Suprasternal gland secretion of male short-tailed opossum induces IP3 generation in the vomeronasal organ

Chemical communication is an important component of mammalian social behaviors. Gray short-tailed opossums (Monodelphis domestica) communicate by scent marking. The male opossum possesses a prominent suprasternal scent gland, extracts of which strongly attract female opossums. This attractivity remains unaltered following repeated lyophilization. The suprasternal gland secretion functions in a sexually dimorphic manner, i.e., it elicits elevated levels of IP₃ in the vomeronasal (VN) sensory epithelium of female opossums, but suppressed the levels of IP₃ in the VN sensory epithelium of male opossums. The elevated levels of IP₃ induced by suprasternal gland secretion in female vomeronasal sensory epithelium is inhibited by the G_i/o specific inhibitor, NF023, but not its inactive analogue, NF007. It is also suppressed by specific antibodies to the alpha subunits of G_i and G_o proteins, by the phospholipase C inhibitor, U73122, as well as by GDPβS. Surprisingly, GDPβS itself enhances basal levels of IP₃ in female VN sensory epithelium. This GDPβS-induced increase in levels of IP₃ is reduced by the PLC inhibitor, U73122, but not by the G_i/o inhibitor, NF023. In addition, GDP also enhances basal levels of IP₃. GDPβS, a known inhibitor of G-protein activation, thus appears to have dual functions: as both stimulator and inhibitor of IP₃ production in the VN sensory epithelium of opossums. In contrast, this nucleotide analogue functions as an inhibitor in the VN sensory epithelium of mice. The mechanism of signal transduction underlying the suprasternal gland secretion-elicited signals in the VN sensory epithelium of opossums appears to involve signals that are generated through activation of G-protein-coupled receptors and transduced via activation of G_i/o-proteins and the effector, phospholipase C, resulting in an increased production of the second messenger, IP₃. The extracellular signals are thus amplified.     

Selective G protein beta gamma-subunit compositions mediate phospholipase C activation in the vomeronasal organ

Chemosensory neurons of the vomeronasal organ (VNO) are supposed to detect pheromones controlling social and reproductive behavior in most terrestrial vertebrates. Recent studies indicate that pheromone signaling in VNO neurons is mediated via phospholipase C (PLC) activation generating the two second messengers inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Since G alpha(i) and G alpha(o) predominantly expressed in VNO neurons are usually not involved in activating PLC, it was explored if PLC activation may be mediated by G beta gamma subunits. It was found that a scavenger for beta gamma dimers reduced the urine-induced IP3 formation in VNO preparations in a dose-dependent manner indicating a role for G beta gamma complexes. Towards an identification of the relevant G beta and G gamma subunit(s), PCR approaches as well as immunohistochemical experiments were performed. It was found that out of the five known G beta subtypes, only G beta2 was expressed in both G alpha(i) as well as G alpha(o) neurons. Experimental approaches focusing on the spatial expression profile of identified G gamma subtypes revealed that G gamma8-positive neurons are preferentially localized to the basal region of the vomeronasal epithelium, whereas G gamma2-reactive cells are restricted to the apical G alpha(i)-positive layer of the sensory epithelium. As IP3 formation induced upon stimulation with volatile urinary compounds was selectively blocked by G gamma2-specific antibodies whereas second messenger formation elicited upon stimulation with alpha2u globulin was inhibited by antibodies recognizing G gamma8, it is conceivable that PLC activation in the two populations of chemosensory VNO neurons is mediated by different G beta gamma complexes.     

