Natural ligands of hamster aphrodisin

The chemical nature of vertebrate pheromones remains largely to be deciphered. Hamster aphrodisin is a rare instance of mammal proteinaceous sexual pheromone. This protein, found in vaginal secretions, facilitates the mounting behaviour of males via activation of a specialized sensory structure named the vomeronasal organ, which activates the accessory olfactory bulb. Since it might carry small pheromonal ligands due to its lipocalin structure, we analysed organic extracts from natural aphrodisin. We identified five predominant compounds specifically bound onto natural aphrodisin as 1-hexadecanol (44.7%), 1-octadecanol (19.5%), Z-9-octadecen-1-ol (18.2%), E-9-octadecen-1-ol (15.4%) and hexadecanoic acid (2.2%). Interestingly, these compounds are also described as part of insect pheromone blends, disclosing the continuing story of amazing coincidences of chemical communication shared by mammals and insects.     

Crystal structure of aphrodisin, a sex pheromone from female hamster

We have solved the crystal structure of aphrodisin, a pheromonal protein inducing a copulatory behaviour in male hamster, using MAD methods with selenium, at 1.63 A resolution. The monomeric protein belongs to the lipocalin family, and possesses a disulfide bridge in a loop between strands 2 and 3. This disulfide bridge is characteristic of a family of lipocalins mainly identified in rodents, and is analogous to the fifth disulfide bridge of the long neurotoxins, such as alpha cobratoxin. An elongated electron density was found inside the buried cavity, which might represent a serendipitous ligand of unknown origin. The analysis of the water accessible surfaces of the side-chains bordering the cavity indicates that Phe76 may be the door for the natural ligand to access the cavity. This residue defines the entry of the cavity as belonging to the consensus for lipocalins. The face bearing Phe76 might also serve for the interaction with the receptor.     

Conspecific and heterospecific proteins related to aphrodisin lack aphrodisiac activity in male hamsters

Three proteins related to the hamster vaginal discharge proteinaphrodisin were purified, subjected to adsorption chromatographyfor removal of volatile compounds and tested for pheromonalactivity in an assay of copulatory behavior exhibited by malehamsters toward a surrogate female. One of the proteins is conspecificto aphrodisin, it is the second most abundant protein in thevaginal discharge. Like aphrodisin it migrates as a relativelyacidic protein in electrophoresis under non-denaturing conditions,and it appears to have an identical monomeric molecular mass(17 kd) in electrophoresis with 0.1% sodium dodecylsulfate.Heterospecific proteins included the female mouse major urinaryprotein (MUP) and ß-lactoglobulin from cow's milk,which have some similarity in amino acid sequence to aphrodisinand belong to the _2u-globulin protein superfamily. In spiteof these chemical relationships, neither the conspecific proteinnor the heterospecific proteins had aphrodisiac activity comparableto that of aphrodisin in the surrogate female behavioral assay.The pheromonal activity of aphrodisin thus appears to be dependenton specific structural features of the protein.     

Pheromonal communication in amphibians

Pheromonal communication is widespread in salamanders and newts and may also be important in some frogs and toads. Several amphibian pheromones have been behaviorally, biochemically and molecularly identified. These pheromones are typically peptides or proteins. Study of pheromone evolution in plethodontid salamanders has revealed that courtship pheromones have been subject to continual evolutionary change, perhaps as a result of co-evolution between the pheromonal ligand and its receptor. Pheromones are detected by the vomeronasal organ and main olfactory epithelium. Chemosensory neurons express vomeronasal receptors or olfactory receptors. Frogs have relatively large numbers of vomeronasal receptors that are transcribed in both the vomeronasal organ and the main olfactory epithelium. Salamander vomeronasal receptors apparently are restricted to the vomeronasal organ. To date, no chemosensory ligands have been matched to vomeronasal receptors or olfactory receptors so it is unknown whether particular receptor types are (1) specialized for detection of pheromones versus other chemosignals, or (2) specialized for detection of volatile, nonvolatile, or water-borne chemosignals. Despite progress in understanding amphibian pheromonal communication, only a small fraction of amphibian species have been examined. Study of additional species of amphibians will indicate which traits related to pheromonal communication are evolutionarily conserved and which traits have diverged over time.     

