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Expression of neuron-specific markers by the vomeronasal neuroepithelium in six species of primates.

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THE ANATOMICAL RECORD PART A 281A:1190 –1200 (2004)
Expression of Neuron-Specific
Markers by the Vomeronasal
Neuroepithelium in Six Species of
Primates
JOHN C. DENNIS,1* TIMOTHY D. SMITH,2,3 KUNWAR P. BHATNAGAR,4
CHRISTOPHER J. BONAR,5 ANNE M. BURROWS,3,6
1
AND EDWARD E. MORRISON
1
Department of Anatomy, Physiology, and Pharmacology, College of Veterinary
Medicine, Auburn University, Auburn, Alabama
2
School of Physical Therapy, Slippery Rock University, Slippery Rock, Pennsylvania
3
Department of Anthropology, University of Pittsburgh, Pittsburgh, Pennsylvania
4
Department of Anatomical Sciences and Neurobiology, University of Louisville
School of Medicine, Louisville, Kentucky
5
Cleveland Metroparks Zoo, Cleveland, Ohio
6
Department of Physical Therapy, Duquesne University, Pittsburgh, Pennsylvania
ABSTRACT
Vomeronasal organ (VNO) morphology varies markedly across primate taxa. Old World monkeys display no
postnatal VNO. Humans and at least some apes retain a vestigial VNO during postnatal life, whereas the strepsirrhines
and New World Monkeys present a morphologically well-defined VNO that, in many species, is presumed to function
as an olfactory organ. Available microanatomical and behavioral studies suggest that VNO function in these species
does not precisely duplicate that described in other mammalian taxa. The questions of which species retain a functional
VNO and what functions they serve require inquiry along diverse lines but, to be functional, the vomeronasal
epithelium must be neuronal and olfactory. We used immunohistochemistry to establish these criteria in six primate
species. We compared the expression of two neuronal markers, neuron-specific ␤-tubulin (BT) and protein gene product
9.5, and olfactory marker protein (OMP), a marker of mature olfactory sensory neurons, in paraffin-embedded VNO
sections from two strepsirrhine and four haplorhine species, all of which retain morphologically well-defined VNOs
during postnatal life. The infant Eulemur mongoz, adult Otolemur crassicaudatus, neonatal Leontopithicus rosalia, and
adult Callithrix jacchus express all three proteins in their well-defined vomeronasal neuroepithelia. The infant Tarsius
syrichta showed some BT and OMP immunoreactivity. We establish that two strepsirrhine species and at least some
New World haplorhines have mature sensory neurons in the VNO. In contrast, at all ages examined, Saguinus geoffroyi
VNO expresses these markers in only a few cells. © 2004 Wiley-Liss, Inc.
Key words: immunohistochemistry; olfaction; olfactory marker protein; primate; ␤-tubulin; PGP 9.5;
vomeronasal
The tetrapod vomeronasal organ (VNO) is bilaterally
symmetric and lies along the ventrorostral aspect of the
nasal septum. Sensory neurons in the vomeronasal neuroepithelium (VNNE) of snakes, rodents, and opossums
detect chemical signals that evoke behavioral and/or physiological changes regarding prey identification, social status, and reproductive state (Wysocki, 1979; Halpern,
1987; Takami, 2002; Halpern and Martı́nez-Marcos,
2003).
The rodent VNO is the best studied in terms of gene
expression during development, its morphological organization, its secondary and higher-order connections, and its
effects on the organism’s behavior and physiology. For
some time, the rodent VNO was thought to epitomize the
©
2004 WILEY-LISS, INC.
mammalian vomeronasal system but VNO function as
exemplified in rodents is not precisely replicated in other
mammalian taxa, namely, ferrets (Weiler et al., 1999;
*Correspondence to: John C. Dennis, Department of Anatomy,
Physiology, and Pharmacology, College of Veterinary Medicine,
Auburn University, Auburn, AL 36849. Fax: 334-844-4542.
E-mail: dennijc@vetmed.auburn.edu
Received 20 May 2004; Accepted 1 July 2004
DOI 10.1002/ar.a.20124
Published online 7 October 2004 in Wiley InterScience
(www.interscience.wiley.com).
