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.     

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.     

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.     

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.     

Differential histochemical localization patterns of reduced nicotinamide adenine dinucleotide phosphate-diaphorase and neuronal nitric oxide synthase during postnatal development of the rat vomeronasal organ

The present study was undertaken to examine the localization patterns of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) by enzyme histochemistry and neuronal nitric oxide synthase (NOS) by immunohistochemistry in the vomeronasal organ of rat from postnatal day 0 for 8 weeks (adult). Nicotinamide adenine dinucleotide phosphate-diaphorase activity was not observed in the sensory epithelium of the vomeronasal organ at postnatal day 0 (the day of birth) and at day 1. At postnatal day 2, NADPH-d activity was observed in several vomeronasal neurons and on the surface of the sensory epithelium. From 25 days through adulthood, the number of vomeronasal neurons having NADPH-d activity increased gradually. On the other hand, neuronal NOS immunoreactivity was not observed in the sensory epithelium of the vomeronasal organ in newborns or in the adult rat. In this study, it is suggested that the nitric oxide pathway in the sensory epithelium of the vomeronasal organ comes into play beyond postnatal day 3. Moreover, it was found that NADPH-d and neuronal NOS are not colocalized in the sensory epithelium of the developing rat vomeronasal organ.     

Chemical properties of a female mouse pheromone that stimulates gonadotropin secretion in males

Female mouse urine contains a pheromone that acts via the vomeronasal organ of conspecific males to stimulate a rapid increase in circulating levels of luteinizing hormone. A bioassay based on this male response was used to test biochemical preparations of female urine. Retention of significant biological activity by the urine after dialysis indicated that the activity is associated with urinary protein. Complete loss of activity from the urine after adsorption chromatography on a neutral polystyrene column suggested that the protein functions as a pheromone carrier. Assay of gel permeation chromatography fractions, before and after degradation of the urinary proteins with proteolytic enzymes, demonstrated that the protein is not necessary for the male response in the bioassay. Its resistance to vigorous proteolytic enzyme treatment further indicates that the pheromone is not a peptide. High biological activity, indistinguishable from that of the unfractionated urine, was isolated in a protein-depleted, presumably low molecular weight fraction containing compounds that are retarded by adsorption on Sephadex. The chemical properties of this female mouse pheromone are markedly different from those of a recently purified female hamster pheromone that also acts via the vomeronasal organ.     

The rodent accessory olfactory system

The accessory olfactory system contributes to the perception of chemical stimuli in the environment. This review summarizes the structure of the accessory olfactory system, the stimuli that activate it, and the responses elicited in the receptor cells and in the brain. The accessory olfactory system consists of a sensory organ, the vomeronasal organ, and its central projection areas: the accessory olfactory bulb, which is connected to the amygdala and hypothalamus, and also to the cortex. In the vomeronasal organ, several receptors—in contrast to the main olfactory receptors—are sensitive to volatile or nonvolatile molecules. In a similar manner to the main olfactory epithelium, the vomeronasal organ is sensitive to common odorants and pheromones. Each accessory olfactory bulb receives input from the ipsilateral vomeronasal organ, but its activity is modulated by centrifugal projections arising from other brain areas. The processing of vomeronasal stimuli in the amygdala involves contributions from the main olfactory system, and results in long-lasting responses that may be related to the activation of the hypothalamic–hypophyseal axis over a prolonged timeframe. Different brain areas receive inputs from both the main and the accessory olfactory systems, possibly merging the stimulation of the two sensory organs to originate a more complex and integrated chemosensory perception.     

Organization of the vomeronasal organ in a plethodontid salamander

Salamanders in the family Plethodontidae show a unique behavior (nose-tapping) and have unique structures (nasolabial grooves) that may be used specifically to convey chemicals to the vomeronasal organ. The nasal structure of Plethodon cinereus was studied to determine if there is enhanced development of the vomeronasal organ compared with other salamander families that would correlate with use of these unique features. The vomeronasal organ in salamanders is found in a ventrolateral diverticulum of each main olfactory organ. P. cinereus has a more anteriorly placed vomeronasal organ within the diverticulum, and the posterior limit of each nasolabial groove is adjacent to the anterior limit of the vomeronasal organs. This suggests that the grooves deliver chemicals preferentially to the vomeronasal organs instead of to the main olfactory organs. In addition, the vomeronasal sensory epithelium is thickest anteriorly and is at its thinnest at about the level corresponding to the location of the vomeronasal organ in other salamander families. These adaptations suggest a specific mechanism of odorant delivery to the vomeronasal organ in plethodontid salamanders not found in other salamander families.     