Identification of glandular (preputial and clitoral) proteins in house rat (Rattus rattus) involved in pheromonal communication

Proteins (18-20 kDa) belonging to lipocalin family have been reported to act as carriers for ligands binding to pheromones in mouse urine, pig saliva, hamster vaginal fluid and human sweat, that are involved in pheromonal communication. As the preputial gland is a major pheromonal source, the present study was aimed to detect the specific protein bands (around 18-20 kDa) in the preputial and clitoral glands of the house rat, R. rattus. The amount of protein was higher in preputial gland of the male than that of female (clitoral) gland. A 20 kDa protein was noted in male and female glands; however, the intensity of the band was much higher in male than in female. In addition, 70, 60, 35 kDa bands, identified in male preputial gland, were absent in females. The presence of higher concentration of glandular proteins in the male preputial gland suggests that male rats may depend more on these glandular proteins for the maintenance of reproductive and dominance behaviours. The results further suggest that these glandular proteins (20 kDa) may act as a carrier for ligand binding.     

Odorant and pheromone binding by aphrodisin, a hamster aphrodisiac protein

Aphrodisin is a soluble glycoprotein of hamster vaginal discharges, which stimulates male copulatory behavior. Natural aphrodisin was purified and its post-translational modifications characterized by MALDI-MS peptide mapping. To evaluate its ability to bind small volatile ligands, the aphrodisiac protein was expressed in the yeast Pichia pastoris as two major isoforms differing in their glycosylation degree, but close in conformation to the natural protein. Dimeric recombinant aphrodisins were equally able to efficiently bind odors (2-isobutyl-3-methoxypyrazine and methyl thiobutyrate) and a pheromone (dimethyl disulfide), suggesting that they could act as pheromone carriers instead of, or in addition to, direct vomeronasal neuron receptor activators.     

Post-translational isoprenylation of tryptophan

Bacillus subtilis and related bacilli produce a post-translationally modified oligopeptide, ComX pheromone, that stimulates natural genetic competence controlled by quorum sensing. The ComX pheromones are formed by geranylation or farnesylation on a tryptophan residue at the 3 position of its indole ring. This results in the formation of a tricyclic structure including, a newly formed five-membered ring, similar to proline. Isoprenylation of ComX to form ComX pheromones is essential for pheromonal activity, and is functionally more crucial than its amino acid sequence. The ComX pheromone is the first example of isoprenoidal modifiations of tryptophan residues in living organisms and post-translational isoprenylation of any amino acid in prokaryotes. Because the presence of geranylated compounds is unusual in primary and secondary metabolites outside the plant kingdom, post-translational geranylation in bacilli is unprecedented in nature.     

Social chemosignals from breastfeeding women increase sexual motivation

Human pheromones, a type of social chemosignal, modulate endocrine function by regulating the timing of ovulation. In animals, pheromones not only regulate ovulation but also female reproductive motivation and behavior. There is no extant evidence that humans produce social chemosignals that affect human sexual motivation or reproductive behavior as occurs in other mammals. Here, we demonstrate that natural compounds collected from lactating women and their breastfeeding infants increased the sexual motivation of other women, measured as sexual desire and fantasies. Moreover, the manifestation of increased sexual motivation was different in women with a regular sexual partner. Those with a partner experienced enhanced sexual desire, whereas those without one had more sexual fantasies. These results are consistent with previous pheromonal effects on endocrine function, and warrant further study of these social chemosignals as candidates for pheromonal processes, including their effects on other aspects of motivation and behavior.     

The primary structure of aphrodisin

Aphrodisin is a protein which is secreted in hamster vaginal discharge and acts via the vomeronasal organ of the accessory olfactory system to elicit copulatory behavior in male hamsters. The complete primary structure of aphrodisin was determined by sequence analysis of intact aphrodisin after unblocking the amino terminus with pyroglutamate aminopeptidase and from peptides generated by trypsin and Lys-C digests. Alignment of the peptides was obtained from sequence analysis of peptides from cyanogen bromide and hydroxylamine cleavages. The protein consists of 151 residues of Mr = 17,000. It has disulfide bonds linking cysteine residues at positions 38 and 42 and at 57 and 149. N-acetylglucosamine residues are linked to asparagines at positions 41 and 69. Based on its similarity to the major urinary proteins in rats and mice, aphrodisin is a putative member of the alpha 2u-globulin superfamily of extracellular proteins.     