1191
EXPRESSION OF NEURON-SPECIFIC MARKERS
TABLE 1. Species, age, sex, source, and procedures used on primates*
Specimen
number
SG4
SG5
MM105
MM101
SG6
LR1
LR3
CJ21
CJ16
P96
EM1
P2855
P2766
P2868
Species
Age
Sex
Source
Saguinus geoffroyi
Saguinus geoffroyi
Saguinus geoffroyi
Saguinus geoffroyi
Saguinus geoffroyi
Leontopithecus rosalia
Leontopithecus rosalia
Callithrix jacchus
Callithrix jacchus
Tarsius syrichta
Eulemur mongoz
Otolemur crassicaudatus
Otolemur crassicaudatus
Otolemur crassicaudatus
n (0)
n (0)
1 month
2 month
2.75 years
n (3)
4 months
21 years
8 years
n (0)
n (0)
9 years
12 years
8 years
F
F
M
F
F
F
F
M
M
?
?
F
F
M
CZ
CZ
CZ
CZ
CZ
CZ
CZ
DM
DM
DPC
DPC
DM
DM
DM
Procedures
BT,
BT,
BT,
BT,
BT,
BT,
BT,
BT,
BT,
BT,
BT,
BT,
BT,
BT,
OMP
PGP,
PGP,
PGP,
PGP,
PGP,
PGP,
PGP,
PGP,
OMP
PGP,
PGP,
PGP,
PGP,
OMP
OMP
OMP
OMP
OMP
OMP
OMP
OMP
OMP
OMP
OMP
OMP
*n, neonatal (days postpartum); CZ, Cleveland Metroparks Zoo; DM, Duke University Medical Center; DPC, Duke University
Primate Center.
Kelliher et al., 2001), swine (Dorries et al., 1997), sheep
(Cohen-Tannoudji et al., 1989), and, in particular, primates (Barrett et al., 1993; Aujard, 1997).
The phylogenetic distribution among primates is confusing in that the VNOs of several New World monkeys
and the Old World lemurs and their kin retain morphological traits that support a presumption of functionality
(Loo and Kanagasuntheram, 1972; Hunter et al., 1984;
Taniguchi et al., 1992; Mendoza et al., 1994; Evans and
Schilling, 1995; Smith et al., 2003). On the other hand, the
Old World monkeys and the great apes, including humans, are not thought to possess a functional VNO. Although chimps and humans retain anatomically identifiable VNOs, these appear, by biochemical and
morphological criteria, to be vestigial in postnatal life
(Meredith, 2001; Smith et al., 2001, 2002).
Ethical considerations mandate various indirect approaches to describe the extent and quality of VNO function in those primates that are presumed to retain functional VNOs. One avenue of inquiry has detailed
differences in lectin binding between species and between
age groups within particular species (Takami, 2002; Halpern and Martı́nez-Marcos, 2003). While those studies contribute to our understanding of the development and microanatomical organization of the vomeronasal epithelium
(VNE) as a tissue, they do not directly identify the differentiated state of VNE cell populations as olfactory and
therefore cannot address the question of function of the
primate VNO as an olfactory organ.
We undertook to compare directly the extent to which
the VNOs of two strepsirrhine and four haplorhine species
contain cells that are both neuronal and olfactory. To that
end, antisera raised against the neuronal markers neuron-specific ␤-tubulin (BT) and protein gene product 9.5
(PGP) and the marker of olfactory identity, olfactory
marker protein (OMP), were applied to tissue sections
from the six species. BT is expressed by neurons throughout the rodent nervous system (Burgoyne et al., 1988). In
particular, BT is expressed in the embryonic, neonatal,
and adult rodent main olfactory epithelium (MOE) (Lee
and Pixley, 1994; Roskams et al., 1998) as well as in the
postnatal rodent VNNE (Hofer et al., 2000; Witt et al.,
2002) and the adult canine VNNE (Dennis et al., 2003).
PGP is a ubiquitin hydrolase first isolated from brain
(Jackson and Thompson, 1981; Wilkinson et al., 1989) and
is a marker of neurons and neuroendocrine cells generally
(Thompson et al., 1983). Specifically, PGP is expressed in
rodent (Johnson et al., 1994) and canine (Dennis et al.,
2003) VNNE as well as rodent, canine, and marmoset
accessory olfactory bulb (Taniguchi et al., 1993; Johnson
et al., 1994; Nakajima et al., 1998a, 1998b, 2003). OMP is
a phylogenetically highly conserved cytoplasmic protein
expressed by mature olfactory chemosensory neurons of
the MOE and VNO (Margolis, 1972, 1980; Farbman and
Margolis, 1980).
