Activation of reproduction in nulliparous and primiparous voles is blocked by vomeronasal organ removal.

Chemical cues from male voles activate reproduction in female prairie voles (Microtus ochrogaster). Twelve hours of contact with a male, followed by exposure to his soiled bedding for 2 days, is sufficient to initiate follicular maturation and induce uterine hypertrophy. Our recent work indicates that the chemosensory vomeronasal organ (VNO) can mediate this response. Here, we examined whether other sensory systems can acquire the ability to activate female reproduction as a result of learning or experience. To explore this issue, the VNO was removed (VNX) from nulliparous and primiparous females who were then exposed to cues from males. In Experiment 1, we found that nulliparous VNX females had lower uterine and ovarian weights than did sham-operated females. In Experiment 2, we determined that sexual experience did not ameliorate the reproductive deficits normally induced by VNX. The present results contrast with those of previous studies suggesting that males of some rodent species, when allowed reproductive experience prior to VNX, can utilize other sensory systems to mediate subsequent reproductive responses. We conclude that the role of the VNO in transducing chemosensory information is crucial for coordinating the reproductive efforts of male and female prairie voles.     

Effects of vomeronasal organ removal on the sperm motility in male mice

Odors play important roles in the communication of house mice. They release behaviors and prime changes of the physiological conditions of other individuals. In our previous study, we showed that sperm motility was lowered in the subordinate mice comparing with dominant mice. Our hypothesis is that the lowered sperm motility was due to some primer effects by odor substances derived from dominant mice. To test the hypothesis, we destroyed the vomeronasal organ (VNO) of male mice (VNX male) at 5 weeks of age and paired them with intact male mice (Experimental Group). As control group males, intact male mice were kept in pairs (Control Group). At 15 weeks of age, the sperm motility and weights of reproductive organs, and social dominance was analyzed. The subordinate VNX males were found to have high sperm motility comparable to the dominant males. It was suggested that there is male-to-male primer effects, mediated by VNO, that suppress sperm motility of the subordinate mice.     

Role of the vomeronasal organ on the estral cycle reduction by pheromones in the rat

The role of he vomeronasal organ on the estral cycle reduction induced by pheromones is studied in adult female wistar rats. The animals were divided in three groups: I, intact rats; II, vomeronasalectomized rats (VNX); and III, sham operated rats (sham). Each group was submitted to another three distinct conditions from the day they were weaned (21 days old): Isolated female rats; with male odors from two adult males of tested sexual potency, and isolated rats again. The isolated intact rats show mainly 5 day length cycles. The groups I and III (intacts and sham) with male odors, show 4 day length cycles. The VNX animals show 5 day cycles in any one experimental conditions. These results support the idea that the vomeronasal organ is the receptor of the male reducing cycle pheromone in the female rat.     

Roles of the main olfactory and vomeronasal systems in the detection of androstenone in inbred strains of mice

We investigated the role of the main olfactory and accessory olfactory systems (MOS and AOS respectively) in the detection of androstenone. We used the following experimental approaches: behavioral, surgical removal of the vomeronasal organ (VNX) followed by histochemical verification and Fos immunohistochemistry. Using a Y-maze paradigm we estimated sensitivity of NZB/B1NJ and CBA/J mice to androstenone. CBA mice were 2,000-fold more sensitive to androstenone than NZB mice. VNX caused a 4- to 16-fold decre...     

Modulatory role of testosterone in alarm pheromone release by male rats

An alarm pheromone released from stressed conspecifics evokes behavioral and autonomic responses in rats. We have previously reported that male Wistar rats show behavioral changes including increased sniffing, walking and rearing, and decreased resting as well as exaggerated response of body temperature to a novel environment [known as stress-induced hyperthermia (SIH)] when they are exposed to an alarm pheromone released from other male rats receiving foot shocks. The purpose of the present study was to examine the role of testosterone in the production and release of the alarm pheromone using these behavioral and autonomic responses in recipient rats. Three groups of alarm pheromone donors were presented, namely, intact males, castrated males, and testosterone-implanted castrated males. The effects of the alarm pheromone on the autonomic responses did not differ among the three groups, regardless of the donor's steroidal milieu, whereas behavioral responses were altered by castrating the donor males and the effects were restored by testosterone implantation. These results suggest that the alarm pheromone released from stressed male rats can be classified into at least two categories according to the androgen dependency of their production and/or release.     