Pheromonal and behavioral cues trigger male-to-female aggression in Drosophila

Appropriate displays of aggression rely on the ability to recognize potential competitors. As in most species, Drosophila males fight with other males and do not attack females. In insects, sex recognition is strongly dependent on chemosensory communication, mediated by cuticular hydrocarbons acting as pheromones. While the roles of chemical and other sensory cues in stimulating male to female courtship have been well characterized in Drosophila, the signals that elicit aggression remain unclear. Here we show that when female pheromones or behavior are masculinized, males recognize females as competitors and switch from courtship to aggression. To masculinize female pheromones, a transgene carrying dsRNA for the sex determination factor transformer (traIR) was targeted to the pheromone producing cells, the oenocytes. Shortly after copulation males attacked these females, indicating that pheromonal cues can override other sensory cues. Surprisingly, masculinization of female behavior by targeting traIR to the nervous system in an otherwise normal female also was sufficient to trigger male aggression. Simultaneous masculinization of both pheromones and behavior induced a complete switch in the normal male response to a female. Control males now fought rather than copulated with these females. In a reciprocal experiment, feminization of the oenocytes and nervous system in males by expression of transformer (traF) elicited high levels of courtship and little or no aggression from control males. Finally, when confronted with flies devoid of pheromones, control males attacked male but not female opponents, suggesting that aggression is not a default behavior in the absence of pheromonal cues. Thus, our results show that masculinization of either pheromones or behavior in females is sufficient to trigger male-to-female aggression. Moreover, by manipulating both the pheromonal profile and the fighting patterns displayed by the opponent, male behavioral responses towards males and females can be completely reversed. Therefore, both pheromonal and behavioral cues are used by Drosophila males in recognizing a conspecific as a competitor.     

Peripheral, central and behavioral responses to the cuticular pheromone bouquet in Drosophila melanogaster males

Pheromonal communication is crucial with regard to mate choice in many animals including insects. Drosophila melanogaster flies produce a pheromonal bouquet with many cuticular hydrocarbons some of which diverge between the sexes and differently affect male courtship behavior. Cuticular pheromones have a relatively high weight and are thought to be -- mostly but not only -- detected by gustatory contact. However, the response of the peripheral and central gustatory systems to these substances remains poorly explored. We measured the effect induced by pheromonal cuticular mixtures on (i) the electrophysiological response of peripheral gustatory receptor neurons, (ii) the calcium variation in brain centers receiving these gustatory inputs and (iii) the behavioral reaction induced in control males and in mutant desat1 males, which show abnormal pheromone production and perception. While male and female pheromones induced inhibitory-like effects on taste receptor neurons, the contact of male pheromones on male fore-tarsi elicits a long-lasting response of higher intensity in the dedicated gustatory brain center. We found that the behavior of control males was more strongly inhibited by male pheromones than by female pheromones, but this difference disappeared in anosmic males. Mutant desat1 males showed an increased sensitivity of their peripheral gustatory neurons to contact pheromones and a behavioral incapacity to discriminate sex pheromones. Together our data indicate that cuticular hydrocarbons induce long-lasting inhibitory effects on the relevant taste pathway which may interact with the olfactory pathway to modulate pheromonal perception.     

Pheromonal control: reconciling physiological mechanism with signalling theory

Pheromones are intraspecific chemical signals. They can have profound effects on the behaviour and/or physiology of the receiver, and it is still common to hear pheromones described as controlling of the behaviour of the receiver. The discussion of pheromonal control arose initially from a close association between hormones and pheromones in the comparative physiological literature, but the concept of a controlling pheromone is at odds with contemporary signal evolution theory, which predicts that a manipulative pheromonal signal negatively affecting the receiver's fitness should not be stable over evolutionary time. Here we discuss the meaning of pheromonal control, and the ecological circumstances by which it might be supported. We argue that in discussing pheromonal control it is important to differentiate between control applied to the effects of a pheromone on a receiver's physiology (proximate control), and control applied to the effects of a pheromone on a receiver's fitness (ultimate control). Critically, a pheromone signal affecting change in the receiver's behaviour or physiology need not necessarily manipulate the fitness of a receiver. In cases where pheromonal signalling does lead to a reduction in the fitness of the receiver, the signalling system would be stable if the pheromone were an honest signal of a social environment that disadvantages the receiver, and the physiological and behavioural changes observed in the receiver were an adaptive response to the new social circumstances communicated by the pheromone.     