Morphological evidence for two types of Mammalian vomeronasal system

The vomeronasal (VN) systems of rodents and opossums are of the segregated type, i.e alpha-subtype G protein Gi2- or Go-expressing VN neurons, which are sensory cells, project discretely to the rostral or caudal region of the accessory olfactory bulb (AOB). Although this zone-specific projection is believed to be a common feature for processing pheromones in mammals, we previously found a uniform-type VN system in goat in which only Gi2-expressing VN axons terminate at the AOB. In most mammals, it remains unclear whether their VN systems are of the segregated or uniform type. Therefore, we investigated morphologically the VN systems of different mammalian species (dog, horse, musk shrew and common marmoset). Consequently, all VN axons of the examined animals were positively stained with immunohistochemistry for Gi2 in the same way as that in the goat. On the other hand, we observed immunoreactivities against Go in the olfactory axons, but not in the VN axons. These results suggest that many mammals have uniform-type VN systems, and at least two types of VN systems exist in terrestrial mammals. This morphological evidence will help us determine the processing function of VN systems.     

Signal transduction in the vomeronasal organ of garter snakes: ligand-receptor binding-mediated protein phosphorylation.

The vomeronasal (VN) system of garter snakes plays an important role in several species-typical behaviors, such as prey recognition and responding to courtship pheromones. We (X.C. Jiang et al., J. Biol. Chem. 265 (1990) 8736-8744 and Y. Luo et al., J. Biol. Chem. 269 (1994) 16867-16877) have demonstrated previously that in the snake VN sensory epithelium, the chemoattractant ES20, a 20-kDa glycoprotein derived from electric shock-induced earthworm secretion, binds to its receptor which is coupled to PTX-sensitive G-proteins. Such binding results in elevated levels of IP3. We now report that ES20-receptor binding regulates the phosphorylation of two membrane-bound proteins with molecular masses of 42- and 44-kDa (p42/44) in both intact and cell-free preparations of the VN sensory epithelium. ES20 and DAG regulate the phosphorylation of p42/44 in a similar manner. ES20-receptor binding-mediated phosphorylation of p42/44 is rapid and transient, reaching a peak value within 40 seconds and decaying thereafter. Phosphorylation of p42/44 appears to be regulated by the countervailing actions of a specific membrane-bound protein kinase and a protein phosphatase. The phosphorylation of these membrane-bound proteins significantly reduces the activity of G-proteins as evidenced by a decrease in GTPase activity, but has little effect on ligand-receptor binding. These findings suggest that p42/44 play a role in modulating the signal transduction induced by ES20 in the vomeronasal system.     

Firing properties of accessory olfactory bulb mitral/tufted cells in response to urine delivered to the vomeronasal organ of gray short-tailed opossums

In comparison with many mammals, there is limited knowledge of the role of pheromones in conspecific communication in the gray short-tailed opossum. Here we report that mitral/tufted (M/T) cells of the accessory olfactory bulb (AOB) of male opossums responded to female urine but not to male urine with two distinct patterns: excitation followed by inhibition or inhibition. Either pattern could be mimicked by application of guanosine 5'-O-3-thiotriphosphate and blocked by guanosine 5'-O-2-thiodiphosphate, indicating that the response of neurons in this pathway is through a G-protein-coupled receptor mechanism. In addition, the inhibitor of phospholipase C (PLC), U73122, significantly blocked urine-induced responses. Male and female urine were ineffective as stimuli for M/T cells in the AOB of female opossums. These results indicate that urine of diestrous females contains a pheromone that directly stimulates vomeronasal neurons through activation of PLC by G-protein-coupled receptor mechanisms and that the response to urine is sexually dimorphic.     

Neurogenesis in the vomeronasal epithelium of adult garter snakes: 3. Use of H3-thymidine autoradiography to trace the genesis and migration of bipolar neurons

