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Endocrinology Vol. 138, No. 8 3497-3504
Copyright © 1997 by The Endocrine Society

ARTICLES

Pheromone Regulated Production of Inositol-(1, 4, 5)-Trisphosphate in the Mammalian Vomeronasal Organ1

Kennedy S. Wekesa and Robert R. H. Anholt

Department of Zoology, North Carolina State University, Raleigh, North Carolina 27695

Address all correspondence and requests for reprints to: Dr. Robert R. H. Anholt, Department of Zoology, Box 7617, North Carolina State University, Raleigh, North Carolina 27695.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Social behaviors of most mammals are profoundly affected by chemical signals, pheromones, exchanged between conspecifics. Pheromones interact with dendritic microvilli of bipolar neurons in the vomeronasal organ (VNO). To investigate vomeronasal signal transduction pathways, microvillar membranes from porcine VNO were prepared. Incubation of such membranes from prepubertal females with boar seminal fluid or urine results in an increase in production of inositol-(1, 4, 5)-trisphosphate (IP3). The dose response for IP3 production is biphasic with a GTP-dependent component at low stimulus concentrations and a nonspecific increase in IP3 at higher stimulus concentrations. The GTP-dependent stimulation is mimicked by GTP{gamma}S and blocked by GDPßS. Furthermore, the GTP-dependent component of the stimulation of IP3 production is sex specific and tissue dependent. Studies with monospecific antibodies reveal a G{alpha}q/11-related protein in vomeronasal neurons, concentrated at their microvilli. Our observations indicate that pheromones in boar secretions act on vomeronasal neurons in the female VNO via a receptor mediated, G protein-dependent increase in IP3. These observations set the stage for further investigations on the regulation of stimulus-excitation coupling in vomeronasal neurons. The pheromone-induced IP3 response also provides an assay for future purification of mammalian reproductive pheromones.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MOST MAMMALS use pheromones to coordinate reproduction (1). These chemical signals can be classified into two categories: those with short-term effects on the behavior of the recipient (signaling pheromones), and those with long term effects on the physiology of the recipient (priming pheromones) (1). For example, signaling pheromones in urine or glandular secretions play a role in the initiation of copulatory behavior (2), whereas priming pheromones are responsible for puberty acceleration (3, 4, 5, 6, 7, 8) and reproductive activation (9, 10). Although the majority of studies on reproductive pheromones have been done on rodents, pheromone-dependent effects on reproduction have been documented also for sheep (11), cattle (12), and pigs (5, 13, 14).

One of the most extensively studied pheromonal effects is the acceleration of puberty in the female house mouse (3, 4, 6, 7). The presence of an adult male, or his urine, accelerates the onset of puberty in prepubertal female mice as evident from a rapid and dramatic increase in uterine weight (3, 4, 6, 7). The induction of the puberty accelerating pheromone is androgen dependent because urine from prepubertal males, castrated adult males, or adult females fails to accelerate puberty (4). Pheromone-dependent puberty acceleration is not unique to rodents but occurs also in pigs (5, 13), sheep (11), and cows (12).

Whereas the perception of signaling pheromones may be mediated by the main olfactory system, the physiological effects of most priming pheromones are initiated in the vomeronasal organ (VNO; 1, 15–17). The VNOs are paired, cartilage-encased elongated organs associated with the vomer bone in the rostral nasal cavity. The VNO contains a lumen that communicates via a duct with the oral (most mammals, including pigs) or nasal (e.g. horses) cavity (15, 16). Chemical stimuli in urine and glandular secretions of conspecifics act upon the dendritic microvilli of bipolar chemosensory neurons in the VNO. The VNO is the chemoreceptive organ of the accessory olfactory system, which is functionally and anatomically distinct from the main olfactory system (16, 18, 19). The main olfactory bulb sends projections to the primary olfactory cortex, the nucleus of the lateral olfactory tract, the olfactory tubercle, and the periamygdaloid region, while afferent neurons from the VNO project to the accessory olfactory bulb, from which secondary neurons extend into the bed nucleus of the stria terminalis, the medial amygdala, and the hypothalamus, enabling pheromones to influence reproductive physiology and behavior (16, 18). Removal of the VNO or the accessory olfactory bulb impairs reproductive behavior, whereas lesions of the main olfactory epithelium or main olfactory bulb that result in anosmia do not impair pheromonal effects (17, 19, 20). Thus, whereas the main olfactory bulb analyses odors in the environment, the accessory olfactory bulb is specialized to detect conspecific chemical signals that activate stereotypic instinctive behaviors via the neuroendocrine system.