The crystal structure of a cockroach pheromone-binding protein suggests a new ligand binding and release mechanism

Pheromone-binding proteins (PBPs) are small helical proteins found in sensorial organs, particularly in the antennae, of moth and other insect species. They were proposed to solubilize and carry the hydrophobic pheromonal compounds through the antennal lymph to receptors, participating thus in the peri-receptor events of signal transduction. The x-ray structure of Bombyx mori PBP (BmorPBP), from male antennae, revealed a six-helix fold forming a cavity that contains the pheromone bombykol. We have identified a PBP (LmaPBP) from the cockroach Leucophaea maderae in the antennae of the females, the gender attracted by pheromones in this species. Here we report the crystal structure of LmaPBP alone or in complex with a fluorescent reporter (amino-naphthalen sulfonate, ANS) or with a component of the pheromonal blend, 3-hydroxy-butan-2-one. Both compounds bind in the internal cavity of LmaPBP, which is more hydrophilic than BmorPBP cavity. LmaPBP structure ends just after the sixth helix (helix F). BmorPBP structure extends beyond the sixth helix with a stretch of residues elongated at neutral pH and folding as a seventh internalized helix at low pH. These differences between LmaPBP and BmorPBP structures suggest that different binding and release mechanism may be adapted to the hydrophilicity or hydrophobicity of the pheromonal ligand.     

Behavioral assay and chemical characters of female sex pheromones in the hermit crab <Emphasis Type="Italic">Pagurus filholi</Emphasis>

Males of the hermit crab Pagurus filholi perform assessment behavior toward females, as a preliminary step of precopulatory guarding, during the reproductive season. It is known that such behavior is elicited by female sex pheromones, but the compounds involved have never been characterized in this species. Several experiments were conducted to develop a reliable bioassay along with purification procedures to identify potential compounds with pheromonal activity in Pagurus filholi. We developed a bioassay protocol to assess pheromonal activity by using an empty shell with cotton containing either artificial seawater (control) or test water. We measured and compared the time duration of male assessment behavior toward each shell if the test water contained female sex pheromones. Ultra-filtering of seawater samples potentially containing pheromones showed that the compound was <1 kDa in molecular weight. Males showed precopulatory assessment behavior toward “female conditioned” water samples treated with open column purification and eluted with MeOH, suggesting that compounds triggering male behavior were low polar molecules. Molecules with pheromonal activity were not volatile after freeze drying, effective even after heating to 90 °C, and remained active in seawater at 12 °C even after 6 days from sample collection, which suggests a rather stable characteristic of the female sex pheromones of this species.     

Phytotoxicity of pheromonal chemicals to fruit tree foliage: chemical and physiological characterization.

Some recent high-load, low-density pheromone-release devices emit an ethanolic blend of pheromone directly onto crop foliage to control insect pests by mating disruption. This study characterized the phytotoxicity associated with deposition of some pheromonal compounds in concentrated drops on the foliage of trees bearing aerosol release devices. The relative toxicity of straight-chained alkanes, alcohols, aldehydes, and acetates with chain lengths varying from C-2 to approximately C-20 was quantified in the laboratory by the severity of necrotic lesions. The order of severity for phytotoxicity caused by pheromonal compounds was alkanes < acetates = aldehydes < or = alcohols. Within compound classes tested, pheromones with chain lengths of 6-13 carbons were the most phytotoxic. Phytotoxicity was not detectable at dosages <1 mg administered in 10 microl of ethanol. Phytotoxicity of pheromones was highly correlated with presence of both a hydrophilic and lipophilic molecular domain. We postulate nonspecific membrane disruption as a likely mode of action for pheromonal phytotoxicity. Limited attempts to remediate this effect by changing carrier solvents or adding surfactants, spreaders, or nonvolatile diluents were not successful. Because the toxic action of pheromones upon plant tissues appears relatively benign, and growers have not been adverse to localized phytotoxicity to foliage and fruits on two trees per 0.4 ha, we propose that limited phytotoxicity associated with first-generation aerosol dispenser technology can be viewed as non-threatening.     

Aphrodisin: pheromone or transducer?

Aphrodisin, the major soluble protein in hamster vaginal discharge,is detected by receptors within the vomeronasal organ of themale hamster, and stimulates copulatory behavior. The loss ofthis effect on behavior after degradation of the protein withheat or proteolytic enzymes shows that the polypeptide chainis an essential part of the pheromone. Furthermore, attemptsto remove small molecules from the protein have provided littleindication of the presence of a transported ligand. However,the chemical and physical properties of the protein itself indicatethat it could bind low molecular weight, water-insoluble compounds.The abundance, size, charge, and the primary structure of aphrodisin,when considered together, all indicate that it is a member ofthe recently recognized -2u-globulin superfamily of extracellularproteins, some of which, such as serum retinol-binding proteinand odorant-binding protein, are known to bind smaller molecules.Preliminary results from a study of the effects of bacterialaphrodisin, produced by molecular cloning in E.coli, on behaviorindicate that the polypeptide backbone is only partially activeand that post-translational modifications of the protein orthe presence of an as yet undetected ligand may be necessaryfor full activity.     

