Table Of ContentZOOLOGICAL SCIENCE 10: 287-294 (1993) © 1993 Zoological SocietyofJapan
Identification and Immunological Properties of an Olfactory
System-Specific Protein in Kokanee Salmon
{Oncorhynchus nerka)
Munetaka Shimizu1'2 Hideaki Kudo1'3 Hiroshi Ueda1*,
, ,
Akihiko Hara4 Kenji Shimazaki2 and Kohei Yamauchi3
,
lToya Lake Stationfor Environmental Biology, Faculty ofFisheries, Hokkaido
University, Abuta 049-57, 2Research Institute ofNorth Pacific Fisheries, Faculty
ofFisheries, Hokkaido University, Hakodate 041, 3Department ofBiology,
Faculty ofFisheries, Hokkaido University, Hakodate 041, and 4Nanae
Fish Culture Experimental Station, Faculty ofFisheries, Hokkaido
University, Nanae 041-11, Hokkaido, Japan
—
ABSTRACT An olfactory system-specific protein of24kDa (N24) was identified in kokanee salmon
(Oncorhynchus nerka) by the comparison of proteins restricted to the olfactory system (olfactory
epithelium, olfactory nerve and olfactory bulb) with those found in other parts of the brain
(telencephalon, optic tectum, cerebellum, hypothalamus) and hypophysis. A polyclonal antibody to
N24 was raised in a rabbit, and the specificity of the antiserum was examined by Western blotting
analysis; the antiserum recognizedonlyone 24kDaband inthe olfactorysystem but not inotherparts
ofthebraininkokaneesalmon,sockeyesalmon(O. nerka),masusalmon(O. masou)andchumsalmon
(O. keta). Immunocytochemical analysisrevealed that positive immunoreactivityoccurred exclusively
in the olfactory nerve and in some olfactory neuroepithelial cells. Both at the time of imprinting of
maternal stream odorants and at the time ofhoming to the maternal stream, the immunoreactivity of
N24infish in the maternal streamwasstrongerthanthat inseawaterfish. The presentstudyindicates
that an olfactory system-specific protein may be importantly related to both imprinting and homing
mechanisms in salmonids.
system plays important roles in imprinting of the
INTRODUCTION
maternal stream odorants during downstream
The imprinting and homing behaviorsofsalmon migration, and that in adult salmon the early
in relation to their maternal stream constitute one olfactoryexposureallowsrecognitionofthe mater-
of the most interesting phenomena in salmon nal stream odorants during upstream spawning
biology. Since Hasler and Wisby [10] introduced migration (homing). However, few attempts have
the olfactory hypothesis for anadromous salmo- been made to investigate biochemical aspects of
nids, olfactoryimprinting and homingmechanisms the olfactory system in any salmonid species,
have been studiedin manybehavioralexperiments There are numerous literatures indicating a
(e.g., [9, 24]). Several neurophysiological ap- close relation between olfactory-specific proteins
proaches using electrophysiological techniques and recognition and discrimination of odorants in
have also pointed to the olfactory recognition of higher vertebrates[l, 13, 16]. Several olfactory-
maternal stream water during spawning migration specific proteins have been identified and used as
in several salmonids [8, 26]. It is now widely molecular markers to study olfactory function,
accepted that in juvenile salmon the olfactory such as the olfactory marker protein in the
Accepted February 2, 1993 mammalian olfactory bulb [11, 14], carnosin in
Received December 14, 1992 tetrapod olfactory systems [3, 15], and olfac-
* To whom correspondence and reprint requestsshould tomedin in frog olfactory neuroepithelium [23].
be addressed. The present study was desinged to compare
288 M. Shimizu, H. Kudo et al.
—
proteins restricted to the olfactory system (olfac- stored at 30°C and used for gel electrophoresis.
