Chem. Senses 29: 425-430,
2004
Chemical Senses Vol. 29 No. 5 © Oxford University Press 2004; all rights reserved
Natural Ligands of Hamster Aphrodisin
1 Biochimie de l’Olfaction et de la Gustation, Neurobiologie de l’Olfaction et de la Prise Alimentaire, bâtiment 526, INRA, Domaine de Vilvert, F-78352 Jouy-en-Josas Cedex, France and 2 Laboratoire de Neurobiologie Sensorielle, Ecole Pratique des Hautes Etudes, CNRS, 1 avenue des Olympiades, F-91305 Massy, France
Correspondence to be send to: J.-C. Pernollet, Neurobiologie de l’Olfaction et de la Prise Alimentaire, bâtiment 526, Domaine de Vilvert, F-78352, Jouy-en-Josas Cedex, France, Tel: 33 1 34 65 27 50; Fax: 33 1 34 65 27 65; E-mail: pernolle{at}jouy.inra.fr
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Abstract |
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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.
Key words: aphrodisin, hamster, pheromone, vaginal secretion, vomeronasal organ
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Introduction |
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Animals emit chemical information, called pheromones, into their environment via diverse secretion fluids for inter-individual communication among the same species, which favour reproduction and social life organization. In many species, in contrast with the large number of well-documented sex attractants in insects, mammal pheromones are still poorly identified chemical cues (Dulac and Torello, 2003
; Schaal et al., 2003; Brennan and Keverne, 2004). Female hamster vaginal secretions contain aphrodisiac substances that facilitate the mating behaviour of young males. The aphrodisiac effect is related to the presence of a protein, named aphrodisin. Aphrodisin is a 17 kDa soluble glycosylated protein originally isolated from hamster vaginal discharges, in which it occurs in relatively high concentrations (5 µg/mg of discharge) (Singer et al., 1986; Henzel et al., 1988). The aphrodisin gene is expressed in vagina, uterus, Bartholin’s glands and also in female hamster parotid glands (Kruh¿ffer et al., 1997; Mägert et al., 1999; Thavathiru et al., 1999). Aphrodisin stimulates the male copulatory behavior via the vomeronasal organ (Clancy et al., 1984; Kroner et al., 1996; Jang et al., 2001), but occurs in vaginal discharges before females reach fertility, suggesting another unknown function for aphrodisin (Mägert et al., 1999).
Aphrodisin consists of 151 amino acids with two disulfide bonds and a blocked N-terminus (pyroglutamic acid). Each N-glycosylation site (N41 and N69) is linked with only one N-acetylglucosamine residue (Singer et al., 1986). Aphrodisin belongs to the lipocalin superfamily, whose ß-barrel structure is well adapted to bind small hydrophobic molecules, as evidenced with the recombinant protein (Briand et al., 2000a). Moreover, crystallographic studies of recombinant aphrodisin suggest the presence of a serendipitous artifactual ligand in the binding pocket (Vincent et al., 2001). Among lipocalins, aphrodisin is highly homologous (40% sequence identity) with rat odorant-binding proteins OBP-1 and 1F, which are thought to carry volatile odorants towards olfactory receptors through the nasal mucus (Briand et al., 2000b). In insects, another class of pheromone-carrier proteins with roles analogous to vertebrate OBPs has been described. Insect pheromone-binding proteins (PBPs), like general OBPs, are structurally different from lipocalins without evolutionary relationships (Steinbrecht, 1998). In contrast to aphrodisin, which exhibits a classical lipocalin fold made of an eight-stranded anti-parallel ß-barrel, insect PBPs are all-helical disulfide-rich proteins (Sandler et al., 2000; Lartigue et al., 2004; Mohanty et al., 2004).
In spite of numerous studies on the pheromonal properties of aphrodisin, it has still not been definitely determined if aphrodisin has pheromonal activity by itself or needs the combination with low molecular mass ligands to be active (Singer et al., 1984; Singer and Macrides, 1990; Briand et al., 2004). Its homology with OBP suggests a role as a lipophilic compound carrier, but it could also intrinsically act as a pheromone by direct activation of neuron receptors of the vomeronasal organ. Aphrodisin can therefore be compared to other rodent lipocalins, the major urinary proteins (MUPs), occurring at high level in mouse urine, which bind small airborne chemicals, endowed with a pheromonal activity. However, when deprived of ligands, MUPs play an intrinsic pheromonal role in individual recognition (Hurst et al., 2001). Consequently, the question of whether the aphrodisiac properties of aphrodisin are solely borne by the protein or need the presence of bound small pheromones is still to be resolved.