Roles for learning in mammalian chemosensory responses

This article is part of a Special Issue “Chemosignals and Reproduction”.A rich variety of chemosignals have been identified that influence mammalian behaviour, including peptides, proteins and volatiles. Many of these elicit innate effects acting either as pheromones within species or allelochemicals between species. However, even innate pheromonal responses in mammals are not as hard-wired as the original definition of the term would suggest. Many, if not most mammalian pheromonal responses are only elicited in certain behavioural or physiological contexts. Furthermore, certain pheromones are themselves rewarding and act as unconditioned stimuli to link non-pheromonal stimuli to the pheromonal response, via associative learning. The medial amygdala, has emerged as a potential site for this convergence by which learned chemosensory input is able to gain control over innately-driven output circuits. The medial amygdala is also an important site for associating social chemosensory information that enables recognition of conspecifics and heterospecifics by association of their complex chemosensory signatures both within and across olfactory chemosensory systems. Learning can also influence pheromonal responses more directly to adapt them to changing physiological and behavioural context. Neuromodulators such as noradrenaline and oxytocin can plasticise neural circuits to gate transmission of chemosensory information. More recent evidence points to a role for neurogenesis in this adaptation, both at the peripheral level of the sensory neurons and via the incorporation of new neurons into existing olfactory bulb circuits. The emerging picture is of integrated and flexible responses to chemosignals that adapt them to the environmental and physiological context in which they occur.     

Synaptic mechanisms underlying pheromonal memory in vomeronasal system

When female mice are mated, they form a memory of the pheromonal signal of the male with which they mated. Our research objective was to determine the neural mechanisms underlying learning and memory by employing a convenient model of pheromone-induced olfactory memory (pheromonal memory). Formation of pheromonal memory depends on the association between mating and exposure to pheromones. Synaptic plasticity involving this memory occurs in the accessory olfactory bulb (AOB), depending on vaginocervical stimulation at mating. The vaginocervical stimulation at mating reduces the dendrodendritic feedback inhibition of principal neurons (mitral/tufted (MT) cells) in the AOB and enhances their cell activity. The enhancement of activity induces on these plastic changes in dendrodendritic synapses, which in turn enhance GABA-mediated inhibition of MT cell activity. This "self-inhibition" of MT cells activity in response to pheromonal signals of the partner can disrupt its signals at the AOB thereby preventing the signals from reaching the central brain. The formation and maintenance of pheromonal memory is based on this inhibition mechanism.     

Olfactory stress and modification of phagocytosis in peripheral blood cells of adult male mice]

Data on pheromonal influence on phagocytic activity of leukocytes in peripheral blood of adult randombred and CBA male mice have been obtained. The identified mouse pheromone 2,5-dimethylpyrazine was used, which induces some physiological effects associated with reproduction in both mouse males and females. Significant differences in spontaneous level of phagocytosis were between inbred CBA and randombred animals: the frequency of phagocytic cells was lower in CBA males by 1.4 times. The substance tested here induces phagocytosis in randombred (by 1.7 times) males. A low dose of 2,5-dimethylpyrazine (similar to the natural pheromone concentration) induces a higher increase in phagocytic activity by leukocytes. Possible mechanisms of pheromonal action on phagocytosis are discussed with the perspectives of finding highly effective immunomodulators among mammalian pheromones.     

鱼类嗅觉系统和性信息素受体的研究进展

鱼类嗅觉系统包括外部嗅觉器官、嗅神经和嗅球三个部分.嗅觉器官也称为嗅囊,由嗅上皮和髓质组成.气味物质的化学信息主要由嗅上皮上随机分布的嗅觉感受神经元感知,通过嗅神经将嗅觉信息传递到嗅球,嗅球在空间上有不同的功能分区,嗅觉信息经过嗅球各分区整合后分别传入端脑,发挥其生理功能.性信息素在鱼类生殖过程中的作用是通过嗅觉系统来完成的,其中嗅觉感受神经元上的性信息素受体起着重要作用.鱼类性信息素受体的研究主要从两个方面入手,一是从低浓度特异的性信息素引起嗅觉器官电生理反应或行为反应入手,寻找特异的性信息素受体;二是参照哺乳动物嗅觉受体的研究结果,从嗅觉受体基因遗传保守性入手,研究鱼类性信息素受体的结构与功能.