Introduction
Pheromonal signals provide specific information concerning the identity, gender, endocrine, and social status of different members of the population in a variety of Pheromones have been mammals  [1;2]. found in saliva, skin gland secretions, and For example, pheromones in urine urine. excreted from male and female rats induce various changes in gonadal functions such as reflex ovulation in the absence of coitus and mounting, a reduction in the oestrous cycle of female rats from 5 to 4 days, and oestrous synchrony among female rats living together. vomeronasal system is a The chemosensory for receiving system pheromones organized in parallel with the main olfactory system in most terrestrial Protein pheromones The vomeronasal organ in the rat Fig. 1 vertebrates (Fig. 1).    The vomeronasal organ, which is the peripheral chemoreceptor organ of the vomeronasal system, forms a tubular structure lying bilaterally in the ventral part of the nasal cavity.   An autonomically controlled vascular pump can transport nonvolatile and volatile pheromones into the inner lumen of the vomeronasal organ [3]. Pump activation is a prerequisite for normal vomeronasal stimulation in behaving animals. Sensory neurons in the vomeronasal sensory epithelium  project  information  to  the accessory olfactory bulb located on the dorso-caudal surface of the main olfactory bulb. Pheromonal transduction mediated via IP 3 in the mammalian vomeronasal system The interaction of a pheromone with the receptive membrane of vomeronasal sensory neurons initiates a sequential molecular event leading to action potential initiation, which transmits pheromonal information to the accessory  olfactory  bulb. Dialysis  of inositol-1,4,5-trisphosphate (IP 3 ) into rat vomeronasal sensory neurons induced inward Increases in the impulse currents  [4]. frequency of sensory neurons in response to urinary  pheromones  were  blocked  by ruthenium red, an IP3-dependent channel and    U-73122    or    neomycin, inhibitor, phospholipase C inhibitors [5].    Male Wistar urinary pheromones as well as female Wistar and male Donryu urinary pheromones induced IP 3 accumulation in the vomeronasal epithelium of the female Wistar rat [6]. IP 3  accumulation  in  response to pheromones in mammals has also been observed in the hamster and the pig.    That is, aphrodisin, a pheromone of 19 kDa protein, excreted from the female hamster, and semen fluid from the pig induce IP 3 accumulation in preparations of male hamster vomeronasal sensory    epithelium   [7]    and    porcine vomeronasal  epithelium,  respectively. The average  reversal  potential  of  urinary pheromones- induced current was similar to that of the IP 3-induced current in rat vomeronasal sensory neurons [4;8].  These results  indicate  that  the  response  of vomeronasal sensory neurons to pheromones is generated via the IP 3-dependent pathway.
in
Selective pheromone reception vomeronasal sensory neurons Single vomeronasal sensory neurons of female Wistar rats responded selectively to urinary pheromones from male and female Wistar  rats  [9]. Each sensory neuron responded to only one class of urinary pheromones.  The neurons also responded selectively to urinary pheromones from male Donryu and Sprague-Dawley rats.  Thus, vomeronasal sensory neurons discriminate differences in sex and strains in urinary pheromones. Cell bodies of vomeronasal sensory neurons are located at various depths in the cellular layer of sensory epithelium. As shown in Fig. 2, the sensory neurons at the apical and basal layers of the sensory epithelium of marsupial and rodents are immunoreactive to anti-Gi2α and anti-Goα proteins, respectively [10].  Localization of the cell bodies of sensory neurons, which responded to male Wistar and male Donryu urinary pheromones, was examined using female Wistar rats [9].  The preponderance of neurons responding to conspecific male urinary pheromones from rats of the same species were found in the Gjα-positive laminae    of    the    vomeronasal    sensory epithelium, and the preponderance of neurons responding to conspecific female rat urinary pheromones and male urinary pheromones different strain (Donryu from  a or Sprague-Dawley) were found in the G o -positive laminae of the vomeronasal sensory epithelium (Fig. 2).   Therefore, it is likely that responses of sensory neurons in the apical portion of the female rat vomeronasal epithelium to the male Wistar urinary pheromones were mediated via G i , while responses of neurons in the basal portion to the male Donryu urinary pheromones were mediated via G o .