Mice (Mus musculus) lacking a vomeronasal organ can discriminate MHC-determined odortypes

Major histocompatibility complex (MHC) genes in mammals (H-2 in mice) play a major role in regulating immune function. They also bestow individuality in the form of a chemical signature or odortype. At present, the respective contributions of the olfactory epithelium and the vomeronasal organ (VNO) in the recognition of individual odortypes are not well defined. We examined a possible role for the VNO in the recognition of MHC odortypes in mice by first removing the organ (VNX) and then training the mice to distinguish the odors of two congenic strains of mice that differed only in their MHC type. C57BL/6J mice (bb at H-2) and C57BL/6J-H-2(k) (kk at H-2) provided urine for sensory testing. Eight VNX and six sham-operated mice were trained to make the discrimination. Neither the number of training trials-to-criterion nor the rate of learning differed significantly for VNX and sham-operated mice. We conclude that the VNO is not necessary for learning to discriminate between MHC odortypes.     

Vomeronasal organ ablation elicits chemosensory dysfunction and abnormal behavior in mice

This study aimed to examine whether the vomeronasal organ (VNO) is a prerequisite in mice to acquire essential information from various social odors and whether long-term VNO dysfunction can elicit behavioral and physiological changes in mice. We used binary choice tests and habituation–dishabituation tests to measure the abilities of male mice to recognize social odors. We found that males with the VNO ablation failed to show olfactory preferences between the odors of mate versus non-mate females, offspring versus non-offspring pups, or opposite-sex conspecifics versus predators (cats or rats), but were capable of discriminating between the two treatments in each of the paired odors, suggesting that male mice with VNO ablation might smell out the chemical differences of the two types of odors, but could not extract the biological information contained in the odors. Furthermore, prolonged VNO deficiency resulted in a reduction in crossing behavior in a light/dark box, the frequency of urine marking, and the time spent in the center in an open field. These results indicate that chronic VNO dysfunction led to anxiety-like or submissive behavior. In addition, males with VNO ablation had atrophic adrenal glands and hypertrophic preputial glands, suggesting that VNO dysfunction could damage the physiological conditions to buffer the stress and that pheromone perception deficiency might enhance self-odor production in mice.     

Xenopus V1R vomeronasal receptor family is expressed in the main olfactory system

To date, over 100 vomeronasal receptor type 1 (V1R) genes have been identified in rodents. V1R is specifically expressed in the rodent vomeronasal organ (VNO) and is thought to be responsible for pheromone reception. Recently, 21 putatively functional V1R genes were identified in the genome database of the amphibian Xenopus tropicalis. Amphibians are the first vertebrates to possess a VNO. In order to determine at which point during evolution the vertebrate V1R genes began to function in the vomeronasal system, we analyzed the expression of all putatively functional V1R genes in Xenopus olfactory organs. We found that V1R expression was not detected in the VNO but was specifically detected in the main olfactory epithelium (MOE). We also observed that V1R-expressing cells in the MOE coexpressed Gi2, thus suggesting that the V1R-Gi2-mediated signal transduction pathway, which is considered to play an important role in pheromone reception in the rodent VNO, exists in the amphibian MOE. These results suggest that V1R-mediated signal transduction pathway functions in Xenopus main olfactory system.     

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.     