In the main olfactory system, odorants bind to heptahelical, G protein-coupled receptors on the ciliated dendrites of olfactory neurons (21). These odorant receptors are encoded by a diverse array of about 1,000 genes (21, 22). Binding of an odorant to its receptor activates a heterotrimeric G protein (G{alpha}olf; 23), which leads to activation of adenylyl cyclase (22, 24). The resulting increase in cAMP elicits the generator potential by directly opening cyclic nucleotide-gated channels in the ciliary plasma membrane (22, 24, 25, 26).

The VNO and olfactory epithelium both derive from the olfactory placode. Both the VNO and main olfactory epithelium possess bipolar neurons that are functionally replaced from neurogenic precursor cells throughout life (27). In addition, in both systems primary chemosensory neurons form convergent projections onto the main olfactory bulb or accessory olfactory bulb. Similarities in embryonic development, anatomical organization, and chemosensory function initially suggested that transduction mechanisms in the VNO would resemble those in the olfactory epithelium. However, screens of complementary DNA (cDNA) libraries from murine VNO failed to yield cDNAs encoding G{alpha}olf, adenylate cyclase type III, and the {alpha}-subunit of the olfactory cyclic nucleotide-gated channel (28, 29). Studies in rat also indicate that the olfactory epithelium and VNO differ in signal transduction pathways (30). Electrophysiological characterization of chemosensory neurons from the murine VNO failed to detect functional cyclic nucleotide-gated channels in vomeronasal neurons (31, 32). Finally, a family of putative pheromone receptors has been identified in the VNO (33). Although these belong to the superfamily of heptahelical receptors, they do not share motifs characteristic of the family of odorant receptors (21). These studies all support the notion that chemosensory neurons of the main olfactory system and the accessory olfactory system use different signal recognition and transduction pathways (32).

Functional biochemical studies on the VNO that would complement molecular biological approaches have been hampered by the small size of the VNO in most common laboratory animals. To eliminate this problem, we selected the domestic pig (Sus scrofa) as our model system. Pigs have large VNOs that are well separated from the main olfactory system, and their reproductive physiology and behavior, like that of rodents, is regulated by pheromones (5, 13, 14). We have developed a procedure for the preparation of a VNO membrane fraction enriched in dendritic microvillar membranes that allows routine measurements of second messengers. Here we report that pheromones contained in seminal fluid and boar urine stimulate the production of inositol-(1, 4, 5)-trisphosphate (IP3) when applied to microvillar VNO membranes from female juvenile pigs (gilts). This stimulation is dose dependent, GTP dependent, sex dependent, and tissue specific. We show further that this microvillar membrane preparation contains a G protein of the G{alpha}q class, which commonly mediates receptor-activated increases in IP3 (34). Finally, immunohistochemical studies show prominent G{alpha}q/11-immunoreactivity concentrated at the microvilli of vomeronasal neurons, supporting the notion that pheromones in seminal fluid and boar urine stimulate vomeronasal neurons of female pigs via a receptor mediated G{alpha}q/11 coupled IP3 pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Membrane preparations
Freshly collected boar urine, gilt urine, and seminal fluid were provided by Dr. W. L. Flowers from the Animal Science Department at North Carolina State University. The seminal fluid was centrifuged to remove cells. The seminal fluid, boar urine and gilt urine were stored as aliquots under argon at -80 C until used. Pigs were made available immediately following euthanasia by Drs. R. A. Argenzio, N. A. Monteiro-Riviere, and R. A Abdullahi, from the College of Veterinary Medicine at North Carolina State University. VNOs from gilts, up to six months old, were dissected from their crevices in the nasal cavity, removed from the cartilaginous capsule, and frozen on dry ice. The tissues were then minced and crushed with a razor blade and subjected to sonication for 2–5 min in ice-cold PBS in a Bransonic bath sonicator. The resulting suspension was layered on a 45% (wt/wt) sucrose cushion and centrifuged at 4 C for 30 min at 40,000 rpm in a Beckman SW55Ti rotor. The membrane fraction on top of the sucrose was collected and centrifuged as before for 15 min to pellet the membranes. The membranes were resuspended in 100 µl of ice-cold PBS. Protein was then determined according to the method of Lowry et al. (35), using BSA as standard. Membranes from olfactory tissue were prepared according to the same procedure. Membranes from liver, brain, kidney, and lung were prepared by homogenizing the tissue in PBS with a Teflon homogenizer. Membranes were collected by centrifugation, washed once, and resuspended in PBS.

