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.

Surface changes in the rat vomeronasal epithelium during degeneration and regeneration of sensory receptor cells.

To investigate cell turnover in the vomeronasal epithelium we used electron microscopy to obtain quantitative measurements of changes observed at the surface of the sensory epithelium. Receptor cell degeneration was induced by sensory nerve transection and animals were examined at postoperative recovery times of 2, 4, 6, 10, 15, 35 and 60 days. We measured the number and density of receptor and supporting cells, and membrane length at the surface of the sensory epithelium. The number of receptor cells rapidly decreased during the degeneration period, reaching a minimum at 6 days. After 15 days of recovery the number and density of receptor cells returned to control levels. The surface membrane length for regenerated receptor cells was similar to that of controls, however the morphological appearance was characteristic of immature cells. In contrast to the receptor cells, the number and density of supporting cells did not change during degeneration and regeneration. However, there was a significant increase in the length of supporting cell-surface membranes. These results suggest that during receptor cell degeneration, supporting cell membranes compensate for the loss of receptor cells by expanding their surface membrane length to help to maintain the continuity of the epithelial surface. Thus, an important role of vomeronasal supporting cells may be to maintain the structural integrity of the epithelium during turnover of the receptor cell population.

Morphological and histochemical changes in the regenerating vomeronasal epithelium.

Receptor cell degeneration and regeneration within the vomeronasal organ (VNO) of the rat was studied using both electron microscopy and histochemical methods. Electron microscopy was employed to examine the morphological changes along the surface of the sensory epithelium, and histochemical markers were used to monitor the changes in the epithelial cell layers. Transection of the vomeronasal nerves induced selective degeneration of the receptor cells, and within six days, a significant decrease in the number of receptor cells was observed. During the subsequent stage of receptor cell regeneration, cilia and bud-like structures characteristic of a developing sensory epithelium were seen. By day 15, thin microvilli covering the surface of the receptor cells reappeared in the sensory epithelium. The neural cell adhesion molecule (NCAM) and two vomeronasal system-specific lectins; 1) Bandeiraea simplicifolia lectin (BSL-I) and 2) Vicia villosa agglutinin (VVA) were used as the histochemical markers. NCAM immunoreactivity on the surface of the epithelium was observed to be decreased significantly six days after nerve transection, and was restored during receptor cell regeneration (day 15). The reactivity of the two lectins, BSL-I and VVA, was decreased slightly during degeneration, but was still detectable at the time of maximum receptor cell degeneration (day 6). Lectin reactivity was restored to control levels by day 15. These findings suggest that (1) NCAM is a useful marker for vomeronasal receptor cells and that the vomeronasal system-specific lectins may bind to both receptor and supporting cells and (2) degeneration of vomeronasal receptor cells occurs during the first week (day 6) following nerve transection and the receptor cell population begins to recover within 15 days. The morphological changes observed during receptor cell regeneration suggest that the stages of VNO receptor cell regeneration are similar to those observed during development.

The expressed localization of rat putative pheromone receptors.

The localization of pheromone receptors in the rat vomeronasal epithelium was examined by light- and electron-microscopic immunocytochemical analysis, using affinity-purified polyclonal antibodies. The antibodies were raised against a synthetic oligopeptide corresponding to a partial sequence of the rat putative pheromone receptor (VN6). Positive immunoreactivity was observed on the luminal surface of the sensory epithelium, and was abolished when an excess of the antigen peptide was added to the primary reaction solution. On electron microscopy, the immunoreactivity for the VN6 peptide was localized at the dendritic knobs and microvilli of receptor cells, but not in those of the supporting cells. These results show the first evidence of cellular localization of putative pheromone receptors in rat vomeronasal receptor cells.

A developmental study using three antibodies (VOBM1, VOBM2, and VOM2): immunocytochemical and electron microscopical analysis of the luminal surface of the rat vomeronasal sensory epithelium.

The development of the rat vomeronasal organ was studied morphologically and immunocytochemically, using the monoclonal antibodies (MAbs) VOBM1, VOBM2 and VOM2 that react with the luminal surface of the vomeronasal sensory epithelium. Postnatal day (P) 7, 14, 21, 28, 35 and adult animals were examined. The vomeronasal organ and the blood vessel of the organ markedly increased in size and the vomeronasal glands increased in number between P7 and P14. At P35, the shape of the vomeronasal organ was similar to that of the adult but its size was slightly smaller. Electron microscopy showed that only a few scattered microvilli were present on supporting cells, and receptor cells were immature at P7. At P21, well-branched microvilli of the receptor cells and many microvilli of the supporting cells were observed on the luminal surface of the sensory epithelium. At P35, most apical endings of supporting cells and receptor cells were covered with numerous microvilli. Less developed areas were also present at the luminal surface of the epithelium at P35. At P7, immunoreactivities of the three antibodies were observed as discontinuous thin-layered bands only on the luminal surface of the sensory epithelium and no immunoreactivity was observed in other regions of the vomeronasal organ. Immunoreactivities of the VOBM1, VOBM2 and VOM2 increased with age and were observed as continuous thin-layered bands on the luminal surface of the epithelium by P35. These finding suggest that the development of the vomeronasal organ continues after birth and that the organ may reach maturity just before puberty (P42-49).