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.     

The vomeronasal organ and associated structures of the fetal African elephant, Loxodonta africana (Proboscidea, Elephantidae)

The vomeronasal organ (VNO) is a chemosensory structure of the nasal septum found in most tetrapods. Although potential behavioural correlates of VNO function have been shown in two of the three elephant species, its morphology in Loxodonta africana has not been studied. The development of the VNO and its associated structures in the African elephant are described in detail using serially sectioned material from fetal stages. The results show that many components of the VNO complex (e.g. neuroepithelium, receptor‐free epithelium, vomeronasal nerve, paravomeronasal ganglia, blood vessels, vomeronasal cartilage) are well developed even in a 154‐day‐old fetus, in which the VNO opens directly into the oral cavity with only a minute duct present. However, the vomeronasal glands and their ducts associated with the VNO were developed only in the 210‐day‐old fetus. Notably, in this fetus, the vomeronasal–nasopalatine duct system had acquired a pathway similar to that described in the adult Asian elephant; the VNOs open into the oral cavity via the large palatal parts of the nasopalatine ducts, which are lined by a stratified squamous epithelium. The paired palatal ducts initially coursed anteriorly at an angle of 45° from the oral recess and/or the oral cavity mucosa, and merged into the vomeronasal duct. This study confirms the unique characteristics of the elephant VNO, such as its large size, the folded epithelium of the VNO tube, and the dorsomedial position of the neuroepithelium. The palatal position and exclusive communication of the VNO with the oral cavity, as well as the partial reduction of the nasopalatine duct, might be related to the unique elephantid craniofacial morphogenesis, especially the enormous growth of the tusk region, and can be seen as autapomorphies.     

Olfactory contribution to Fos expression during mating in inexperienced male hamsters

Male hamsters are very dependent on chemosensory cues for normal mating behavior. We have previously reported that central, vomeronasal pathways are intensely and selectively activated during mating or pheromonal stimulation. The contribution of main olfactory sensory input to the patterns of c-fos activation was investigated in this study. Sexually inexperienced male hamsters were either made anosmic by intranasal infusion of zinc sulfate or remained intact. Fos protein immunoreactivity was analyzed in main olfactory and vomeronasal pathways of the zinc sulfate-treated, anosmic animals after mating with receptive females for 45 min, and compared with Fos patterns seen in intact mating animals, some of which have been described in a previous publication. The zinc sulfate-treated anosmic males described here all mated when given access to receptive females. Whether mated or unstimulated, anosmic males had little or no Fos expression in main olfactory pathways; significantly less even than in unstimulated intact animals. Mating did not increase Fos expression in main olfactory pathways of intact animals over that of unstimulated intact controls. However, Fos expression in central vomeronasal pathways was significantly higher in mating anosmic males, as in intact males, compared with appropriate non-mating controls. Fos expression was significantly different between intact and zinc sulfate-treated anosmic mating males in only one area studied. The rostral anterior medial amygdala, known to receive a small olfactory terminal field, had significantly lower Fos expression in zinc sulfate-treated anosmic males that mated when compared with intact-mating animals. Thus, functional main olfactory input to the rostral vomeronasal amygdala can be demonstrated but does not appear to be critical for mating behavior in previously inexperienced male hamsters with intact vomeronasal organs. Other main olfactory input appears to have a negligible contribution to Fos-patterns in such animals.      

The nasopalatine ducts and associated structures in the rhesus monkey (Macaca mulatta): topography, prenatal development, function, and phylogeny

Morphological and developmental characteristics of the rhesus monkey nasopalatine duct system and associated primary palatal structures are described along with functional and phylogenetic considerations. Examination of five adult palates and coronal sections of 13 fetal palates together with dissections of a sixth adult specimen and of a 119-day-old fetal palate reveal that the lateral lobes of the tripartate incisive papilla cover clefts leading into the ducts. The ducts pierce the bony palate to enter the nasal fossae in proximity to the incisive suture. The ontogenetic stability of the duct path reflects the retention of ancient duct and primitive choanae relationships and functionally maintains an optimal oral odorant-to-receptor channel. Sixteen timed pregnancy specimens (35-100 days) provided histological material for documenting rostral nasopalatal development. Duct primordia, identified at 35 days, had by 40 days formed the medial duct walls (conjoined septum-papilla from the primary medial palatal component), the lateral duct walls (maxillary processes), and the rostral walls (fused maxillary-intermaxillary components). The caudal walls derive from the fusion of palatal shelves with the papilla (45 days), thus distinguishing primary and secondary fusion modes. Duct epithelial maturation occurs between 70 and 100 days. The absence of a vomeronasal system is attributed to reduction of olfaction in reproductive behavior, while the presence of the coevolved nasopalatine ducts is linked to the persistence of epiglottal-velar valving. The ducts serve as oral food-odor conduits in otherwise functionally separated respiratory and digestive tracts.     