Second messenger assays
Forskolin and nucleotides were purchased from Boehringer Mannheim (Indianapolis, IN), [3H] cAMP and [32P]-{alpha}-ATP were from Amersham Radiochemical Corporation (Arlington Heights, IL). Adenylate cyclase activity was measured according to the method of Salomon et al. (36), in the presence of 10 µM forskolin, boar urine or 10 µM guanosine 5'-0-(3-thiotriphosphate (GTP{gamma}S). For IP3 assays, reactions were incubated for 1 min at 37 C in 25 mM Tris-acetate buffer, pH 7.2, 5 mM Mg-acetate, 1 mM dithiothreitol, 0.5 mM ATP, 0.1 mM CaCl2, 0.1 mg/ml BSA, 10 µM GTP, and 20 µg VNO membrane protein. Reactions were terminated by adding 1 M trichloroacetic acid. IP3 was measured with a kit from New England Nuclear, Inc. (Boston, MA) according to the manufacturer’s instructions and is based on displacement of [3H] IP3 from a specific IP3 binding protein. Differences between experimental and control animals were analyzed using the Student’s t test.

Western Blotting
VNO membrane samples were subjected to electrophoresis on a 10% SDS-polyacrylamide gel, followed by electrophoretic transfer onto a nitrocellulose membrane. Strips of the membrane, containing approximately 10 µg protein were probed with a 1,000-fold dilution of normal rabbit serum or 1,000-fold dilutions of rabbit antisera against specific G protein subunits (Calbiochem, La Jolla, CA). Bound antibody was visualized via a biotinylated goat-antirabbit secondary antibody complexed with avidin and biotinylated horseradish peroxidase, using Amersham’s chemiluminescent ECL detection system. Migration distances were calibrated with biotinylated low range molecular weight markers (Bio-Rad, Richmond, CA).

Immunohistochemistry
Formalin-fixed and paraffin-embedded, 5-µm thick, coronal sections through the VNO were deparaffinized in xylene and rehydrated through graded alcohols. Sections were blocked with 0.5% casein for 1 h at room temperature. They were then incubated with a 200-fold dilution of either normal rabbit serum or anti-G{alpha}q/11 antiserum in PBS, 0.05% Triton X-100. This was followed by several washes and incubation for 1 h at room temperature with biotinylated goat antirabbit IgG in PBS supplemented with 0.05% Triton X-100 and 1% normal rabbit serum. At this stage, endogenous peroxidase activity was abolished by exposing the sections for 10 min to 0.3% hydrogen peroxide. After addition of streptavidin-biotinylated horseradish peroxidase complex (Zymed Laboratories, Inc., San Francisco, CA), bound antibody was visualized as brown immunoprecipitates using 3,3'diaminobenzidine as chromogenic substrate. Sections were then counterstained with hematoxylin, and viewed and photographed under a Zeiss Axioplan microscope.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dose-dependent and GTP-dependent increases in IP3 levels induced by boar seminal fluid and urine in VNO membranes from gilts
To study transduction pathways activated by pheromonal stimuli from the male, we developed a preparation enriched in microvillar membranes from VNOs of gilts. The tissue is subjected to sonication to detach microvilli from the vomeronasal neuroepithelial surface, and the resulting membranes are then collected by centrifugation on a sucrose cushion. The yield of these membranes is 141 ± 9 µg protein/VNO (n = 11) and this membrane fraction is approximately 3-fold, enriched in both the specific activities of adenylate cyclase and phospholipase C. The basal activity of adenylate cyclase (68 ± 12 pmol/min·mg n = 5) can be readily activated by the nonhydrolyzable GTP analogue, GTP{gamma}S, and forskolin (117 ± 13 and 228 ± 38 pmol/min·mg, respectively, n = 5), but we could not detect stimulation with pheromonal stimuli from either seminal fluid, boar urine, or the known boar pheromone, 5{alpha}-androst-16-en-3-one (Sigma Chemical Company, St. Louis, MO). Vomeronasal adenylate cyclase activity is approximately 50-fold lower than activities observed in olfactory cilia preparations (37, 38) and is not affected by calcium/calmodulin (38). This is in line with observations by Berghard and Buck which indicate that the vomeronasal adenylate cyclase is the calmodulin-insensitive type II isoform rather than the adenylate cyclase type III found in olfactory receptor cells (29).