4种两栖爬行动物嗅器和犁鼻器的显微结构比较

用光镜观察了4种两栖爬行动物嗅器和犁鼻器的组织结构.结果显示,北方山溪鲵(Batrachuperus tibetanus)鼻囊内开始分化出犁鼻器,犁鼻器位于嗅器的腹外侧,但犁鼻器还不发达;隆肛蛙(Feirana quadranus)犁鼻器与嗅器虽然共同位于鼻囊内,但犁鼻器较为发达且其周围有发达的犁鼻腺,犁鼻器通过一细小管道与嗅器相通;秦岭蝮(Gloydius qinlingensis)和菜花烙铁头(Trimeresurus jerdonii)犁鼻腔与鼻腔已经完全分离形成两个独立的囊,而且鼻腔又进一步分化为嗅部与呼吸部.说明犁鼻器从有尾两栖动物开始出现,至无尾两栖类开始分化,到蛇类高度发达且成为一个独立器官.犁鼻器的形成是脊椎动物适应陆地生活的直接结果,是四足动物的特征之一.     

Pre-exposure to female chemosignals or intracerebral GnRH restores mating behavior in naive male hamsters with vomeronasal organ lesions

Chemosensory input is essential for mating in male hamsters and the vomeronasal organ is critical to mating in naive males. In studies to investigate the convergence of vomeronasal chemosensory input and the neurohormone gonadotrophin releasing hormone (GnRH), we have unexpectedly found that pre-exposure to pheromone-containing chemosignals from female hamsters will also eliminate mating deficits normally seen in naive male hamsters with vomeronasal organs removed (VNX). In the present studies, naive-intact and naive-VNX male hamsters were given intracerebroventricular injections of GnRH or saline and exposed to female pheromones found in hamster vaginal fluid (HVF) or to water 40 min prior to a 5 min mating test. VNX males given saline injections and exposed to water had severe mating deficits, but VNX males given saline injections and exposed to HVF mated normally. As shown previously, males given GnRH injections and exposed to water also mated normally. HVF exposure prior to a mating test apparently acted to compensate for the lack of vomeronasal input in these males.     

Tongue manipulation of the palate assists estrous detection in the bovine

To understand the mechanisms for introducing urine or vaginal secretions into the vomeronasal organ, we used 16 mm cinematography and a freeze frame/slow motion technique to analyze the mouth and tongue movements of Brahman bulls while they examined the vulvas of restrained, estrogen-primed cows. Prior to flehmen, the mouth slowly opened, the curled tip of the tongue compressed the hard palate and the body of the tongue protruded from the mouth. The tongue maintained this form and moved forward. Once the tip of the tongue reached the incisive papilla, the body of the tongue retracted and the tip of the tongue relaxed. This tongue compression stroke (TCS) of the hard palate occurred 2 to 6 times, lasting 1 4 to 1 2 sec/stroke. Pressure changes in the vomeronasal organ are assumed to occur during and following TCSs, resulting in aspiration of any liquid in the incisive pit into the incisive and vomeronasal ducts. Such aspiration probably does not occur during flehmen because the tongue is relaxed and on the floor of the mouth.     

Effect of the odour of vaginal secretion on non-copulatory behaviour of male hamsters (Mesocricetus auratus)

Male hamsters were exposed to female vaginal secretion (in the absence of the female). When subsequently tested with females, their behaviour was determined by whether the test female matched or did not match the vaginal secretion. Less time was spent with mismatching females and less sniffing and licking was directed towards them. The effect of vaginal odour was present up to 3–4 h after the last contact with the secretion. The number of encounters with the vaginal odour and the spacing between them was important in determining the response to mismatching females. Contact with the vaginal secretion as opposed to its volatile components alone was necessary for the discrimination to occur. Discussion centres around the role of the vomeronasal organ and possible physiological pathways underlying the effect.     