Nursing behavior in lactating rats--the role of the vomeronasal organ

The vomeronasal organ (VNO) of female Wistar-Imamichi rats was removed completely. The females were impregnated and gave birth to young. The question addressed was whether such dams would show deficits in nursing behavior. The data indicated that removal of the VNO severely depressed nursing behavior. We conclude that the VNO contributes to the nursing behavior of lactating rats.     

Comparative anatomic studies of the vomeronasal complex and the rostral palate of various mammals

The structures of the rostral palate in regard to the vomeronasal complex of different species of mammals were studied. In all cases, we find a very interesting system of furrows which preserves a connection between the nasopalatine ducts and the preoral surroundings. For rodents, lagomorphs, Solenodon, Setifer, and Echinops, we find a special situation in this part of the palate. Here the incisors are not separated by a diastema nor the oral openings of the nasopalatine ducts are overgrown by a bipartite caudal branch of the rhinarium. The results of the anatomic studies of the vomeronasal complex and the rostral palate of the mammals investigated are discussed: First of all, some elements of the vomeronasal complex needed to be analysed in regard to structure and nomenclature, specifically the cartilago paraseptalis with its outer bar, the cartilago ductus nasopalatini and the cartilago palatina. Because of 2 criterions, the vomeronasal complex could be classified as either primitive or progressive. We find a primitive one in Didelphis, Tupaia, Solenodon, Oryctolagus, and all rodents. In contrast, the other insectivores studied and all primates show progressive structures at their vomeronasal complex. Finally, conclusions in regard to the function of the organs of Jacobson are derived from these studies. The significance of the "flehmen" mechanism for the functioning of the organs is questioned.     

Suggestions for the functional relationship between differently located taste buds and vomeronasal olfaction in several mammals

In almost all mammals a well developed, paired and blind ending vomeronasal Organ (VNO) situated within the basement of the nasal septum, communicates with the oral cavity. This contact is established by two nasopalatine ducts, which penetrate the rostral palate close to the incisors. These ducts open orally into the sulcus which moulds the palatine papilla. In several mammals taste buds were found in the epithelium of the patatine papilla located within the nasopalatine ducts or close to their oral openings. Presumably these taste buds interact with the vomeronasal olfaction. It is likely that they are leading to a chemosensory sensation comparable to the combination of normal taste and smell. As not all mammals with a functionable VNO possess taste buds in this position, an inspection of the rostral part of the tongue which touches the palatine papilla presented an interesting situation concerning the distribution of taste buds. This region of the tongue is almost completely free of taste buds in species like Tupaia glis and Didelphis marsupialis virginiana, which have taste buds in the epithelium of their palatine papilla. In Lemur catta however, where the palatine papilla is lacking taste buds, the respective tongue part is densely covered with them. In this case it appears likely that they in a way of substitution functionally are connected with vomeronasal olfaction.     

Comparative anatomical studies of the vomeronasal complex and the rostral palate of various mammals. I

The anatomy of the vomeronasal complex and, in connection with this, the structures of the rostral palate were studied in different species of mammals, namely members of the order Marsupialia, Scandentia, Insectivora, Primates, Rodentia, and Lagomorpha. The following results were obtained: The organs of Jacobson of all forms studied are well-developed. The organ of Jacobson is situated at the base of the nasal septum and opens rostrally, always closely connected to the nasopalatine duct. Even in rodents, lagomorphs and Solenodon, where the openings of the organs are positioned rostral to the ductus, both systems are nevertheless connected by means of special furrows. Accordingly the organs of Jacobson are functionally much more closely related to the oral cavity than to the nasal cavity, which they actually belong to. This can be emphasized by the peculiar structures of the rostral palate inclosing the papilla palatina and with it the oral openings of the nasopalatine ducts. In all species studied, the anterior part of the upper jaw presents a very interesting situation because the median furrow of the rhinarium communicates directly or indirectly with the sulcus papillae palatinae, thus forming a very distinct system of grooves which preserves a connection between the nasopalatine ducts and the preoral surroundings. In rodents, lagomorphs, and Solenodon, we find in this part of the palate a special situation because of their unusually arranged incisors, which are not separated by a diastema. However, also in these cases, there are distinct connecting passages between the papilla palatina and the extraoral surroundings. The conditions found in Ratufa bicolor and in early stages of the rat demonstrate that the extraordinary topography of the rostral palate in rodents is a secondary formation by means of ontogeny and phylogeny. Cebus apella, a platyrrhine simian, shows already a clear reduction of palatal structures compared to those found in prosimians. In Setifer setosus and Echinops telfairi, we find the papilla palatina and with it the oral openings of the nasopalatine ducts overgrown by a bipartite caudal branch of the rhinarium. The neonate Setifer allows us to reconstruct the mechanism of this overgrowing procedure. We find a similar situation in Erinaceus, where the papilla palatina remains uncovered, however. Because of contradictory bibliographical data, some elements of the vomeronasal complex in mammals needed to be carefully analysed in regard to structure and nomenclature: in many species the paraseptal cartilage bifurcates rostrally into a dorsal and a ventral branch.(ABSTRACT TRUNCATED AT 400 WORDS)     