Molecular biology of pheromone perception in mammals

In mammals, olfactory sensory perception is mediated by two anatomically and functionally distinct sensory organs: the main olfactory epithelium (MOE) and the vomeronasal organ (VNO). Pheromones activate the VNO and elicit a characteristic array of innate reproductive and social behaviors, along with dramatic neuroendocrine responses. Recent approaches have provided new insights into the molecular biology of sensory transduction in the vomeronasal organ. Differential screening of cDNA libraries constructed from single sensory neurons from the rat VNO has led to the isolation of a family of genes which are likely to encode mammalian pheromone receptors. The isolation of these receptors from the vomeronasal organ might permit the analysis of the molecular events which translate the bindings of pheromones into innate stereotypic behaviors and help to elucidate the logic of pheromone perception in mammals.     

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.     

Removal of the vomeronasal organ reduces reproductive performance and aggression in male prairie voles

Several short-duration tests have demonstrated that the surgicalremoval of the vomeronasal organ (VNX) from sexually-inexperiencedmale rodents results in a reduction in copulatory behavior,compared to the effects of sham surgery (SHAM). We extendedthese studies to adult male prairie voles, Microtus ochrogaster,and substantially increased the duration of the tests. Duringthe initial interactions with females, VNX males spent significantlyless time with their noses in close proximity to the femalesthan did SHAM males. Moreover, only two of nine VNX males siredoffspring after having been paired with females for 8 weeks,whereas nine of 12 SHAM males sired offspring in that interval.We also found that VNX and SHAM males were equivalently non-aggressiveto an anesthetized stimulus-male prior to being paired withfemales. However, after spending 2 weeks paired with a female,the VNX males were significantly less aggressive than were theSHAM males, possibly as a result of having copulated less often.In a later test, nearly all of the VNX and SHAM males that siredoffspring were vigorously aggressive to a stimulus male. Weconclude that the stimulation of the vomeronasal system in sexually-inexperiencedmale prairie voles is important for miximal reproductive performanceand that the VNX-induced impairment in reproduction is associatedwith a decrease in inter-male aggression. The possible sensoryeffects of the vomeronasal system on the neural and endocrinecontrol of reproduction and behavior are discussed.     

Conditioned odor aversion induces social anxiety towards females in wild‐type and TrpC2 knockout male mice

Female‐emitted pheromonal inputs possess an intrinsic rewarding value for conspecific males, promoting approach and investigation of the potential mating partner. In mice these inputs are detected mainly by the vomeronasal organ (VNO) and the main olfactory epithelium (MOE). We investigated the role of VNO‐mediated inputs in experience‐dependent plasticity of reproductive responses. We applied a sex‐specific conditioned odor aversion (COA) paradigm on adult, wild‐type (WT) male mice and on male mice impaired in VNO‐mediated signal transduction (TrpC2^?/?). We found that WT males, which underwent COA to female‐soiled bedding, lost their innate preference to female odors and presented lower motivation to approach a sexually receptive female. COA also abolished the testosterone surge normally seen following exposure to female odors. Moreover, the conditioned males displayed impairments in copulatory behaviors, which lasted for several weeks. Surprisingly, these males also exhibited phobic behaviors towards receptive females, including freezing and fleeing responses. In contrast, WT males which underwent COA specifically to male pheromones showed no change in olfactory preference and only a marginally significant elevation in intermale aggression. Finally, we show that TrpC2^?/? males were able to acquire aversion to female‐soiled bedding and presented similar behavioral alterations following COA in their responses to female cues. Our results demonstrate that the intrinsic rewarding value of female pheromones can be overridden through associative olfactory learning, which occurs independently of VNO inputs, probably through MOE signaling.     

Alarm pheromone that aggravates stress-induced hyperthermia is soluble in water

We previously reported that stressed male Wistar rats released alarm pheromone from the perianal region, which aggravated stress-induced hyperthermia and increased Fos expression in the mitral/tufted cell layer of the accessory olfactory bulb in recipient rats. In this study, we attempted to obtain this pheromone in water using these responses as bioassay parameters. Water droplets were collected from the ceiling of a box in which no animal was placed, or from a box in which an anesthetized donor rat was given electrical stimulation to either the neck or perianal regions in order to induce neck odor or alarm pheromone release, respectively. Then we placed one of the three kinds of water-containing filter papers on the wall of a recipient's home cage and observed heart rate, body temperature and behavioral responses, as well as Fos expression in the main and accessory olfactory bulbs of the recipient. The water collected from the box containing the alarm pheromone was found to generate a reproduction of all of the responses seen in the animal that had been directly exposed to alarm pheromone in our previous studies. These results suggest that the alarm pheromone is soluble in water.     