Putative receptor for pheromones
Two families of vomeronasal G-protein coupled receptors (GPCRs) unrelated to olfactory GPCRs were cloned from the rat and mouse vomeronasal epithelium [11]. The human genome contains a vomeronasal GPCR gene.  Each family is composed of about one hundred species of vomeronasal In situ hybridization studies GPCR. suggested that a single sensory neuron expressed one type of vomeronasal GPCR. One family consists of the sensory neurons expressing G i2α in the upper layer of the epithelium, and another family consists of the neurons expressing Goα.  It is possible that responses to the male Wistar urinary pheromones are induced via the former type of vomeronasal GPCR, and those to the female Wistar urinary pheromones and the male Donryu urinary pheromones are induced via the latter type of vomeronasal GPCR. Projection of pheromonal information to the accessory olfactory bulb Two populations of sensory neurons in the vomeronasal organ project information to different regions of the glomerular layer of the accessory olfactory bulb [10].  These subregions receive different pheromonal information vomeronasal sensory from Thus, the response to neurons  [12]. pheromones in the male Wistar rat urinary pheromones is selectively transmitted to the rostral part of the accessory olfactory bulb of female Wistar rats. It  is  likely  that information from different pheromones is transmitted to the higher brain through the different regions of the accessory olfactory bulb.
Chemical characterization of pheromones
Pheromones have been found to be proteins and low molecular weight molecules At present, pheromones that [13-17]. induce behavioral and endocrinological changes in rats have not been identified. We have characterized active components stimulating the rat vomeronasal organ in urine by examining excitation of neurons in the accessory olfactory bulb.  The activity of the component in male urinary expression  of pheromones to induce Fos-immunoreactivity, which is correlated with neural activity, in the caudal region of the accessory olfactory bulb of female rats was abolished by papain treatment, while that in the rostral region was not [18].  The pronase treatment of male urine abolished the expression of immunoreactivity in the rostral region as well as in the caudal region, suggesting that at least two urinary peptides (papain-sensitive and -insensitive ones) with the ability to stimulate the vomeronasal organ of female rats are contained in male Wistar Exposure of the female rat rat urine. vomeronasal organ to either the dialyzed urine preparation (< 500 Da) or the remaining substances (> 500 Da) of male rats did not induce  expression Fos- of immunoreactive cells in the accessory olfactory bulb, whereas exposure to a mixture of these preparations did induce expression [19].  This suggests that a combination of low and high molecular weight substances is necessary for the increases in Fos-immunoreactivity in the accessory olfactory bulb. Similar results have been obtained in the mouse and hamster.  Thus, the application of   urine-derived   compounds   of   low molecular weight such as 2,3-dehydro-exo- brevicomin induces only hyperpolarizing responses, that is, inhibitory responses, in the mouse vomeronasal sensory neuron and does not induce c-fos mRNA expression in the mouse accessory olfactory bulb.  However, 2,3-dehydro-exo-brevicomin in combination with major urinary proteins (~19 kDa) induced significantly greater c-fos mRNA The female golden hamster expression. produces a 17-kDa protein (aphrodisin) that is emitted in vaginal discharge and stimulates sexual behavior in the male hamster.  Gel filtration experiments indicated that a mixture of ligands may be present in the purified aphrodisin.    Analysis of the crystal structure of aphrodisin revealed the electron density for a small linear ligand in the cavity of the β-barrel [20].    At present, it is still not clear whether aphrodisin itself or its combination with a low molecular weight ligand is necessary for pheromonal activity. Decreases in urinary pheromonal activities in male mice after exposure to environmental pollutant Many classes of environmental pollutants, which are found at significant levels in the environment, affect  the reproductive functions and endocrine systems.   Exposure of female rats to 3-methylchoranthrene (3-MC)     or 4-tert-octylphenol, typical environmental pollutants, has been found to be associated with a significant increase in estrus cycle length.  The gonadal functions various animals are regulated by of pheromones excreted from mating partners as described above. Some volatile and nonvolatile pheromones exhibit a strong dependence on the endocrine status of the animals, suggesting that the production of pheromones is controlled by hormones.    We have shown that 3-MC interferes with the activity of male urine to stimulate female vomeronasal sensory neurons 1 day after intraperitoneal  administration  of  3-MC (unpublished data). Intraperitoneal administration of 3-MC also decreased the levels of mRNA encoding major urinary proteins (MUPs), which are thought to be candidate pheromones themselves or possible carrier proteins of pheromones in urine (Nukazuka et al., submitted for publication).  SDS analyses showed that the protein levels in the urine from males 1 day after administration of 3-MC were similar to those from non administered mice, while the levels of a protein with an apparent molecular mass of 19 kDa in urine from 3 and 10 days after the administration were lower than those in the control mice (unpublished data).  These results suggest that 3-MC blocks chemical communication between male and female mice by reducing pheromonal activities without changing the total amount of MUPs in male mice 1 day after administration.