The vomeronasal organ of the male ferret.

The vomeronasal organ (VNO) is known to play a major role in sexual behavior in many mammals. This study is the first report that the adult male ferret has a VNO, which is considerably smaller and morphologically different from the usually crescent-shaped epithelium in several mammalian species, particularly rodents. There were no differences in the size or structure of the ferret VNO between the mating season in spring and the sexually quiescent season in autumn, although plasma testosterone, testis size and brain size are dramatically increased in spring and behavior changes significantly. The histological data suggest that the VNO might be not as important a structure in male ferret sexual behavior as in rodents.     

A nose by any other name (should smell as sweetly)

The standard view that the control of mating behavior by pheromones is mediated by the vomeronasal organ, and not by the main olfactory epithelium, has recently been called into question. In this issue of Cell, two independent studies (Boehm et al., 2005; Yoon et al., 2005) examine the inputs from each of these olfactory pathways to a population of neurons that plays a central role in mating behavior.     

On the cue for male territorial site selection in the dragonfly,Nannophya pygmaea: A field experiment

A field observation of the dragonflyNannophya pygmaea revealed that males prefer some territorial sites to others, and that these same sites attract more females than others (Tsubaki & Ono, 1986, 1987). In this paper, we asked if males choose territorial sites in response to female dispersion or distribution of resources. We conducted 3 types of removal experiments to test the following 2 hypotheses; (1) a male may assess the territory quality by the female encounter rate at his site (learning), (2) a male may assess the resource quality (or quantity) in the territory. The results of our experiments show that males discriminate attractive and less attractive territorial sites without any mating experience within the study area. Moreover, the territorial site preference of males was not affected by the mating experience. Therefore, males probably choose territorial sites by resource quality rather than by female dispersion.     

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.     

Sexual experience does not compensate for the disruptive effects of zinc sulfate--lesioning of the main olfactory epithelium on sexual behavior in male mice

Recent studies point to an important role for the main olfactory epithelium (MOE) in regulating sexual behavior in male mice. We asked whether sexual experience could compensate for the disruptive effects of lesioning the MOE on sexual behavior in male mice. Male mice, which were either sexually naive or experienced, received an intranasal irrigation of either a zinc sulfate solution to destroy the MOE or saline. Sexual behavior in mating tests with an estrous female was completely abolished in zinc sulfate-treated male mice regardless of whether subjects were sexually experienced or not before the treatment. Furthermore, zinc sulfate treatment clearly disrupted olfactory investigation of both volatile and nonvolatile odors. Destruction of the MOE by zinc sulfate treatment was confirmed by a significant reduction in the expression of Fos protein in the main olfactory bulb following exposure to estrous female urine. By contrast, vomeronasal function did not seem to be affected by zinc sulfate treatment: nasal application of estrous female urine induced similar levels of Fos protein in the mitral and granule cells of the accessory olfactory bulb (AOB) of zinc sulfate- and saline-treated males. Likewise, the expression of soybean agglutinin, which stains the axons of vomeronasal organ neurons projecting to the glomerular layer of the AOB, was similar in zinc sulfate- and saline-treated male mice. These results show that the main olfactory system is essential for the expression of sexual behavior in male mice and that sexual experience does not overcome the disruptive effects of MOE lesioning on this behavior.     