tory epithelium, olfactory nerve and olfactory
Gel electrophoresis
bulb) with those found in other parts of the brain
(telencephalon, optic tectum, cerebellum, hypo- Sodium dodecyl sulfate polyacrylamide gel elec-
thalamus) and hypophysisofseveral salmonid spe- trophoresis (SDS-PAGE) [12] was performed us-
cies. In addition, two immunological analyses ing a Protean II slab gel system (Bio-Rad, South
(Western blotting and immunocytochemistry) Richmond, CA, USA). Samples containing 50fig
were conducted to investigate the possible role of protein, as determined by Lowry's method, were
an olfactory system-specific protein in olfactory heated at 100°C for 2min in double volumes of
functions and to evaluate its possible usage as a SDS sample buffer containing 2-mercaptoethanol,
molecular marker in the study of olfactory and applied to slab gels consisting of 7.5-17.5%
mechanisms. logarithmic gradient of polyacrylamide and 5%
stacking gel. Electrophoresis was carried out at
mA mA
MATERIALS AND METHODS 12.5 for stacking gel followed at 25 for
gradient gel. Molecular weight markers (Pharma-
cia Fine Chemicals, Uppsala, Sweden) for deter-
Fish mination of low molecular weight were run in a
Male and female mature kokanee salmon separate lane. Gelswere fixed andstainedfor2hr
(Oncorhynchus nerka; land-locked sockeye sal- in 0.1% Coomassie Brilliant Blue in 5% ethanol-
mon), 3 to 5 years old, were caught in Lake Toya 5% acetic acid solution, destained overnight in
from October 22 to November 19, 1991. Mature 10% acetic acid, soakedin20% ethanol-5% acetic
sockeye salmon (O. nerka) of both sexes, 3 to 5 acid-2.5% glycerin, and dried with gel dryer.
years old, were collected from the Chitose Branch Two-dimmensional (isoelectric-SDS) PAGE
ofthe Hokkaido Salmon Hatchery on October29, (2D-PAGE) [19] wasalso carriedouttoobtain the
1991. Adult chum salmon (O. keta) ofboth sexes, evidence whether an olfactory system-specific pro-
3 to 6 years old, were captured at sea and in fresh tein appeared single spot or not.
water at two different stages of sexual maturation
(pre-spawning fish about 10-20 days before final Antiserumproduction
A
maturation and spawning fish) on September 25, protein band corresponding to an olfactory
1991. Wildone-year-oldjuvenile masusalmon (O. system-specific protein, designated as N24, was
masou) were caught once amonth in the Shakotan prepared from olfactory bulbs of mature kokanee
River from November 21, 1991 to May 25, 1992. salmon of both sexes. The band was excised
Fish were anesthetized with 10% ethyl p- carefully with scissors, and eluted from gel slices
aminobenzoate, and their heads were removed by using an Electro-Eluter (Bio-Rad, South Rich-
decapitation andplacedon ice. Underadissecting mond, CA, USA) following the manufacturer's
microscope, olfactory organs and brains were dis- instructions. An elution buffer was constituted
mM mM
sected into olfactory epithelium, olfactory nerve, with 25 Tris, 192 glycine and 0.1% SDS,
olfactory bulb, telencephalon, optic tectum, cere- and elution was done at 10mA for 2 hr.
A
bellum, hypothalamus and hypophysis. Each tis- polyclonal antiserum was raised in a rabbit
sue was washed quickly in ice-cold salmon Ringer using lymph node injections. Initial injection of35
(0.15 M NaCl, 0.31 mM KC1, 0.03 mM MgS04- figeluted N24was made intoinguinal lymph nodes
7H20, 0.34mMCaCl2-2H20, 0.4mMglucose, 0.4 in Freund'scomplete adjuvant. Subsequent boost-
mM Hepes buffer, pH 7.5) containing aprotinin (1 erinjectionsof90jugN24emulsified with Freund's
figlm\), phenylmethylsulfonyl fluoride (PMSF; 1 incomplete adjuvant were made intradermally into
mM) andethylenediaminetetraaceticacid (EDTA; the back of rabbit at four weekly intervals. One
1 mM). The tissue was homogenized in about 1.5 week after the final booster injection, a test bleed-
volumes of salmon Ringer and centrifuged at ingwastaken fromthe earvein, andtheserumwas
10,000Xg for lOmin. The supernatant fluid was assayed by Western blotting analysis.