We show here that, in the age groups sampled, all six
primate species examined express both neuronal markers
and OMP. In one species, Saguinus geoffroyi, the pattern
of marker expression by VNNE cells differs significantly
from the other five species.
MATERIALS AND METHODS
The histological sample included four Leontopithecus
rosalia (three neonates, one juvenile), six Saguinus geoffroyi (three neonates, two infants, and one 2.75-year-old
adult), two adult Callithrix jacchus, one neonatal Tarsius
syrichta, two adult Otolemur crassicaudatus, and one infant Eulemur mongoz (Table 1). All specimens were sectioned similarly (Smith et al., 2003). Briefly, tissues were
dissected free of the nasal chambers after fixation in 10%
buffered neutral formalin (Fisher Scientific, Pittsburgh,
PA), decalcified using a formic acid-sodium citrate solution, dehydrated in a graded series of ethanols, and embedded in paraffin. Blocks were sectioned serially at
10 –12 ␮m and every fifth section was stained alternately
with Gomori trichrome or hematoxylin-eosin procedures.
Intervening sections were saved for immunohistochemistry.
Immunohistochemistry
Mounted tissue sections were deparaffinized in Hemo-D
(Scientific Safety Products) and hydrated to distilled water (dH2O). To abolish endogenous peroxidase-like activity, the sections were incubated in absolute methanol
made to 0.9% hydrogen peroxide (H2O2) for 20 min at
1192
DENNIS ET AL.
Fig. 1. Antineuron-specific ␤-tubulin immunoreactivity. A: Otolemur
crassicaudatus, adult. BT immunoreactivity is restricted to the VNNE,
fascicles of the VNN, and nerves associated with vasculature. VL, vomeronasal lumen. Scale bar ⫽ 100 ␮m. B: Otolemur crassicaudatus, adult.
BT immunoreactivity in an individual different than that shown in A is
expressed differentially by sensory neuron somata (arrows) and dendrites (arrowheads) in the VNNE. BT immunoreactivity also appears in
the axon fascicles (Ax) of the VNN. Scale bar ⫽ 20 ␮m. C: Eulemur
mongoz, neonate. BT immunoreactivity is most prominent in the medial
VNNE and VNN fascicles in the lamina propria. Some immunoreactivity
is present in the NSE (arrowheads). Scale bar ⫽ 200 ␮m. D: Tarsius
syrichta, neonate. BT immunoreactivity is present but the signal is light
in this preparation. Clusters of immunoreactive cells (arrowheads) occur
throughout the VNNE. Axon fascicles are labeled with intensity similar to
that expressed by the BT⫹ VNNE cell somata. Scale bar ⫽ 50 ␮m.
room temperature (23.5–25°C). Subsequently, the tissues
were washed in dH2O, then in 10 mM phosphate-buffered
saline (PBS; 2.7 mM KCl, 137 mM NaCl; Sigma). Tissues
to be analyzed by epifluoresence microscopy were not incubated in H2O2/methanol but transferred from dH2O directly to PBS. All tissues were incubated 20 min in the
appropriate blocking solution [5% normal serum (Sigma)
of the species in which the secondary antibody was made
and 2.5% BSA (Sigma) in PBS], then washed briefly in
PBS. Primary antibody appropriately diluted in blocking
solution was applied and the tissue sections were left
overnight at room temperature. For double-label BT/PGP
assays, anti-BT (Covance) and anti-PGP (Chemicon) were
applied as a cocktail. For OMP/BT double-label assays,
sections were incubated overnight in anti-OMP (gift of Dr.