Ontogenetic and phylogenetic transformations of the vomeronasal complex and nasal floor elements in marsupial mammals

Histological sections and three-dimensional reconstructions of section-series were used to document the anatomy of the vomeronasal complex and other aspects of the ethmoidal region in representatives of 13 families and six orders of marsupial mammals, including for the first time Microbiotheria. The changes during growth of several features were examined in ontogenetic series. Marsupials are very conservative in comparison with eutherians regarding the vomeronasal complex. All have a vomeronasal organ and a nasopalatine duct, have no nasopalatine duct cartilage, have no (or just an incipient) palatine cartilage, and the overall construction of the nasal floor is uniform across species. Most features examined show a high degree of homoplasy (e.g. presence of glandular ridges, isolated dorsal process of the paraseptal cartilage), and their systematic value is confined to low taxonomic levels. Significant ontogenetic changes occur in features usually discussed in the systematic/taxonomic literature. Amongst the didelphids examined, Caluromys philander shows several autapomorphies. It is hypothesized that the opening of the VNO into the upper end of the nasopalatine duct was present in the marsupial groundplan. Most marsupials have a large and horizontal anterior transverse lamina, the plesiomorphic condition, which becomes oblique in diprotodontians. Some features are autapomorphies of well-supported monophyletic groups of marsupials, e.g. the conspicuous internasal communication of perameliformes and the 'tube-like' or ring-shaped paraseptal cartilage of vombatiformes. An outer bar joining the middle (and not the dorsal-most portion) of the paraseptal cartilage characterizes Australasian marsupials and Dromiciops, with the exclusion of perameliformes, and evolved independently in Caluromys philander.     

Conversion of testosterone to estradiol may not be necessary for the expression of mating behavior in male Syrian hamsters (Mesocricetus auratus)

Male sexual behavior is mediated in part by androgens, but in several species, mating is also influenced by estradiol formed locally in the brain by the aromatization of testosterone. The role of testosterone aromatization in the copulatory behavior of male Syrian hamsters is unclear because prior studies are equivocal. Therefore, the present study tested whether blocking the conversion of testosterone to estradiol would inhibit male hamster sexual behavior. Chronic systemic administration of the nonsteroidal aromatase inhibitor Fadrozole (2.0 mg/kg/day) for 5 or 8 weeks did not significantly increase mount latency or reduce mount frequency, intromission frequency, ejaculation frequency, or anogenital investigation relative to levels shown by surgical controls. However, Fadrozole effectively inhibited aromatase activity, as evidenced by the suppression of estrogen-dependent progesterone receptor immunoreactivity in the male hamster brain. The JZB39 anti-progesterone receptor antibody labeled significantly more neurons in brains of sham-treated hamsters than in brains of Fadrozole-treated hamsters. These data suggest that aromatization of testosterone to estradiol is not necessary for normal mating behavior in Syrian hamsters.     

HRP uptake by olfactory and vomeronasal receptor neurons: use as an indicator of incomplete lesions and relevance for non-volatile chemoreception

The transport of HRP (horseradish peroxidase) from the nasalcavity to the brain by intact olfactory receptor axons was usedto investigate the effectiveness of methods commonly used inbehavioral studies for deafferenting nasal chemoreceptor systems.The HRP experiments demonstrated that routine intranasal lavagewith zinc sulfate solution fails to destroy all olfactory receptorneurons in hamsters, in spite of the distinct behavioral deficitthat this treatment can cause in the male hamster. The intracranialdeafferentation of the accessory olfactory bulb by surgicalsection of the vomeronasal nerves was generally effective butthere was much incidental damage to main olfactory nerves thatwould probably not be detected without the HRP tracer. The distribution pattern of HRP molecules introduced into themammalian nasal cavity, as shown by the uptake of HRP by nasalchemoreceptors and its transport to the brain, was also usedto identify potential pathways for non-volatile stimulus moleculeswithin the nose. HRP reaction product was reliably detectedin the glomeruli of the main olfactory bulb after HRP was depositedat the nostril, demonstrating that nonvolatile materials, oncethey have entered the nasal cavity, can reach the main olfactoryreceptor neurons in the posterior nasal epithelium. Significantamounts of HRP reaction product were never observed in the accessoryolfactory bulbunlessa large dose of epinephrine had been givento activate the vomeronasal organ pumping mechanism, which drawssubstances into the vomeronasal organ lumen. Thus, it seemsthat stimulus access to vomeronasal receptor neurons is controlledindependently of access to main olfactory receptor neurons.     