In contrast to the vomeronasal adenylate cyclase, incubation of microvillar membranes from gilts with seminal fluid results in a robust, dose-dependent increase in IP3 (Fig. 1Go). The dose-response curve is biphasic. At lower stimulus concentrations a component of the response saturates at the level of stimulation observed with GTP{gamma}S. At higher stimulus, concentrations an additional nonsaturable, nonspecific increase in IP3 is observed. The response seen at low concentrations of seminal fluid (up to 1.5% vol/vol) is mimicked by GTP{gamma}S and blocked by GDPßS (Fig. 2Go). Similar responses are elicited with urine from the boar, but here higher stimulus concentrations (up to 3% vol/vol) are required to resolve the GTP-dependent component of the IP3 response. No increases in IP3 were observed in response to 5{alpha}-androst-16-en-3-one up to concentrations of 100 mM. This is consistent with previous studies that suggest that androstenone is a signaling pheromone which mediates its effects via the main olfactory rather than the accessory olfactory system (39). We conclude from these observations that female VNO membranes respond to stimuli in boar seminal fluid and urine with an increase in IP3 via a G protein coupled pathway.



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  		Figure 1. Dose dependence of the production of IP3 by seminal fluid in female VNO membranes. The dose-response curve for activation of IP3 is biphasic with a specific GTP-dependent component at lower concentrations of seminal fluid and a nonspecific GTP-independent component at higher concentrations. Each data point represents the mean of four to six independent experiments, each consisting of duplicate measurements. SEs are within 18% of the mean for all measurements.

 


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  		Figure 2. GTP dependence of the production of IP3 by seminal fluid in female VNO membranes. Reactions were performed without stimulus, in the presence of 1.5% seminal fluid (vol/vol), 10 µM GTP{gamma}S, or 1.5% seminal fluid together with 100 µM GDPßS. Significant stimulation compared with basal activity is observed in the presence of seminal fluid and GTP{gamma}S (*, P < 0.05) and not in the presence of seminal fluid together with GDPßS.

 
Sex specificity and tissue specificity of the IP3 response
Relatively little information is available about the chemical nature of mammalian pheromones. Because the active components in boar seminal fluid and urine have not been identified, we deemed it important to establish whether the observed IP3 responses were physiologically relevant. Previous studies on rodents have shown that production of reproductive pheromones by the male is androgen dependent (4). We reasoned, therefore, that only boar urine, but not urine from prepubertal females, should elicit an increase in IP3 in VNO membranes from gilts. The results presented in Fig. 3Go demonstrate that this prediction is correct. Whereas boar urine elicits a robust increase in IP3, experiments using urine samples from gilts failed to activate IP3 production above the basal level. To further document specificity of the observed IP3 response, we investigated the tissue specificity of seminal fluid-induced increases in IP3 by testing the effect of 1.5% seminal fluid side-by-side on microvillar VNO membranes and membranes obtained from olfactory tissue, brain, lung, liver, and kidney. Basal activity of phospholipase C was detected in all samples and was particularly high in membranes from olfactory tissue (Fig. 4Go). However, whereas seminal fluid caused a 2-fold increase in IP3 production in VNO membranes, no significant stimulation above the basal level was observed in membranes from olfactory tissue, liver, lung, and kidney. Besides the VNO, significant increases in the level of IP3 upon exposure to seminal fluid were observed only in membranes from brain. We attribute this stimulation to the presence of neuroactive substances in seminal fluid.



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  		Figure 3. Sex dependence of the production of IP3 in female VNO membranes. Reactions were performed without stimulus, in the presence of 3% (vol/vol) gilt urine, 3% (vol/vol) boar urine, 1.5% (vol/vol) seminal fluid, and 10 µM GTP{gamma}S. All assays were done in the presence of 10 µM GTP. Significant stimulation compared with basal activity is observed in the presence of boar urine, seminal fluid, and GTP{gamma}S (*, P < 0.05). Levels of IP3 production by boar urine, seminal fluid, and GTP{gamma}S are not statistically different. There is also no significant difference between the baseline control and stimulation by gilt urine.