To identify aphrodisin natural ligands, we prepared crude natural aphrodisin extracted from vaginal secretions, avoiding any contact with plastic, which may contaminate carrier protein (Lartigue et al., 2004). We extracted the protein and identified five non-covalent compounds, specifically bound onto natural aphrodisin.
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Materials and Methods |
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Collection of hamster vaginal secretions
To complete natural aphrodisin extraction, two separate collections of Syrian golden hamster (Mesocricetus auratus) vaginal secretions were made from five mature animals, three times a week over a period of 4 months. Hamsters (120–160 g) were housed individually in glass cages with fresh bedding so they had no contact with plastic. All experiments followed the rules of French Ministries (Agriculture; Research and Technology). Vaginal discharges were obtained by tactile genital stimulation of slightly anesthetized females using a cotton swab impregnated with MilliQ H2O. Cotton swabs obtained from five females were then placed in a 5 ml glass tube with 1 ml MilliQ and frozen immediately at –20°C until used.
Preparation of crude aphrodisin from vaginal secretions
Immediately prior to use, diluted vaginal secretions corresponding to 115 samples (
35 ml) were defrosted and pooled. All procedures were carried out at 4°C. Cotton swabs were removed and the resulting solution clarified by centrifugation at 10 000 x g for 20 min. After filtration (0.22 µm), diluted vaginal secretions were concentrated to 1 ml using a YM-10 ultrafiltration membrane (Amicon). The experiments were duplicated separately on the two independent vaginal secretion collections.
Electrophoresis, N-terminal sequencing and peptide mass finger printing
SDS/PAGE (16% acrylamide) was performed using a Mini-Protean II system (Bio-Rad, France). The molecular mass calibration kits low range and polypeptides (Bio-Rad) were used and the proteins stained with Serva blue G. The broad electrophoretic band corresponding to aphrodisin was excised and submitted to N-terminal sequencing using a Procise Applied Biosystems sequencer after passive transfer onto PVDF membrane. The excised electrophoretic band was also subjected to trypsinolysis for peptide mass fingerprinting using a Perkin Elmer Voyager DE-STR MALDI-TOF spectrometer. This approach confirmed that the crude preparation contained essentially aphrodisin.
Determination of aphrodisin natural ligands
Ligand extraction
As controls for putative contaminants, 1 mg of bovine 
-lactalbumin (Sigma) and of recombinant aphrodisin were prepared in 35 ml of 25 mM ammonium acetate buffer pH 8.0, treated and concentrated like crude aphrodisin. For ligand extraction, crude aphrodisin, recombinant aphrodisin and -lactalbumin were concentrated to 50 µl using a YM-3 Microcon centrifugal unit (Amicon). As estimated by SDS-PAGE, 2.5 mg of natural aphrodisin and control proteins (50 µl) were first denatured with 100 µl of 9 M GuCl by 21-h incubation in a sealed tube at 35°C. Proteins were then digested with 750 µl of 30% (wt/wt) proteinase K (Sigma) in 50 mM phosphate buffer, pH 8.0 by overnight incubation at 35°C. The reaction mixtures were then placed on ice and extracted with 50 µl of CHCl3. After centrifugation at 10 000 x g for 30 min at 4°C, the extracts were analysed by GC/MS. An aliquot of ammonium acetate buffer (50 µl) was treated with the same denaturation, proteolysis and extraction procedures and the chloroform extract was analysed by GC/MS as a blank.
GC/MS identification
Analyses were conducted on a gas chromatograph (6890N; Agilent Technologies Inc.) coupled to a mass spectrometer detector (5973N; Agilent Technologies Inc.). Samples (2 µl) were injected, via a purged-splitless injection system, on a non-polar capillary column (HP-5MS, 30 m x 0.25 mm, 0.25 µm film thickness; Agilent Technologies Inc.). The oven temperature gradient applied was 25°C for 5 min, then 5°C/min to 240°C and held there for 10 min. The carrier gas was helium at 1 ml/min. The column was directly connected to the mass-sensitive detector by an interface heated at 280°C. The electron impact energy was set at 70 eV and mass spectra were recorded in the range of 20–800 atomic mass units. Pheromones were identified by comparison of their retention time in gas chromatography and fragmentation in mass spectroscopy with mass spectra of NIST98 library. Candidate ligands were further confirmed by matching the retention times and mass spectra with those of commercially available synthetic material purchased from Sigma. The relative abundance of each ligand was determined from their integrated peak areas. Similar results were obtained with crude aphrodisin from the second vaginal secretion collection.