Use of H3-thymidine autoradiography and unilateral vomeronasal (VN) axotomy has permitted us to demonstrate directly the existence of VN stem cells in the adult garter snake and to trace continuous bipolar neuron development and migration in the normal VN and deafferentated VN epithelium in the same animal. The vomeronasal epithelium and olfactory epithelium of adult garter snakes are both capable of incorporating H3-thymidine. In the sensory epithelium of the vomeronasal organ, H3-thymidine-labeled cells were initially restricted to the base of the undifferentiated cell layer in animals surviving 1 day following H3-thymidine injection. With increasing survival time, labeled cells progressively migrated vertically within the receptor cell column toward the apex of the bipolar neuron layer. In both the normal and denervated VN epithelium, labeled cells were observed through the 56 days of postoperative survival. In the normal epithelium, labeled cells were always located within the matrix of the intact receptor cell columns. However, labeled cells of the denervated epithelium were always located at the apical front of the newly formed cell mass following depletion of the original neuronal cell population. In addition, at postoperative days 28 and 56, labeled cells of the denervated VN epithelium achieved neuronal differentiation and maturation by migrating much farther away from the base of the receptor cell column than the labeled cells on the normal, unoperated contralateral side. This study directly demonstrates that basal cells initially incorporating H3-thymidine are indeed stem cells of the VN epithelium in adult garter snakes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vomeronasal phenotype and behavioral alterations in G alpha i2 mutant mice

Several social and reproductive behaviors are under the influence of the vomeronasal (VN) organ; VN neurons detect odorous molecules emitted by individuals of the same species. There are two types of VN neurons, and these differ in their expression of chemosensory receptors and G protein subunits. The significance of this dichotomy is largely unknown. VN neurons express high levels of either G alpha i2 or G alpha o. A mouse line carrying a targeted disruption of the G alpha i2 gene offered the opportunity for studying the effects of a lack of receptor signaling through the heterotrimeric Gi2 protein in one VN cell type. As a consequence of this deficiency, the number of VN neurons that normally express G alpha i2 is decreased by half. These residual neurons are defective in eliciting a response in their target neurons in the accessory olfactory bulb. Moreover, G alpha i2 mutant mice show alterations in behaviors for which an intact VN organ is known to be important. Display of maternal aggressive behavior is severely blunted, and male mice show significantly less aggression toward an intruder. However, male mice show unaltered sexual-partner preference. This suggests that the two types of VN neurons may have separate functions in mediating behavioral changes in response to chemosensory information.     

Type-specific inositol 1,4,5-trisphosphate receptor localization in the vomeronasal organ and its interaction with a transient receptor potential channel,TRPC2

The vomeronasal organ (VNO) is the receptor portion of the accessory olfactory system and transduces chemical cues that identify social hierarchy, reproductive status, conspecifics and prey. Signal transduction in VNO neurons is apparently accomplished via an inositol 1,4,5-trisphosphate (IP3)-activated calcium conductance that includes a different set of G proteins than those identified in vertebrate olfactory sensory neurons. We used immunohistochemical (IHC) and SDS-PAGE/western analysis to localize three IP3 receptors (IP3R) in the rat VNO epithelium. Type-I IP3R expression was weak or absent. Antisera for type-II and -III IP3R recognized appropriate molecular weight proteins by SDS-PAGE, and labeled protein could be abolished by pre-adsorption of the respective antibody with antigenic peptide. In tissue sections, type-II IP3R immunoreactivity was present in the supporting cell zone but not in the sensory cell zone. Type-III IP3R immunoreactivity was present throughout the sensory zone and overlapped that of transient receptor potential channel 2 (TRPC2) in the microvillar layer of sensory epithelium. Co-immunoprecipitation of type-III IP3R and TRPC2 from VNO lysates confirmed the overlapping immunoreactivity patterns. The protein-protein interaction complex between type-III IP3R and TRPC2 could initiate calcium signaling leading to electrical signal production in VNO neurons.     

Immunocytochemical study of G(i)2alpha and G(o)alpha on the epithelium surface of the rat vomeronasal organ

To investigate in detail the distribution of G protein subtypes G(i)2alpha and G(o)alpha along the surface of the vomeronasal epithelium, we used double labeling immunocytochemical methods and electron microscopy. We examined the immunoreactivity of these surface structures with antibodies against G(i)2alpha and G(o)alpha. G(i)2alpha- and G(o)alpha-positive cells were observed at the epithelial surface and were evenly distributed. Electron microscopy revealed that strong immunoreactivities to both antibodies were observed on the microvilli and knob-like surface structures of receptor cells. No immunoreactivity was found on the microvilli or surface membranes of supporting cells. This expression pattern is similar to that reported for putative pheromone receptors. These data confirm that there are two distinct classes of vomeronasal receptor cells expressed at the surface of the epithelium. These two classes of receptors correspond to the same G(i)2alpha- and G(o)alpha-positive cells distributed in cell body layers of the epithelium and in the axon terminals in the accessory olfactory bulb.     