 


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  		Figure 4. Tissue specificity of the production of IP3 by seminal fluid in female VNO membranes. Reactions were performed using 20 µg of membrane protein and 1.5% seminal fluid (vol/vol) as the stimulus. There were no significant differences between the basal level of IP3 (open bars) and its level in the presence of seminal fluid (closed bars) with membranes from olfactory tissue (OLF), kidney, liver, or lung. Significant differences between control and stimulated IP3 levels are observed in VNO membranes and brain membranes (*, P < 0.05).

 
The experiments described above demonstrate a GTP-dependent and dose-dependent increase in the formation of IP3, but not cAMP, in female VNO membranes upon exposure to boar seminal fluid or urine. This stimulation shows tissue specificity and sex dependence. Together, these observations indicate that pheromones from the male activate the production of IP3 in the female VNO via specific G protein-coupled receptors.

Identification of a G{alpha}q/11 related G protein on the microvillar surface of the VNO
Previously, immunohistochemical studies identified {alpha}-subunits of Gi2 and Go in distinct subpopulations of vomeronasal neurons. Although we cannot exclude that either of these two G proteins could mediate the observed stimulation of phospholipase C, we decided to investigate whether our VNO membrane preparation contains a G protein of the G{alpha}q class, known to cause activation of at least the ß1-isoform of phospholipase C (34). Using subunit-specific rabbit antisera against unique peptide sequences of G{alpha}s, G{alpha}i1/G{alpha}i2, G{alpha}i1, G{alpha}i3/G{alpha}o, G{alpha} i3, G{alpha}q/11, and Gß, we confirmed in our microvillar membrane preparation the presence of G{alpha}i2 and G{alpha}o, as reported previously (29, 40; Fig. 5Go). In addition, we observe G{alpha}s, and a single ß-subunit at 35 kDa. A G{alpha}i3/G{alpha}o specific antiserum sometimes reveals a doublet of immunoreactive bands, suggesting the presence of both G{alpha}i3 and G{alpha}o. Of greatest interest, however, is the observation of a prominent, previously unreported, immunoreactivity revealed by an antiserum against the G{alpha}q/11 protein (Fig. 5Go).



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  		Figure 5. Identification of G protein subunits in vomeronasal membranes. Each strip contained 10 µg of VNO membrane protein and was probed either with 1,000-fold dilution of normal rabbit serum (NRS) or monospecific rabbit antisera against subunits of G proteins, as indicated. The arrow indicates the 45-kDa G{alpha}s subunit, identified by the G{alpha}s-antiserum. A single ß-subunit is detected which migrates at 35 kDa. The immunoreactive bands identified by antisera against G{alpha}i3 and G{alpha}i3/G{alpha}o can sometimes be resolved as doublets, suggesting the presence of both G{alpha}i3 and G{alpha}o. Note also the presence of G{alpha}i2 and the prominent presence of a polypeptide immunoreactive with anti-G{alpha}q/11.

 
We investigated whether the G{alpha}q/11 protein is localized to the microvillar surface of the VNO. The apical region of porcine vomeronasal neurons contains a dense group of microvilli in contrast to supporting cells that carry smaller groups of microvilli (41). Immunohistochemical staining of coronal sections through the VNO with the G{alpha}q/11 antiserum reveals intense staining of microvillar tufts at the surface of the vomeronasal lumen (Fig. 6AGo). Cell bodies of vomeronasal neurons and dendritic processes are also stained, but the staining here is lighter, and it is obvious that immunoreactivity is concentrated primarily at the microvillar surface. To verify the specificity of this staining, adjacent sections were incubated either without the primary antibody or with normal rabbit serum at the same concentration. Under these conditions, no staining was detected (Fig. 6BGo). Thus, G{alpha}q/11, which links receptor mediated responses to activation of phospholipase C, is expressed by vomeronasal neurons and concentrated at the microvillar surface, where the pheromone first encounters the chemosensory neurons.