Frank Margolis) and then in anti-BT for 1 hr at room
temperature. Sections to be analyzed with bright field
optics were treated with biotinylated secondary antibodies
(Vector) diluted 1:200, then with ABC Elite reagent (Vector), reacted with diaminobenzidine (Vector), dehydrated,
and mounted with VectaMount (Vector). Sections to be
analyzed using epifluoresence microscopy were incubated
1 hr with appropriate Alexa-conjugated secondaries (Molecular Probes) diluted at 1:500, mounted with VectaShield (Vector), and sealed with clear nail polish. Tissues
were examined with a Nikon Eclipse E600 microscope
equipped with epifluoresence. Images were made with an
RT Slider digital camera (Diagnostic Instruments) using
EXPRESSION OF NEURON-SPECIFIC MARKERS
1193
Fig. 2. Antineuron-specific ␤-tubulin immunoreactivity. A: Saguinus
geoffroyi, neonate. BT signal is present in a small number of isolated
cells in the thin VNNE (arrowheads). Scale bar ⫽ 50 ␮m. B: Saguinus
geoffroyi, adult. The VNNE is thicker than the neonatal epithelium and
more bipolar BT⫹ cells (arrowhead) are present. The small scattered BT⫹
axon fascicles reflect the small number of BT⫹ cells in the VNNE. Scale
bar ⫽ 50 ␮m. C: Leontopithecus rosalia, neonate. The VNNE is several
cells deep and contains numerous BT⫹ cells occurring in clusters (ar-
rowhead). Heavily labeled axon fascicles occur immediately below the
VNNE. Scale bar ⫽ 50 ␮m. D: Leontopithecus rosalia, juvenile. After 4
months of development, the VNNE contains many BT⫹ cells, a few of
which are intensely labeled (arrow). Label in the axon fascicles is not
uniformly heavy (arrowhead). Scale bar ⫽ 50 ␮m. E: Callithrix jacchus,
adult. The vomeronasal lumina are defined by sensory epithelia. The BT⫹
cell layers are two or three cells thick in the basal compartment. NS,
nasal septum. Scale bar ⫽ 200 ␮m.
Spot Advanced software and processed with Photoshop 7.0
(Adobe).
is not uniform in that not all cell bodies and dendrites are
BT⫹ (Fig. 1B). The BT expression pattern manifest in the
VNNE is reflected in the nonuniform labeling within and
among the VNN axon fascicles. The VNNE and VNN axon
bundles of neonatal E. mongoz are intensely BT⫹ (Fig.
1C). In contrast, fewer VNNE cell bodies of T. syrichta are
BT⫹ (Fig. 1D). The signal is much less intense in both
RESULTS
Antineuron-Specific ␤-Tubulin
The VNNE and associated axon fascicles of adult O.
crassicaudatus are BT⫹ (Fig. 1A). The immunoreactivity
1194
DENNIS ET AL.
Fig. 3. Antineuron-specific ␤-tubulin and antiprotein gene product
9.5 immunoreactivity. A: Eulemur mongoz, neonate. The VNNE cell
somata label heavily with PGP (green) in this preparation. Some cell
bodies express immunoreactivity to both antibodies (yellow/orange) and
a few are BT⫹ (red)/PGP⫺. Most dendrites are BT⫹/PGP⫹ or BT⫹ only. A
few dendrites are BT⫺/PGP⫹. The NSE is predominantly negative for
either antibody. Scale bar ⫽ 50 ␮m. B: Saguinus geoffroyi, neonate. The
VNO at this level is almost entirely BT⫺. A small axon bundle above the
basement membrane is BT⫹/PGP⫹ (arrowhead). Most PGP⫹ cell somata
occur in the apical epithelial compartment. Scale bar ⫽ 50 ␮m. C:
Saguinus geoffroyi, 2 months. The VNNE is relatively thick compared to
that of the neonate. Labeled cell somata are BT⫹ (red) or PGP⫹ (green)
as well as BT⫹/PGP⫹. Double-labeled axon bundles are present in the
VNNE (arrow) and a few are present below the VNNE (arrowhead). Scale
bar ⫽ 50 ␮m. D: Saguinus geoffroyi, 2 months. A cluster of labeled cells
in the VNNE of the same individual as B shows variable signal intensity
from cell to cell. Some cell somata are BT⫹ (red) or PGP⫹ (green;
arrows). A few show degrees of double labeling (arrowhead). Scale bar ⫽
20 ␮m. E: Leontopithicus rosalia, 4 months. Many cells in the VNNE are
either BT⫹ or PGP⫹ or both. Cytoplasm of double-labeled cells varies
from yellow through red-orange. Dendrites are BT⫹/PGP⫹ or BT⫹/PGP⫺
(arrowhead). Small axon fascicles (arrow) are BT⫹. Scale bar ⫽ 20 ␮m.