A classification schema for the vomeronasal organ in humans

The vomeronasal organ is a chemoreceptive structure located at the base of the nasal septum with direct axonal connections to the accessory olfactory bulb in many terrestrial vertebrates. Pheromones presumably bind to the vomeronasal organ and exert behavioral or physiologic responses, thereby allowing chemical communication between animals of the same species. The presence and function of the vomeronasal organ in humans is debated. A phenotypic classification schema for the human vomeronasal organ is described and applied to 253 human subjects who underwent nasal examination. Of these subjects, only 6 percent possessed a vomeronasal organ with 64 percent unilateral and 36 percent bilateral in appearance. No difference existed in gender, age, or race between those subjects with or without a vomeronasal organ. There is no evidence supporting involutional senescence of this structure. Future investigations should use this phenotypic schema for the vomeronasal organ to allow accurate comparisons of study populations.     

The structure of the nasal chemosensory system in squamate reptiles. 2. Lubricatory capacity of the vomeronasal organ

The vomeronasal organ is a poorly understood accessory olfactory organ, present in many tetrapods. In mammals, amphibians and lepidosaurian reptiles, it is an encapsulated structure with a central, fluid-filled lumen. The morphology of the lubricatory system of the vomeronasal organ (the source of this fluid) varies among classes, being either intrinsic (mammalian and caecilian amphibian vomeronasal glands) or extrinsic (anuran and urodele nasal glands). In the few squamate reptiles thus far examined, there are no submucosal vomeronasal glands. In this study, we examined the vomeronasal organs of several species of Australian squamates using histological, histochemical and ultrastructural techniques, with the goal of determining the morphology of the lubricatory system in the vomeronasal organ. Histochemically, the fluid within the vomeronasal organ of all squamates is mucoserous, though it is uncertain whether mucous and serous constituents constitute separate components. The vomeronasal organ produces few secretory granules intrinsically, implying an extrinsic source for the luminal fluid. Of three possible candidates, the Harderian gland is the most likely extrinsic source of this secretion.     

Encoding pheromonal signals in the mammalian vomeronasal system

The past few years have delivered substantial progress in understanding the molecular logic of the mammalian vomeronasal system. Selective expression of vomeronasal receptors and high response selectivity of vomeronasal receptor neurons suggest that pheromones are encoded by labeled lines at the level of the vomeronasal organ: each pheromonal compound is represented by the activation of a small and exclusive subset of receptor neurons. Labeled lines might be transferred to the accessory olfactory bulb through convergent connections. The key challenges ahead will be to identify the pheromonal ligands of the receptors and unravel the functional connectivity from the vomeronasal organ to the hypothalamus.     

Functional Architecture of the Vomeronasal Organ of the Frog (Genus Rana)

Abstract The vomeronasal organ in the frog, genus Rana, is composed of three interconnected cavities; superior, middle and inferior, which are separated from and anterior to the principal olfactory cavity. The superior cavity is found just underneath the external naris and forms a vestibule both for the principal olfactory organ and the vomeronasal organ. The vomeronasal sensory epithelium is located in the medial region of the inferior cavity and contains ciliated cells and microvillous receptor cells. Inspection of microscopic sections of frogs that had been swimming in fluorescent colorants revealed fluorescence on the surface of the vomeronasal organ, but not on that of the olfactory organ. Observations in vivo show that water enters via the external naris by two fissures, one on each side of the movable nasal lid, passes the middle cavity to flow via the sensory epithelium of the inferior cavity. The design of the frog nose makes it possible for this amphibious animal to sample the chemical composition of its environment; above water the frog can inhale air and expose its olfactory organ to volatile substances; in water the vomeronasal organ samples water-borne substances. These new findings are discussed in relation to the air/water interface and the position of the amphibians in the evolution of terrestrial vertebrates.     

Vomeroglandin/CRP-Ductin is strongly expressed in the glands associated with the mouse vomeronasal organ: identification and characterization of mouse vomeroglandin

Vomeroglandin, a subform of mouse CRP-ductin, is a protein strongly expressed in the glands of mouse vomeronasal system. Both the proteins contain several of scavenger receptor cysteine-rich and CUB domains and one ZP domain. This domain arrangement is similar to those of rat Ebnerin, human DMBT1, and rabbit hensin. In situ hybridization analysis shows strong expression of vomeroglandin mRNA in the glands of vomeronasal system. Immunological analyses detect both membrane-bound and secreted forms of vomeroglandin. The secreted protein seems to be localized in the lumen of the vomeronasal organ, playing a certain role in the pheromone perception.     