Adolescents and androgens, receptors and rewards

Adolescence is associated with increases in pleasure-seeking behaviors, which, in turn, are shaped by the pubertal activation of the hypothalamo-pituitary-gonadal axis. In animal models of naturally rewarding behaviors, such as sex, testicular androgens contribute to the development and expression of the behavior in males. To effect behavioral maturation, the brain undergoes significant remodeling during adolescence, and many of the changes are likewise sensitive to androgens, presumably acting through androgen receptors (AR). Given the delicate interaction of gonadal hormones and brain development, it is no surprise that disruption of hormone levels during this sensitive period significantly alters adolescent and adult behaviors. In male hamsters, exposure to testosterone during adolescence is required for normal expression of adult sexual behavior. Males deprived of androgens during puberty display sustained deficits in mating. Conversely, androgens alone are not sufficient to induce mating in prepubertal males, even though brain AR are present before puberty. In this context, wide-spread use of anabolic-androgenic steroids (AAS) during adolescence is a significant concern. AAS abuse has the potential to alter both the timing and the levels of androgens in adolescent males. In hamsters, adolescent AAS exposure increases aggression, and causes lasting changes in neurotransmitter systems. In addition, AAS are themselves reinforcing, as demonstrated by self-administration of testosterone and other AAS. However, recent evidence suggests that the reinforcing effects of androgens may not require classical AR. Therefore, further examination of interactions between androgens and rewarding behaviors in the adolescent brain is required for a better understanding of AAS abuse.     

Sexual differentiation of pheromone processing: Links to male-typical mating behavior and partner preference

Phoenix et al. (Phoenix, C., Goy, R., Gerall, A., Young, W., 1959. Organizing actions of prenatally administered testosterone propionate on the tissues mediating mating behavior in the female guinea pig. Endocrinology 65, 369–382.) were the first to propose an essential role of fetal testosterone exposure in the sexual differentiation of the capacity of mammals to display male-typical mating behavior. In one experiment control male and female guinea pigs as well as females given fetal testosterone actually showed equivalent levels of mounting behavior when gonadectomized and given ovarian steroids prior to adult tests with a stimulus female. This finding is discussed in the context of a recent, high-profile paper by Kimchi et al. (Kimchi, T., Xu, J., Dulac, C., 2007. A functional circuit underlying male sexual behaviour in the female mouse brain. Nature 448, 1009–1014.) arguing that female rodents possess the circuits that control the expression of male-typical mating behavior and that their function is normally suppressed in this sex by pheromonal inputs that are processed via the vomeronasal organ (VNO)-accessory olfactory nervous system. In another Phoenix et al. experiment, significantly more mounting behavior was observed in male guinea pigs and in females given fetal testosterone than in control females following adult gonadectomy and treatment with testosterone. Literature is reviewed that attempts to link sex differences in the anatomy and function of the accessory versus the main olfactory projections to the amygdala and hypothalamus to parallel sex differences in courtship behaviors, including sex partner preference, as well as the capacity to display mounting behavior.     

Papilla palatina, nasopalatine duct and taste buds of young and adult rats

The morphology of the papilla palatina, the nasopalatine ducts and the taste buds situated within these ducts was studied in pups and adult rats using light and electron microscopy. During development, the papilla palatina grew in width and depth, becoming a protuberance in weanlings and adults. The nasopalatine ducts enlarged and two folds of the lateral walls of the ducts differentiated, reducing the width of the tubes in the region of the oral openings. Taste buds appeared postnatally. Light, dark, and perigemmal cells were found in all stages studied, but light cells were scarce up to 8 days of age. The taste pore appeared between 11 and 13 days of age; it lacked electron-dense material of cellular origin. Synaptic-like images could be found only in relation to dark cells. The papilla palatina, the nasopalatine ducts and the taste buds were fully developed by the 3rd week of life.     

Female snake sex pheromone induces membrane responses in vomeronasal sensory neurons of male snakes

The vomeronasal organ (VNO) is important for activating accessory olfactory pathways that are involved in sexually dimorphic mating behavior. The VNO of male garter snakes is critically important for detection of, and response to, female sex pheromones. In the present study, under voltage-clamp conditions, male snake VNO neurons were stimulated with female sexual attractiveness pheromone. Thirty-nine of 139 neurons exhibited inward current responses (reversal potential: -10.6 +/- 2.8 mV). The amplitude of the inward current was dose dependent, and the relationship could be fitted by the Hill equation. Under current-clamp conditions, application of pheromone produced membrane depolarizing responses and increases in firing frequency. These results suggest that the female pheromone directly affects male snake VNO neurons and results in opening of ion channels, thereby converting the pheromone signal to an electrical signal. The response to female pheromone is sexually dimorphic, that is, the pheromone does not evoke responses in VNO neurons of female snakes. An associated finding of the present study is that the female sex pheromone, which is insoluble in aqueous solutions, became soluble in the presence of Harderian gland homogenate.