EXPRESSION OF NEURON-SPECIFIC MARKERS
Fig. 4. Olfactory marker protein immunoreactivity. A: Eulemur mongoz, neonate. OMP⫹ cells vary in label intensity. Axon fascicles are more
lightly labeled but are above background. The NSE is OMP⫺. Scale
bar ⫽ 100 ␮m. B: Tarsius syrichta, neonate. A few sensory cells are
OMP⫹ but just above background (arrowheads). C, vomeronasal cartilage. Scale bar ⫽ 100 ␮m. C: Otolemur crassicaudatus, adult. The VNNE
and axon fascicles are OMP⫹. The NSE is OMP⫺. Scale bar ⫽ 100 ␮m.
D: Callithrix jacchus, adult. Cell bodies, dendrites, and axon fascicles are
1195
strongly OMP⫹. Scale bar ⫽ 50 ␮m. E: Saguinus geoffroyi, 1 month. The
VNNE is largely OMP⫺ but does contain a small number of scattered
OMP⫹ cells that label above background. Axon fascicles are above
background. Scale bar ⫽ 100 ␮m. F: Leontopithicus rosalia, juvenile. The
VNNE is OMP⫹ and, in this section, roughly equals the length of the NSE.
OMP⫹ cells are present among the axon fascicles below the basement
membrane (arrowhead). Scale bar ⫽ 100 ␮m.
1196
DENNIS ET AL.
somata and axon fascicles and the VNE displays some
segregation of labeled cells to the medial side (Fig. 1D).
The VNO of neonatal S. geoffroyi is almost completely
BT⫺ with few BT⫹ cell bodies and small BT⫹ axon bundles
(Fig. 2A). As individuals develop, BT expression increases
somewhat. An adult S. geoffroyi VNO contains more BT⫹
cells compared to the neonate but a small number relative
to the entire VNO epithelial volume (Fig. 2B). Small axon
fascicles are BT⫹ and are present both in the VNE and in
the lamina propria. Cell bodies of neonatal L. rosalia VNO
are BT⫹ but not pervasive (Fig. 2C). Axon fascicles of the
VNN are BT⫹. After 4 months of development, most of the
L. rosalia VNNE is BT⫹ (Fig. 2D). In adult C. jacchus
VNO, BT⫹ cells occur around the entire circumference of
the VNO (Fig. 2E). These BT⫹ cells are interrupted by
regions of BT⫺ cells, many of which are cytokeratin⫹ and
are glandular duct cells (data not shown). Axon bundles in
the lamina propria are BT⫹.
Antineuron-Specific ␤-Tubulin and Protein
Gene Product 9.5 Double Labeling
A VNO section from the same E. mongoz individual
shown in Figure 1C was double-labeled with anti-BT and
anti-PGP antibodies (Fig. 3A). In this preparation, most of
the sensory cell somata are BT⫺/PGP⫹, although a small
number are clearly double-labeled. Double-labeled dendrites are more numerous than double-labeled cell bodies
and BT⫹/PGP⫺ dendrites are more prevalent than BT⫹/
PGP⫺ cell bodies. In the relatively thin neonatal S. geoffroyi VNO, PGP⫹ cells are present in the apical compartment but BT expression by cell bodies is largely absent
(Fig. 3B). After 1 month of postnatal development, the
VNNE has thickened and the number of BT⫹ cells has
increased compared to the neonate (Fig. 3C). Cells expressing immunoreactivity to either or both antibodies are
present largely in the basal compartment, although occasional cell clusters span the thickness of the epithelium
(Fig. 3D). Double-labeled axon fascicles are present both
above the basement membrane and below it (Fig. 3C and
D). In contrast, the 4-month L. rosalia VNNE (Fig. 2D)
contains many BT⫹/PGP⫹ cells across the thickness of the
epithelium, although cells negative for either antibody are
present in the middle region of the epithelium (Fig. 3E).
The PGP signal intensity varies among cell nuclei and the
relative expression of BT and PGP in the cytoplasm varies
among cell somata and some dendrites as inferred from
the range of color from yellow to dark orange and red.
Olfactory Marker Protein
Neonatal E. mongoz VNNE is clearly defined by a population of OMP⫹ cells and some dendrites (Fig. 4A).
