discovery of a specific high-affinity binding protein for the fungal sex steroid pheromone antheridiol in the cytosol of Achlya ambisexualis male cells. In this report, we describe the fractionation of the binding protein from the cytosol by ammonium sulfate precipitation, the optimization of in vitro conditions for radioligand binding assays, and some of the biochemical properties of the binding protein. In the presence of sodium molybdate, the macromolecule has a sedimentation coefficient of 8.3 S in sucrose gradients of low ionic strength, a Stokes radius of 56.6 A (Sephacryl S-300 columns), a molecular weight of approximately 192,000, a frictional ratio of 1.5, and an axial ratio of 8.9. The binding protein can be eluted with 0.24 M KCl as a single peak from DEAE-Sephadex A-25 columns. These results indicate that this steroid-binding protein from a primitive eukaryotic microbe has in vitro biochemical properties that are similar to those of other known steroid receptors in higher organisms.

Effect of culture medium composition on pheromone receptor levels in Achlya ambisexualis

Sexual reproduction in the eukaryotic fungi Achlya is controlled by two steroid pheromones. Antheridiol is the steroid released by female cells that induces male sexual differentiation. The antheridiol-induced response of male cells has been shown to be influenced by the composition of the culture medium. The present study was designed to determine if the composition of the culture media might also affect the levels of antheridiol binding protein in the cytosol of male cells. The mycelial content of cytosolic steroid pheromone binding sites in Achlya ambisexualis E87 males was measured at daily intervals during 6 days of suspension culture in media containing different nitrogen sources. Levels of binding sites increased during the first 2 days in culture to a plateau that was maintained for the next 2–3 days. During the first 3 days in culture, levels were much lower in mycelia cultured in an enriched medium containing lactalbumin hydrolysate compared to mycelia cultured in defined media containing glutamic acid as the nitrogen source. The level of binding sites increased rapidly when mycelia were transferred from an enriched medium to a nutrient-free salt solution and decreased when mycelia were transferred from a defined to an enriched medium. The relative differences in cytosolic binding measured by in vitro radioligand saturation analysis were confirmed by in vivo uptake studies. It is concluded that the mycelial content of antheridiol binding sites can be experimentally manipulated by variations in the composition of the culture medium and/or the time period of incubation in the medium.

Aggression during sexual encounters between hormone-treated gonadectomized pigs in the presence or absence of boar pheromones

Aggressive and sexual behaviour was studied in 5-min paired encounters between castrated prepubertal boars (N = 14) and normal (N = 2) or bulbectomized (N = 2) oestrogen-treated ovariectomized gilts. The boars were made sexually active by treatment with 17β-oestradiol + 5α-dihydrotestosterone. Half of the boars were sprayed on the snout with a solution containing a mixture of the boar pheromones 3α-androstenol and 5α-androstenone immediately before testing, and the remainder were treated with the vehicle solution. Aggression was almost always initiated by the gilts, and neither bulbectomy nor pheromone treatment of the boars reduced its occurrence. The results suggest that the display of aggression by gilts towards boars of similar body size is unlikely to be suppressed by the steroidal pheromones present in boar saliva.

Detection of a pheromone-binding protein in the aquatic fungus Achlya ambisexualis

Sexual reproduction among the eukaryotic fungi of the genus, Achlya, is controlled by two steroid pheromones. Antheridiol is the pheromone constitutively produced by female cells that induces male sexual differentiation and development. A biologically active tritium-labeled derivative of antheridiol, [1,2- 3H]7-deoxy-7-dihydro-antheridiol ([ 3H]7dA), has been synthesized. Radioligand-binding studies have revealed the presence of a specific binding protein in the cytosol of male cells that may represent the endogenous receptor for antheridiol. Binding to this macromolecule was characterized by an apparent equilibrium dissociation constant and maximum binding capacity of approx. 7 × 10 −10 M and 1 100–2 000 fmoles/mg protein, respectively. Sedimentation analysis in sucrose gradients revealed that the binding protein distributes in the 8S region under low ionic strength and sodium molybdate-stabilized conditions. Under conditions of high ionic strength, in the presence or absence of 10 mM sodium molybdate, the binding site distributes in the 3.6S region of the gradient. Analysis of radioligand binding in the presence of other steroids and steroid hormones revealed that the binding is specific for antheridiol and its analog.

A note on the significance of boar salivary pheromones to the male-effect on puberty attainment in gilts

ABSTRACTEighteen Large White gilts were allocated to one of three treatments so that there were six gilts on each treatment. The treatments were (1) daily exposure to a nasal spray of boar saliva reinforced with 16-androstene steroid pheromones, (2) daily exposure to a nasal spray of saline and a mature boar for 20 min, and (3) daily exposure to a nasal spray of saline only.The experiment began when the gilts were between 158 and 165 days of age, and was terminated when they reached 240 to 247 days of age, the gilts were slaughtered soon afterwards.The results indicate that if salivary pheromones operate in the overall effect of the male on puberty attainment in gilts, the effect is of minor significance. Exposure to a mature boar confirmed the phenomenon of the 'male-effect' on puberty attainment in gilts.