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Results |
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To identify putative molecules bound onto natural aphrodisin, we collected vaginal secretions from mature Syrian golden hamsters (M. auratus), avoiding any contact with plastic, which may contaminate carrier proteins (Lartigue et al., 2004
). As already described, hamster vaginal secretions mainly contain aphrodisin, with some other proteins and small peptides in minute amounts (Briand et al., 2000a). Thus, in order to avoid ligand release in the course of purification, we limited purification to a crude aphrodisin preparation. SDS–PAGE analysis of the preparation (Figure 1) revealed a broad electrophoretic band migrating around 17 kDa. The excised electrophoretic band was submitted to trypsinolysis for peptide mass fingerprinting. This approach confirmed that the crude preparation contained essentially aphrodisin. N-Terminal sequencing comfirmed the presence of aphrodisin, partially N-terminal unblocked, without protein contaminant in appreciable amount. So, this preparation, devoid of free small molecules, did not contain proteins in significant amount other than aphrodisin and was therefore called ‘crude aphrodisin’. Using a calibrated solution of recombinant aphrodisin produced by the yeast Pichia pastoris (Briand et al., 2000a), the amount of prepared crude aphrodisin was estimated to be 2.5 mg.
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To release non-covalently bound ligands, the protein was denatured and thoroughly proteolyzed. The same procedure was applied to recombinant aphrodisin produced by P. pastoris, which is similar to the natural aphrodisin (Briand et al., 2000a
), but did not make contact with the vaginal secretions and to -lactalbumin, a protein that does not bind hydrophobic molecules. Chloroform extracts of digested proteins were analysed by gas chromatography coupled with mass spectrometry. We identified five predominant compounds specifically present in natural aphrodisin, but not in control proteins, 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%) (Figure 2). These ligands were further confirmed by matching the retention times and mass spectra with those of commercially available synthetic material (Figure 3). The relative abundance of each compound revealed that 45% aphrodisin was bound with ligands. Dibutylphthalate, a contaminant originating from unavoidable plasticware, was also found in all protein extracts in equivalent trace amounts.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Up to now, all attempts to identify the endogenous ligand of native aphrodisin have been unsuccessful (Singer et al., 1986
; Henzel et al., 1988; Singer, 1991; Petrulis and Johnston, 1995; Ramoni et al., 2001), although identification of lipocalin-bound compounds based on mass spectroscopy and X-ray structure has been achieved in the past, for example in the case of MUPs, in which four pheromonal components were identified as copurified ligands (Böcskei et al., 1992) and bovine OBP, in which a product of rumination, 1-octen-3-ol has been observed (Ramoni et al., 2001). In the present study, owing to the large amounts of collected aphrodisin in vaginal secretions and to our mode of crude aphrodisin purification, we report the identification of five compounds bound into natural aphrodisin. Moreover, the complexes aphrodisin/ligands were well preserved during the preparation, since 45% of aphrodisin was found with bound ligands. Interestingly, these components are also described in insects, especially in the Lepidoptera (for example, 1-hexadecanol in Chilo zacconius and 1-octadecanol and hexadecanoic acid in Myelois cribrella), as part of female pheromone blends to attract males (Arn et al., 1992). This is not the only case of coincident substrates for sexual chemical communication shared in common by mammals and insects (Kelly, 1996). For instance, two Asian elephant pheromones are also described as insect pheromones. Z-7-dodecen-1-yl acetate from the pre-ovulatory urine of female Asian elephants has been identified as a sex attractant for moths (Rasmussen et al., 1996) and frontalin (1,5-dimethyl-6,8-dioxabicyclo[3.2.1]octane), a bicyclic ketal, released from the temporal gland, is a well-studied pheromone in insects (Rasmussen and Greenwood, 2003). The present findings, demonstrating the presence, on natural aphrodisin, of ligands, which are pheromones in unrelated species, raises the question of the intrinsic aphrodisiac activity of unliganded aphrodisin. Thus, aphrodisin would be either a pheromone carrier protein or, like rat MUP, would have a pheromone transport function, in addition to its own pheromonal role (Krieger et al., 1999). Interestingly, the sex pheromone present in the pre-ovulatory urine of female Asian elephants has been demonstrated to interact with a PBP, abundant in the mucus of the trunk, which is homologous to vertebrate OBPs (Lazar et al., 2002). The high homology of aphrodisin with OBPs suggests that at least one function of aphrodisin would be to transport the reported lipophilic ligands through the aqueous mucus to the vomeronasal receptor cells. We now aim at defining the pheromonal function of the blend or single components extracted from aphrodisin, complexed or not with recombinant aphrodisin. These investigations will provide novels insights into the communication responsible for the fascinating behavioural effects of pheromones in mammals.
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Acknowledgements |
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The authors are grateful to Claire Schlegel for her precious help during collection of hamster vaginal secretions.
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References |
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Accepted April 6, 2004
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