OMP⫹ axon fascicles of variable size occur in the lamina
propria. The axon bundles do not label strongly but the
label is above background. Neonatal T. syrichta VNO contains a few cells that label above background in this preparation (Fig. 4B). Compared to neonatal E. mongoz, the
VNNE of adult O. crassicaudatus contains a prominent
population of OMP⫹ cells and more strongly OMP⫹ axon
fascicles (Fig. 4C). Adult C. jacchus shows a heavily OMP⫹
VNNE (Fig. 4D). The smaller lightly OMP⫹ axon fascicles
of the adult C. jacchus contrast with relatively larger and
more heavily OMP⫹ axon fascicles in the lamina propria of
O. crassicaudatus (Fig. 4C). One-month-old S. geoffroyi
contains a small number of OMP⫹ cells relegated in the
TABLE 2. Summary of immunohistochemical results*
Species
Saguinus geoffroyi
Saguinus geoffroyi
Saguinus geoffroyi
Saguinus geoffroyi
Leontopithecus rosalia
Leontopithecus rosalia
Callithrix jacchus
Tarsius syrichta
Eulemur mongoz
Otolemur crassicaudatus
Age
BT
PGP
OMP
n
1 month
2 month
Adult
n
4 month
Adult
n
n
Adult
⫹
⫹
⫹
⫹
⫹
⫹⫹
⫹⫹
⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
ND
⫹⫹
⫹⫹
⫺
⫹
⫹
⫹
⫺
⫹⫹
⫹⫹
⫹
⫹⫹
⫹⫹
*n, neonate; ⫹⫹, many reactive cells; ⫹, few reactive cells; ⫺,
no reactive cells; ND, data not available.
main to the basal compartment (Fig. 4E). In the VNOs of
the two 1-month-old individuals that were studied, lightly
labeled axon fascicles are present but not numerous (not
shown). Four-month-old L. rosalia VNNE contains a
strongly OMP⫹ cell population in the basal compartment
and, apically, OMP⫹ dendrites (Fig. 4F). Axon fascicles in
the lamina propria are less strongly OMP⫹. In this section, OMP⫹ cells observed among the axon bundles are
below the basement membrane.
Antineuron-Specific ␤-Tubulin Olfactory
Marker Protein Double Labeling
The adult O. crassicaudatus VNNE contains cell bodies
positive for either or both BT and OMP immunoreactivity
and the nonsensory epithelium (NSE) negative for either
antibody (Fig. 5A). The axon fascicles in the lamina propria immediately below the basement membrane are
largely BT⫹/OMP⫺. Adult C. jacchus contains double-labeled cell somata and largely BT⫹ dendrites (Fig. 5B). A
section of C. jacchus main olfactory epithelium doublelabeled for BT and OMP is shown for comparison with
VNO labeling in all species represented (Fig. 5C). The
VNE of neonatal L. rosalia is OMP⫺ and largely, but not
completely, BT⫺ (Fig. 5D). Scattered BT⫹ cell bodies and
dendrites are present and BT⫹ axon bundles are present
in the lamina propria. The 4-month-old L. rosalia VNNE
is BT⫹/OMP⫹ and is segregated from an NSE that is
OMP⫺ (Fig. 5E). Intensely BT⫹ axon fascicles of variable
size occur in the lamina propria. At higher magnification,
immunoreactive cells in the VNNE are labeled with either
antibody and, in a few cases, with both antibodies (Fig.
5F). The axon fascicles are BT⫹/OMP⫺. In contrast to
neonatal and 4-month-old L. rosalia, 1-month-old S. geoffroyi contains BT⫹ cells and axon bundles in the VNNE
(Fig. 5G). The OMP⫹ signal is slight compared with other
species (Fig. 5A, B, E, and F), but the signal is above
background. Adult S. geoffroyi VNO contains few cells
immunopositive for either antibody but many small BT⫹
axon fascicles are located in the lamina propria immediately below the basement membrane (Fig. 5H). The few
OMP⫹ cells are bipolar and resemble OMP⫹ cells of E. mongoz, O. crassicaudatus, and L. rosalia VNNE. The remaining
OMP signal is diffuse and resembles that observed in the
1-month-old individual shown in Figure 5G. Immunohistochemical observations are summarized in Table 2.
DISCUSSION
Data from this study demonstrate that the VNE of all
species examined express immunoreactivity to the neu-
Figure 5.
1198
DENNIS ET AL.