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  		Figure 6. Immunohistochemical localization of G{alpha}q/11 to the microvillar surface of the VNO. A, Section stained with a 200-fold dilution of antiserum against G{alpha}q/11. Note the deposition of brown reaction product on microvillar tufts along the microvillar surface of the neuroepithelium (arrows) and in the cell bodies of the vomeronasal neurons (arrowheads). B, Adjacent section incubated with normal rabbit serum. Scale bar, 100 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results suggest that in the porcine VNO IP3 is the principal second messenger that mediates pheromonal signal transduction. This is in contrast to the main olfactory system, where cAMP appears to be the dominant second messenger that regulates olfactory transduction (22, 24, 25, 26). Although we cannot exclude a role for cAMP in vomeronasal signal transduction, we were unable to detect effects of either boar urine or boar seminal fluid on vomeronasal adenylate cyclase. Failure to detect message for G{alpha}olf, adenylate cyclase type III and the {alpha}-subunit of the olfactory cyclic nucleotide gated channel in cDNA libraries from VNO (29, 30) further accentuates the differences between chemosensory transduction in olfactory and vomeronasal neurons.

Involvement of IP3 has also been implicated in signal transduction in the reptilian VNO. In garter snakes, a polypeptide purified from secretions of earthworms, the snake’s prey, induces a G protein-dependent increase in IP3 and a decrease in cAMP, suggesting interactions between both signal transduction pathways (42, 43). Furthermore, patch-clamp studies on vomeronasal neurons from the turtle have shown that intracellular injection of IP3 elicits a membrane conductance (44), although effects of cAMP have also been reported in this system (45). Thus, the role of IP3 in vomeronasal signal transduction may be universal among vertebrates, whereas the influence of cAMP in vomeronasal signaling remains to be evaluated further.

Despite behavioral, electrophysiological, and molecular biological studies on the mammalian VNO, no direct measurements of dose-dependent, GTP-dependent increases in second messengers upon exposure to pheromones have been reported until now. Evaluation of the dose dependence of vomeronasal stimulation is especially important in light of the large nonspecific component we observed. In light of the previously observed diversity of putative pheromone receptors (33), it may seem puzzling that in our case specific G protein-dependent production of IP3 in response to boar seminal fluid approaches the level of IP3 production observed with GTP{gamma}S. However, this observation can be explained, if we assume that boar seminal fluid contains a cocktail of pheromones and that the high stimulus concentrations used in our study activate the vast majority of pheromone receptors, coupled to the IP3 pathway. Although the chemical nature of the pheromonal stimuli in boar urine and boar seminal fluid are not known, we believe that the IP3 responses we report reflect the physiological response of the vomeronasal neuron to pheromones because: 1) the response shows a saturable dose-dependent component; 2) this component is GTP dependent, indicating the involvement of a G protein, and by inference, G protein-coupled receptors (33); 3) the response is sex dependent, as expected from pheromones produced only by the male and intended to affect the female; 4) the response shows tissue-specificity, in agreement with the notion that it is mediated via receptors expressed selectively in the VNO; and 5) a G{alpha}q/11 type G protein, classically involved with receptor-mediated activation of phospholipase C (34), is expressed in vomeronasal neurons and concentrated on their chemosensory microvilli.

The procedure used for the preparation of microvillar membranes is modeled after well established methods for harvesting olfactory cilia from olfactory neuroepithelium (46, 47). Sonication of olfactory membranes results not only in the detachment of olfactory cilia but also in the detachment of microvilli from sustentacular cells and plasma membrane fragments from other components of the neuroepithelium (46, 47). Electron microscopic examination of these preparations revealed membrane vesicles, axonemal structures devoid of a plasma membrane, and axonemal structures associated with membrane fragments (47). The membrane preparation we refer to as "microvillar membranes" is, therefore, likely to contain contaminants derived from other components of the VNO, including microvillar membranes from supporting cells, and it is difficult to estimate the purity of this preparation precisely. However, our preparation appears to be sufficiently enriched in chemosensory membranes for the purpose of our studies. This assessment is based on the fact that the observed pheromone induced responses are tissue specific, sex dependent, and G protein mediated. It is further supported by the notion that the prominent expression of G{alpha}q/11 immunoreactivity at the microvillar surface of the neuroepithelium (Fig. 6Go) mirrors the prominent visualization of G{alpha}q/11 immunoreactivity observed in the microvillar membrane preparation on Western blots (Fig. 5Go).