Reference List
1) Halpern M. The organization and function of the vomeronasal system. Annu. Rev. Neurosci. (1987) 10: 325-362. 2) Kashiwayanagi M. Molecular recognition and intracellular  transduction  mechanisms in olfactory and vomeronasal systems, In Hormones, Brain and Behavior, Vol. 2, Academic Press, New York (D. Pfaff, ed), (2002) pp. 1-16. 3) Meredith M, Marques DM, O'Connell RJ, Stern EL. Vomeronasal pump: Significance for male hamster sexual behavior. Science (1980) 207 : 1224-1226. 4) Inamura K, Kashiwayanagi M, Kurihara K. Inositol-1,4,5-trisphosphate induces responses in receptor neurons in rat vomeronasal sensory slices. Chem. Senses (1997) 22: 93-103. 5) Inamura K, Kashiwayanagi M, Kurihara K. Blockage of urinary responses by inhibitors for IP3-mediated pathway in rat vomeronasal sensory neurons. Neurosci. Lett. (1997) 233: 129-132. Androgen steroids associated with boar odour as an aid to the detection of oestrus in pig artificial insemination. Br. Vet. J. (1971) 127: 497-502. 6) Sasaki K, Okamoto K, Inamura K, Tokumitsu Kashiwayanagi M. Inositol-1,4,5- Y, trisphosphate accumulation induced by urinary pheromones in female  rat vomeronasal epithelium. Brain Res. (1999) 823 : 161-168. 15) Goodwin M, Gooding KM, Regnier F. Sex pheromone in the dog. Science (1979) 203: 559-561. 7) Kroner C, Breer H, Singer AG, O'Connell RJ. Pheromone-induced second messenger signaling in the hamster vomeronasal organ. Neuroreport (1996) 7: 2989-2992. 16) Novotny M, Schwende FJ, Weisler D, Jorgenson JW, Carmack M. Identification of a testosterone-dependent volatile uniue constituent of male mouse urine: 7-exo-ethyl-5- methyl-6,8-dioxabicyclo[3.2.1]-3-octene. Experientia (1984) 40: 217-219. 8) Inamura K, Kashiwayanagi M. Inward current urinary  substances responses to in  rat vomeronasal sensory neurons. Eur. J. Neurosci. (2000) 12: 3529-3536. 17) Singer AG Macrides F, Clancy AN, Agosta Purification and analysis WC. of a proteinaceous aphrodisiac pheromone from hamster vaginal discharge. J. Biol. Chem. (1986) 261 : 13323-13326. 9) Inamura K, Matsumoto Y, Kashiwayanagi M, Kurihara K. Laminar distribution of pheromone-receptive neurons in rat vomeronasal epithelium. J. Physiol. Lond. (1999) 517: 731-739. Tsujikawa Kashiwayanagi 18) K, M. Protease-Sensitive Urinary Pheromones Induce Region-Specific Fos- Expression in Rat Accessory Olfactory Bulb. Biochem. Biophys. Res. Commun. (1999) 260: 222-224. 10) Halpern M, Shapiro LS, Jia C. Differential localization of G proteins in the opossum vomeronasal system. Brain Res. (1995) 677: 157-161. 19)  Yamaguchi T, Inamura K, Kashiwayanagi M. Increases in Fos-immunoreactivity after exposure to a combination of two male urinary components in the accessory olfactory bulb of the female rat. Brain Res. (2000) 876: 211-214. 20) Vincent F, Lobel D, Brown K, Spinelli S, Grote P, Breer H, Cambillau C, Tegoni M. Structure of Aphrodisin, a Crystal Sex Pheromone from Female Hamster. J. Mol. Biol. (2001) 305: 459-469. 11) Dulac C, Axel R. A novel family of genes encoding putative pheromone receptors in mammals. Cell (1995) 83: 195-206. 12) Inamura K, Kashiwayanagi M, Kurihara K. Regionalization of Fos immunostaining in rat accessory olfactory bulb when the vomeronasal organ was exposed to urine. Eur. J. Neurosci. (1999) 11: 2254-2260. 13) Brownlee RG, Silverstein RM, Muller-Schwarze D, Singer AG. Isolation, identification chief and  function of the component of the male tarsalscent in black-tailed deer. Nature (1969) 221 : 284-285. 14) Melrose DR, Reed HC, Patterson RL. Communicated by Hiroshi Ueno