Heterogeneity in the accessory olfactory system

The mammalian accessory olfactory bulb (AOB) is chemoarchitecturally heterogeneous in that it stains differentially with a number of markers; the receptor cells that project to the AOB are similarly heterogeneous. What is the significance of this heterogeneity? We have found that the AOB of the gray, short-tailed opossum, Monodelphis domestica, stains differentially with a number of 'markers': antibodies to olfactory marker protein (OMP) and the alpha subunit of the G protein Gi2, the lectin of Vicia villosa and NADPH-diaphorase. These markers stain the rostral AOB more strongly than the caudal AOB whereas, the G protein subunit G(o) alpha is located predominantly in the posterior subdivision of the AOB. This heterogeneity in the chemoarchitecture of the AOB may reflect a fundamental organizational dichotomy within the vomeronasal system that corresponds to a functional dichotomy. The vomeronasal sensory epithelium also exhibits a chemoarchitectural heterogeneity: receptor cells in the basal third are G(o) alpha-immunoreactive whereas the cells in the middle third are Gi2 alpha-immunoreactive. Tracing studies using WGA-HRP demonstrate that the neurons in the middle third of the vomeronasal sensory epithelium project their axons to the anterior AOB whereas those in the basal third appear to project to the posterior AOB.      

Projection pattern of vomeronasal neurons to the accessory olfactory bulb in goats

Goats have a well-developed vomeronasal (VN) system and exhibit pheromone-induced reproductive facilitation, but there are no reports on the projection pattern of VN neurons in this species. Rodent, guinea pig and opossum accessory olfactory bulbs (AOBs) have been shown to have a segregated pattern of projection of the VN neurons, which express the two alpha-subtypes of the G-protein, namely Gi2 and Go, to the rostral and caudal regions of the AOB, respectively. In this study we investigated the projection pattern of VN nerve terminals by immunocytochemical staining of the goat vomeronasal organ (VNO) and the AOB with antibodies to Gi2 and Go. Gi2-immunoreactivity was found on the luminal surface of the sensory epithelium of the VNO, and in the VN nerve and glomerular layer throughout the AOB. On the other hand, Go-immunoreactivity was not identified in either the VNO or the VN nerve layer of the AOB. These results indicate that the projection pattern of VN neurons from the VNO to the AOB in the goat is considerably different from that in rodents which show a distinct segregated pattern.     

Sex- and strain-specific expression and vomeronasal activity of mouse ESP family peptides

Male mice secrete exocrine-gland-secreting peptide 1 (ESP1) from the extraorbital lacrimal gland into tear fluid [1]. Other mice detect ESP1 through sensory neurons in the vomeronasal organ (VNO), a secondary olfactory system that senses pheromonal information, including sex, strain, and species. ESP1 is now known to be a member of a multigene family that encodes peptides of various lengths. We herein performed genomic and expression analyses of the ESP family. The ESP family consists of 38 members in mice and 10 members in rat but is absent from the human genome, suggesting rapid molecular evolution. In addition to the male-specific ESP1, we discovered one, which we designated ESP36, that, in adult BALB/c mice, is expressed only in the female extraorbital lacrimal gland. The sexually dimorphic expression is ensured by the release of testosterone after puberty. However, we observed dramatic differences in the expression levels of ESPs between strains. Finally, all ESPs elicited an electrical response in the vomeronasal epithelium but not in the main olfactory epithelium. Multielectrode recording of VNO activity demonstrated that ESP1 induces action potentials in vomeronasal neurons, leading to an increase in the spike firing rate, and that ESP1 is recognized by narrowly tuned vomeronasal sensory neurons. Sexual dimorphism and strain differences of ESPs and their reception in the VNO suggest that the ESP family can convey information about sex and individual identity via the vomeronasal system. The chemosensation of this nonvolatile peptide family by direct contact appears to be one of strategies for sociosexual communication in rodent species.     