Identification and characterization of new protein chemoattractants in the frog skin secretome

The vomeronasal organ is a chemosensory organ present in most vertebrates and involved in chemical communication. In the last decade, the deciphering of the signal transduction process of this organ has progressed. However, less is known about the vomeronasal organ ligands and their structure-function relationships. Snakes possess a highly developed vomeronasal system that is used in various behaviors such as mating, predator detection, or prey selection, making this group a suitable model for study of the vomeronasal chemoreception. In this work, we used a proteomics approach to identify and characterize proteins from frog cutaneous mucus proteome involved in prey recognition by snakes of the genus Thamnophis. Herein we report the purification and characterization of two proteins isolated from the frog skin secretome that elicit the vomeronasal organ-mediated predatory behavior of Thamnophis marcianus. These proteins are members of the parvalbumin family, which are calcium-binding proteins generally associated to muscular and nervous tissues. This is the first report that demonstrates parvalbumins are not strictly restricted to intracellular compartments and can also be isolated from exocrine secretions. Purified parvalbumins from frog muscle and mucus revealed identical chemoattractive properties for T. marcianus. Snake bioassay revealed the Ca(2+)/Mg(2+) dependence of the bioactivity of parvalbumins. So parvalbumins appear to be new candidate ligands of the vomeronasal organ.     

Development of the nasal chemosensory organs in two terrestrial anurans: the directly developing frog, Eleutherodactylus coqui (Anura: Leptodactylidae), and the metamorphosing toad, Bufo americanus (Anura: Bufonidae)

Nearly all vertebrates possess an olfactory organ but the vomeronasal organ is a synapomorphy for tetrapods. Nevertheless, it has been lost in several groups of tetrapods, including aquatic and marine animals. The present study examines the development of the olfactory and vomeronasal organs in two terrestrial anurans that exhibit different developmental modes. This study compares the development of the olfactory and vomeronasal organs in metamorphic anurans that exhibit an aquatic larva (Bufo americanus) and directly developing anurans that have eliminated the tadpole (Eleutherodactylus coqui). The olfactory epithelium in larval B. americanus is divided into dorsal and ventral branches in the rostral and mid-nasal regions. The larval olfactory pattern in E. coqui has been eliminated. Ontogeny of the olfactory system in E. coqui embryos starts to vary substantially from the larval pattern around the time of operculum development, the temporal period when the larval stage is hypothesized to have been eliminated. The nasal anatomy of the two frogs does not appear morphologically similar until the late stages of embryogenesis in E. coqui and the terminal portion of metamorphosis in B. americanus. Both species and their respective developing offspring, aquatic tadpoles and terrestrial egg/embryos, possess a vomeronasal organ. The vomeronasal organ develops at mid-embryogenesis in E. coqui and during the middle of the larval period in B. americanus, which is relatively late for neobatrachians. Development of the vomeronasal organ in both frogs is linked to the developmental pattern of the olfactory system. This study supports the hypothesis that the most recent common ancestor of tetrapods possessed a vomeronasal organ and was aquatic, and that the vomeronasal organ was retained in the Amphibia, but lost in some other groups of tetrapods, including aquatic and marine animals.     

Olfactory receptors and signalling elements in the Grueneberg ganglion

The Grueneberg ganglion (GG) is a cluster of neurones present in the vestibule of the anterior nasal cavity. Although its function is still elusive, recent studies have shown that cells of the GG transcribe the gene encoding the olfactory marker protein (OMP) and project their axons to glomeruli of the olfactory bulb, suggesting that they may have a chemosensory function. Chemosensory responsiveness of olfactory neurones in the main olfactory epithelium (MOE) and the vomeronasal organ (VNO) is based on the expression of either odorant receptors or vomeronasal putative pheromone receptors. To scrutinize its presumptive olfactory nature, the GG was assessed for receptor expression by extensive RT-PCR analyses, leading to the identification of a distinct vomeronasal receptor which was expressed in the majority of OMP-positive GG neurones. Along with this receptor, these cells expressed the G proteins Go and Gi, both of which are also present in sensory neurones of the vomeronasal organ. Odorant receptors were expressed by very few cells during prenatal and perinatal stages; a similar number of cells expressed adenylyl cyclase type III and G(olf/s), characteristic signalling elements of the main olfactory system. The findings of the study support the notion that the GG is in fact a subunit of the complex olfactory system, comprising cells with either a VNO-like or a MOE-like phenotype. Moreover, expression of a vomeronasal receptor indicates that the GG might serve to detect pheromones.