Expression of candidate pheromone receptor genes in vomeronasal neurons

In mammals, olfactory sensory perception is mediated by two anatomically and functionally distinct organs: the main olfactory epithelium (MOE) and the vomeronasal organ (VON). Pheromones activate the VNO and elicit a characteristic array of innate reproductive and social behaviors, along with dramatic neuroendocrine responses. Recent approaches have provided new insights into the molecular biology of sensory transduction in the VNO. Differential screening of cDNA libraries constructed from single sensory neurons from the rat VNO has led to the isolation of a family of genes which are likely to encode mammalian pheromone receptors. The isolation of these receptors from the VNO might permit the analysis of the molecular events which translate the bindings of pheromones into innate stereotypic behaviors and help to elucidate the logic of pheromone perception in mammals.      

Neuropilin-2 mediates axonal fasciculation, zonal segregation, but not axonal convergence, of primary accessory olfactory neurons

The mechanisms that underlie axonal pathfinding of vomeronasal neurons from the vomeronasal organ (VNO) in the periphery to select glomeruli in the accessory olfactory bulb (AOB) are not well understood. Neuropilin-2, a receptor for secreted semaphorins, is expressed in V1R- and V3R-expressing, but not V2R-expressing, postnatal vomeronasal neurons. Analysis of the vomeronasal nerve in neuropilin-2 (npn-2) mutant mice reveals pathfinding defects at multiple choice points. Vomeronasal sensory axons are severely defasciculated and a subset innervates the main olfactory bulb (MOB). While most axons of V1R-expressing neurons reach the AOB and converge into distinct glomeruli in stereotypic locations, they are no longer restricted to their normal anterior AOB target zone. Thus, Npn-2 and candidate pheromone receptors play distinct and complementary roles in promoting the wiring and patterning of sensory neurons in the accessory olfactory system.     

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.     

Expression of glycoproteins in the vomeronasal organ reveals a novel spatiotemporal pattern of sensory neurone maturation

The main olfactory and the accessory olfactory systems are both anatomically and functionally distinct chemosensory systems. The primary sensory neurones of the accessory olfactory system are sequestered in the vomeronasal organ (VNO), where they express pheromone receptors, which are unrelated to the odorant receptors expressed in the principal nasal cavity. We have identified a 240 kDa glycoprotein (VNO(240)) that is selectively expressed by sensory neurones in the VNO but not in the main olfactory neuroepithelium of mouse. VNO(240) is first expressed at embryonic day 20.5 by a small subpopulation of sensory neurones residing within the central region of the crescent-shaped VNO. Although VNO(240) was detected in neuronal perikarya at this age, it was not observed in the axons in the accessory olfactory bulb until postnatal day 3.5. This delayed appearance in the accessory olfactory bulb suggests that VNO(240) is involved in the functional maturation of VNO neurones rather than in axon growth and targeting to the bulb. During the first 2 postnatal weeks, the population of neurones expressing VNO(240) spread peripherally, and by adulthood all primary sensory neurones in the VNO appeared to be expressing this molecule. Similar patterns of expression were also observed for NOC-1, a previously characterized glycoform of the neural cell adhesion molecule NCAM. To date, differential expression of VNO-specific molecules has only been reported along the rostrocaudal axis or at different apical-basal levels in the neuroepithelium. This is the first demonstration of a centroperipheral wave of expression of molecules in the VNO. These results indicate that mechanisms controlling the molecular differentiation of VNO neurones must involve spatial cues organised, not only about orthogonal axes, but also about a centroperipheral axis. Moreover, expression about this centroperipheral axis also involves a temporal component because the subpopulation of neurones expressing VNO(240) and NOC-1 increases during postnatal maturation.