Behavioral and reproductive responses of female opossums to volatile and nonvolatile components of male suprasternal gland secretion

Female gray short-tailed opossums (Monodelphis domestica) lack an estrous cycle and are induced into estrus by exposure to a pheromone in male scent marks. Behavioral and physiological responses of females to the volatile and nonvolatile components of scent marks were examined in two experiments. Young females (n = 9) were tested prior to and during their first estrus for behavioral responses to scent marks, collected on a 7-ml glass vial rubbed over the suprasternal gland of a mature male. The response to volatile components of the scent mark, recorded when marked and unmarked vials were covered with a perforated shield, was compared to the response to these vials when unshielded. Estrous females nuzzled the shields over marked vials (55.8 ± 8.5 nuzzles/10 min) more than the shielded clean vial (10.9 ± 2.4) (P < 0.05); a similar response was observed in anestrous females. Nuzzling of unshielded, scent-marked vials was higher (P < 0.05) during anestrus than in the same females when in estrus. The role of nonvolatile pheromones in reproductive activation was tested in adult females (n = 11) exposed for up to 14 days to a shielded, marked vial or to an unshielded, marked vial in a crossover design. All females exposed to unshielded vials expressed estrus, and 10 copulated. Only 2 females expressed estrus (significantly fewer, P < 0.05), when exposed to shielded marked vials, and neither copulated. These results demonstrate that females detect and respond behaviorally to both volatile and nonvolatile components of male suprasternal gland secretion, but the estrus-inducing pheromone in these secretions is nonvolatile.     

Insulin-like growth factor-1 induces an inositol 1,4,5-trisphosphate-dependent increase in nuclear and cytosolic calcium in cultured rat cardiac myocytes

In the heart, insulin-like growth factor-1 (IGF-1) is a pro-hypertrophic and anti-apoptotic peptide. In cultured rat cardiomyocytes, IGF-1 induced a fast and transient increase in Ca(2+)(i) levels apparent both in the nucleus and cytosol, releasing this ion from intracellular stores through an inositol 1,4,5-trisphosphate (IP(3))-dependent signaling pathway. Intracellular IP(3) levels increased after IGF-1 stimulation in both the presence and absence of extracellular Ca(2+). A different spatial distribution of IP(3) receptor isoforms in cardiomyocytes was found. Ryanodine did not prevent the IGF-1-induced increase of Ca(2+)(i) levels but inhibited the basal and spontaneous Ca(2+)(i) oscillations observed when cardiac myocytes were incubated in Ca(2+)-containing resting media. Spatial analysis of fluorescence images of IGF-1-stimulated cardiomyocytes incubated in Ca(2+)-containing resting media showed an early increase in Ca(2+)(i), initially localized in the nucleus. Calcium imaging suggested that part of the Ca(2+) released by stimulation with IGF-1 was initially contained in the perinuclear region. The IGF-1-induced increase on Ca(2+)(i) levels was prevented by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, thapsigargin, xestospongin C, 2-aminoethoxy diphenyl borate, U-73122, pertussis toxin, and betaARKct (a peptide inhibitor of Gbetagamma signaling). Pertussis toxin also prevented the IGF-1-dependent IP(3) mass increase. Genistein treatment largely decreased the IGF-1-induced changes in both Ca(2+)(i) and IP(3). LY29402 (but not PD98059) also prevented the IGF-1-dependent Ca(2+)(i) increase. Both pertussis toxin and U73122 prevented the IGF-1-dependent induction of both ERKs and protein kinase B. We conclude that IGF-1 increases Ca(2+)(i) levels in cultured cardiac myocytes through a Gbetagamma subunit of a pertussis toxin-sensitive G protein-PI3K-phospholipase C signaling pathway that involves participation of IP(3).     