Fig. 5. Olfactory marker protein and neuron-specific ␤-tubulin double-label immunoreactivity. A: Otolemur crassicaudatus, adult. The
VNNE is strongly OMP⫹ (green) and BT⫹ (red) but the NSE is not
immunoreactive. The axon fascicles below the VNNE are predominantly
BT-labeled. Scale bar ⫽ 100 ␮m. B: Callithrix jacchus, adult. The cytoplasm in a cluster of VNNE sensory cells (arrowhead) is double-labeled
(yellow-orange) but the dendrites are predominantly BT⫹. Scale bar ⫽ 20
␮m. C: Callithrix jacchus, adult MOE. OMP (green)/BT (red) labeling in
the main olfactory epithelium for comparison with immunoreactivity in
the VNO. Most cell somata are OMP⫹/BT⫺, but some are doublelabeled. Dendrites are double-labeled or OMP⫺/BT⫹. Axon bundles vary
in the relative amount of OMP/BT label. N, nasal space. Scale bar ⫽ 20
␮m. D: Leontopithecus rosalia, neonate. The VNNE contains a few BT⫹
(red) cells and BT⫹ axon bundles are located in the lamina propria near
the epithelium. No OMP⫹ labeling is present. Scale bar ⫽ 100 ␮m. E:
Leontopithecus rosalia, 4 months. OMP⫹ and BT⫹ cells occur in the
basal compartment throughout most of the VNNE and strongly BT⫹ axon
bundles occur in the lamina propria. The NSE contains some BT signal.
Scale bar ⫽ 100 ␮m. F: Leontopithecus rosalia, 4 months. A detail of the
VNNE from the individual shown in D demonstrates a range of labeling in
the cell bodies and dendrites. Some cell bodies are OMP⫹ (green) only
or BT⫹ (red) only and several are OMP⫹/BT⫹ (yellow-orange). Axon
bundles are predominantly BT⫹. Scale bar ⫽ 40 ␮m. G: Saguinus geoffroyi, 1 month. Some cells (arrows) express OMP immunoreactivity and
three or four cells (arrowhead) are BT⫹ in the VNNE. Small axon fascicles
below the VNNE and NSE are BT⫹. Scale bar ⫽ 20 ␮m. H: Saguinus
geoffroyi, adult. OMP (green) and BT (red) labeling is sparse in the VNNE
but a few bipolar OMP⫹ cells are present (arrowhead). BT⫹ axon bundles
are present below the basement membrane. Scale bar ⫽ 40 ␮m.
ronal marker BT and the specific olfactory marker OMP
at some point during postnatal life. The VNE of the two
strepsirrhine (E. mongoz and O. crassicaudatus) and
three haplorhine (C. jacchus, L. rosalia, S. geoffroyi)
species examined also expressed PGP immunoreactiv-
ity. We presume PGP expression in the neonatal tarsier
VNE, Tarsius syrichta, but tissue was limited and we
did not confirm PGP expression by assay. Of these species, S. geoffroyi showed the least expression, by relative numbers of labeled cells in the VNE, of the three
EXPRESSION OF NEURON-SPECIFIC MARKERS
marker proteins and that expression was greatest in the
1-month age group but, by adulthood, very few VNNE
cells were positive for any of the markers.
Among the neonatal individuals examined, a large number of cells in E. mongoz VNE expressed OMP, indicating
that at birth, mature sensory neurons are present. The
tarsier was intermediate between E. mongoz and the haplorhine species in that a few cells were labeled with BT
and OMP antibodies at levels above background. Neonatal
L. rosalia expressed BT but not OMP immunoreactivity
and neonatal S. geoffroyi expressed immunoreactivity to
neither antibody but did express PGP immunoreactivity.
In preliminary assays of neonatal C. jacchus, no reactivity
to BT or OMP antibodies was observed (data not shown).
These observations suggest that, at nativity, the VNE is
mostly populated by nonneuronal cells and/or neuronal
cells that have not differentiated to the stage in which this
set of markers is expressed. Therefore, by immunohistochemistry, and accepting the supposition that OMP expression is required in the VNO for olfactory signaling,
only E. mongoz could possess a functional VNO at birth
among the species considered here.