Immunohistochemical studies on opossum (40) and in situ hybridization studies in mouse (29) revealed that a population of neurons in the apical layer of the vomeronasal neuroepithelium expresses G{alpha}i2. These neurons project to the anterior region of the accessory olfactory bulb (40). In contrast, a population of neurons located in the base of the vomeronasal neuroepithelium expresses G{alpha}o (29) and projects to the posterior region of the accessory olfactory bulb (40). Both {alpha}-subunits of these G proteins are also detected in the VNO of the pig. It remains to be determined whether these G proteins play roles directly in pheromonal transduction or in signaling processes that coordinate the growth and differentiation of vomeronasal neurons. In an extensive screen of a rat VNO cDNA library, Berghard and Buck (29) detected several cDNAs encoding G{alpha}11. It seems, therefore, reasonable to presume that the G{alpha}q/11 immunoreactivity we detect most likely represents G{alpha}11. Because the frequency of clones encoding G{alpha}11 in the library screened by Berghard and Buck (29) was low relative to cDNAs encoding G{alpha}i2 and G{alpha}o, it appears that low levels of message are produced for the G{alpha}11 protein, which may reflect a slower turnover than G{alpha}i2 and G{alpha}o. Although a direct link between pheromone detection and activation of G{alpha}11 must still be documented, the uniform presence of this G protein on all microvillar tufts suggests a role for this G protein in pheromonal signaling in all mature vomeronasal neurons. Localization of G{alpha}q/11 to the neuronal compartment of the VNO is supported by the observation that neuronal cell bodies of the vomeronasal epithelium also stain and is in agreement with immunohistochemical observations at the electron microscopic level by Menco et al. (48), who reported presence of G{alpha}q immunoreactivity on axons of vomeronasal neurons.

Pheromone-induced increases in IP3 imply a role for calcium in vomeronasal signal transduction (49). Although the mechanisms that underlie transduction-excitation coupling in the VNO remain to be elucidated, several lines of circumstantial evidence support a role for calcium in this process. High levels of three calcium binding proteins, calretinin, calbindin-D28k, and parvalbumin are found in vomeronasal neurons (50, 51). In addition, patch-clamp studies identified both an L-type and T-type calcium current in rat vomeronasal neurons, indicating a role for calcium in neuronal excitation (31). Electron microscopic studies show that, in all species thus far examined, including pigs, the apical dendritic domes of vomeronasal neurons are densely populated with intracellular vesicles (41). It is tempting to speculate that these vesicles may serve as calcium storage depots, from where calcium can be released by IP3. It will be of interest to determine in future studies whether IP3 receptors are located on these vesicular membranes. In addition, we have observed that incubation of microvillar membranes with phorbol esters results in protein phosphorylation (data not shown). Thus diacylglycerol formed together with IP3 may also play a role in pheromonal signaling through activation of protein kinase C.

Although behavioral and physiological effects of pheromones have been well documented, mostly in rodents (1, 2, 3, 4, 6, 7, 8, 9, 10), but also in pigs (5, 13, 14), unambiguous identification of pheromones from urine has been problematic. Several laboratories have reported the identification of putative pheromones (6, 7), but independent confirmation of these claims has been notoriously lacking. Hitherto, identification of pheromones has depended on laborious and time-consuming bioassays that involve many animals maintained under controlled environmental conditions to limit individual variation. This renders systematic isolation of pheromones extremely difficult and virtually impossible if the biological response depends on a blend of pheromones, of which individual components may separate during fractionation. In addition to setting the stage for further biochemical studies on regulation of pheromonal signal transduction pathways and transduction-excitation coupling in the VNO, our experiments provide a biochemical assay, i.e. a robust GTP-dependent increase in IP3, for the future identification of mammalian pheromones.


   Acknowledgments
 
We thank Marjory Gray, Martha Amstrong, Drs. R. A. Argenzio, N. A. Monteiro-Riviere, and R. A. Abdullahi, from the College of Veterinary Medicine at North Carolina State University for enabling us to obtain fresh porcine vomeronasal organs and Dr. W. L. Flowers from the Department of Animal Science at North Carolina State University for providing us with gilt urine, boar urine, and seminal fluid. We thank Yee-Lut Kwok and Sundip Patel for technical assistance. We would also like to thank Dr. John G. Vandenbergh for valuable discussions and critical reading of the manuscript.


   Footnotes
 
1 This work was supported by a grant from the North Carolina Biotechnology Center (9605-ARG-0015) and partially by grants from the National Institutes of Health (DC-02485) and the U.S. Army Research Office (DAAH04–96-I–0096). Back

Received March 17, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Vandenbergh JG 1994 Pheromones and mammalian reproduction. In: Knobil E, Neill D (eds) Physiology of Reproduction. Vol. 2, Raven Press, New York, pp 343–359
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