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.     

Immunolocalization of water channel aquaporins in the vomeronasal organ of the rat: expression of AQP4 in neuronal sensory cells

The vomeronasal organ comprises a pair of narrow tubes in the mammalian nasal septum, serving as a chemosensory system for pheromones. We examined the expression and localization of water channel aquaporins (AQPs) in the rat vomeronasal organ. AQP1 was localized in blood vessels, being particularly abundant in cavernous tissues of the nonsensory mucosa. AQP5 was found in the apical membrane of the gland acinar cells in the vomeronasal organ. AQP3 was detected in the basal cells of the nonsensory epithelium, whereas it was absent in the sensory epithelium. AQP4 was found in both the sensory and the nonsensory epithelia. Interestingly, AQP4 was highly concentrated in the sensory cells of the sensory epithelium. Immunoelectron microscopic examination clearly showed that AQP4 was localized at the plasma membrane in the cell body and lateral membrane of the dendrite, except for the microvillous apical membrane. Nerve fiber bundles emanating from neuronal sensory cells were positive for AQP4, whereby the plasma membrane of each axon was positive for AQP4. These observations clearly show that neuronal sensory cells in the vomeronasal organ are unique in that they express abundant AQP4 at their plasma membrane. This is in marked contrast to the olfactory and central nervous systems, where AQPs are not detectable in neurons, and instead, AQP4 is abundant in the supporting cells and astrocytes surrounding them. The present findings suggest a unique water-handling feature in neuronal sensory cells in the vomeronasal organ.     

Transposition and Intermingling of Gαi2 and Gαo Afferences into Single Vomeronasal Glomeruli in the Madagascan Lesser Tenrec Echinops telfairi

The vomeronasal system (VNS) mediates pheromonal communication in mammals. From the vomeronasal organ, two populations of sensory neurons, expressing either Gαi2 or Gαo proteins, send projections that end in glomeruli distributed either at the rostral or caudal half of the accessory olfactory bulb (AOB), respectively. Neurons at the AOB contact glomeruli of a single subpopulation. The dichotomic segregation of AOB glomeruli has been described in opossums, rodents and rabbits, while Primates and Laurasiatheres present the Gαi2-pathway only, or none at all (such as apes, some bats and aquatic species). We studied the AOB of the Madagascan lesser tenrec Echinops telfairi (Afrotheria: Afrosoricida) and found that Gαi2 and Gαo proteins are expressed in rostral and caudal glomeruli, respectively. However, the segregation of vomeronasal glomeruli at the AOB is not exclusive, as both pathways contained some glomeruli transposed into the adjoining subdomain. Moreover, some glomeruli seem to contain intermingled afferences from both pathways. Both the transposition and heterogeneity of vomeronasal afferences are features, to our knowledge, never reported before. The organization of AOB glomeruli suggests that synaptic integration might occur at the glomerular layer. Whether intrinsic AOB neurons may make synaptic contact with axon terminals of both subpopulations is an interesting possibility that would expand our understanding about the integration of vomeronasal pathways.     

The effects of gonadal hormones on scent marking and related behavior and morphology in female gray short-tailed opossums (Monodelphis domestica)

The hormonal control of scent marking and related behavior and morphology was examined in female gray short-tailed opossums. Females rarely scent marked when intact or following ovariectomy. Testosterone (T) but not estradiol (E) treatment stimulated chest marking while either hormone stimulated head marking in ovariectomized females tested alone. When the same females were tested with males, T-treated females showed little scent marking of any type; E-treated females showed hip marking in significantly more tests than females in the other treatment groups. Suprasternal scent glands (absent in intact females) and phalluses of females that received T were significantly larger than those of animals that received E or control animals. These findings are discussed with respect to similarities and differences between marsupial and eutherian females and between male and female gray opossums in the hormonal control of sexually dimorphic behavior and morphology.