As the postnatal individual develops, expression of all
three markers increases. By 4 months, L. rosalia VNE is
strongly OMP⫹/BT⫹ and, in its expression pattern of immunoreactivity, resembles the adult state demonstrated
for O. crassicaudatus and C. jacchus (compare Fig. 5E and
F with A and B). In the 1- and 2-month-old S. geoffroyi
individuals examined, marker expression was greater
compared to that seen in the neonate but was not comparable to the level seen at 4 months in L. rosalia. The VNE
in both species are about equal thickness and labeled cells
occur in small clusters and are rarely observed in layers
even two cells deep. Of the labeled cells, BT and PGP
signal is most prominent. In S. geoffroyi, OMP signal is
above background but weak compared to the OMP signal
in L. rosalia. Additionally, the VNE does not appear well
organized in as much as axon fascicles occur in the epithelium, although that judgement is based on a limited
number of available specimens and may be sampling artifact. By adulthood, BT and OMP signal is present but in
only a few cells in Saguinus, and more BT⫹ cells are
observed than OMP⫹ cells. These labeled cells occur in
loose clusters, as in the 1-month-old, suggesting a clonal
genesis, but the dearth of OMP⫹ cells suggests that many
of the BT⫹ cells are not in the OMP-expressing lineage or
these cells die before they, or their daughter cohorts, terminally differentiate and begin expressing OMP.
These possibilities suggest that BT and OMP expression, rather than PGP expression, are better indicators of
capacity for VNO function vis-à-vis the VNE. First, not all
cells are PGP⫹, begging the question of the PGP⫺ cells’
identity. Second, although PGP is a neuronal marker, it is
expressed in the MOE and the VNO throughout life but
the expression pattern within a given cell’s life span is
unknown. Finally, PGP is involved with ubiquitin and is
therefore part of the cellular metabolism. Its expression
pattern may be episodic or it may undergo regular
changes, for example, conformational, that make it invisible to the antibody used in any particular study. On the
other hand, the BT expression pattern in the VNO is likely
to conform to the pattern of olfactory differentiation observed in the MOE, wherein cell cohorts in the olfactory
neuron lineage express BT before they express OMP (Lee
and Pixley, 1994). As individual cells begin to express
1199
OMP, BT expression in the cell body subsides but remains
high in the dendrites. BT expression suggests that neurogenesis and the passage of precursors through the olfactory developmental program to terminal differentiation
marked by OMP expression continues in some adults considered here. Expression of growth-associated protein 43,
a marker of new neurons that are extending processes, in
adult O. crassicaudatus VNO (data not shown) lends credence to the supposition of continuous birth and maturation of mature vomeronasal cells in the VNE.
OMP expression confirms that mature sensory neurons
are present in the VNE of the several species considered.
That expression does not affirm the inevitability of vomeronasal function, nor does it address the extent to which
the vomeronasal system may function in these species.
Compared to rodent VNO, the histology of the primate
VNO is more similar to that of goats (Takigami et al.,
2000), sheep (Cohen-Tannoudji et al., 1989), swine (Dorries et al., 1997), and ferrets (Weiler, et al., 1999). In the
last three of these species, the VNO is not involved in all
of the functions mediated by the rodent VNO (CohenTannoudji et al., 1989; Dorries et al., 1997; Weiler, et al.,
1999; Kelliher et al., 2001). Likewise, several behaviors
that in rodents are mediated by the VNO are not solely
VNO-mediated in primates (Barrett et al., 1993; Aujard,
1997; Kraus et al., 1999). The histological similarities to
sheep, swine, and ferret VNO together with the behavioral
observations argue that primate VNO function is likely to
be restricted compared to rodents and more similar to that
of the former species.
The extent to which VNO function resembles, or differs
from, that in the several nonrodent species mentioned
above not withstanding, we establish here that the VNE of
infant E. mongoz, adult O. crassicaudatus, adult C. jacchus, and 4-month-old L. rosalia express mature olfactory
neurons. By that criterion, VNE function as a chemosensory vomeronasal epithelium may be possible in those
species.
ACKNOWLEDGMENTS
The authors are grateful to Dr. Frank Margolis for the
generous gift of the anti-OMP antisera used in this study.
They also thank J.H. Kinzinger and K.L. Shimp for sectioning some of the specimens used in the study and A.B.
Taylor and C.J. Vinyard for providing nasal tissues from
adult marmosets. Supported in part by Federal Aviation
Administration grant 01-6-022 and U.S. Army Robert
Morris Acquisition Center grant N66001-1099-0072 to
(E.E.M.). This is Duke Primate Center publication number 789.
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