Olfactory System-Specific Protein in Salmonids 289
other parts of the brain from mature kokanee
Western blotting salmon were resolved by SDS-PAGE (Fig. 1A).
Protein samples were resolved by SDS-PAGE Several protein bands were observed in extractsof
and transferred to Immobilon PVDF membranes the olfactory system but not in other parts of the
(Millipore, Bedford, MA, USA) by the method of brain or the hypophysis. Among these proteins,
Towbin et al. [25]. The PVDF membranes were the mostclearlyvisible protein band (arrowhead in
incubated for 1 hr with 1% skim milk in Tris- Figure 1A), which had a molecular weight of 24
buffered saline (TBS, pH7.4) to reduce non- kDa, was designated N24, and appeared single
specific protein absorption, and allowed to react spot by the analysis of 2D-PAGE (manuscript in
for 2 hr with polyclonal antiserum against N24 preparation). This protein was eluted from gel
(working dilution from 1 : 100 to 1 :5000) in a slices for antibody generation.
solution of 3% bovine serum albumin and 1% In Western blotting analysis, a polyclonal anti-
normal goat serum in TBS. After rinsing with serum to N24 at dilutions of 1:500 and 1:1000
TBS, the membranes were incubated for 1 hr with recognized onlythe band whose positionwas iden-
horseradish-peroxidase-labeled goat anti-rabbit tical to N24 in extracts of kokanee salmon olfac-
IgG (Cappel Laboratories, West Chester, PA, tory bulb. At a dilution of 1 :100, presumably
USA) at a dilution of 1 :1000 in TBS, and de- weak non-specific reactions were observed in
veloped in 4-chloro-l-naphthol solution (60mg in bands of molecular weight around 40kDa. At a
100ml TBS containing 0.01% H2 2). The reac- dilution of 1 :5000, a faint reaction only was
tion was stopped after 3-5 min by washing in observed in the 24kDa band. At a dilution of
distilledwater, andthe membraneswere air-dried. 1:2500, the antiserum reacted with the 24kDa
protein in the olfactory system, which was absent
Immunocytochemistry
from other parts of the brain and hypophysis in
Theolfactorysystemandotherpartsofthebrain kokanee salmon (Fig. IB).
of mature kokanee salmon were fixed with 4% Electrophoretic patterns of soluble extracts of
M
paraformaldehyde in 0.1 phosphate buffer (pH the olfactory system and telencephalon of chum
M
7.3) for 18hr at 4°C. After rinsing with 0.1 salmon, sockeye salmon and masu salmon were
phosphate buffer containing 10% sucrose, tissues similar to those of kokanee salmon, and a similar
were embedded in Tissue-Tek OCT compound olfactory system-specific protein of molecular
(Miles Laboratories, Elkhart, IN, USA) and weight 24kDa was recognized in all these salmo-
—
stored at 30°C, to be sectioned later at 10-^m nid species (Fig. 2).
thickness in a cryostat. Tissue sections were im- Duringparr-smolt transformation in masu salm-
mersed in 0.3% hydrogen peroxide in methanol on, although there were no distinct changes in
for 30min to eliminate endogenous peroxidase electrophoretic patterns of the olfactory system
activity. After rinsing with phosphate-buffered and telencephalon between parr and smolt, the
saline (PBS), sections were incubated with 1% immunoreactivity of N24 to the antiserum was
normal goat serum in PBS for 15 min, and im- more intense in parr than in smolt (Fig. 3). Simi-
munoreacted with anti-N24 serum (working dilu- larly, during spawning migration of chum salmon
tion 1 :2500 or 1 :5000) for 2hr at room tempera- from coastal sea to maternal river, electrophoretic
ture. The strepto-avidin-biotin complex (sABC) patterns of olfactory nerve, olfactory bulb and
method (Histofine, Nichirei, Tokyo, Japan) was telencephalon showed no prominent changes.
used for the immunocytochemistry. In order to However, the cross-reactivity of anti-N24 serum
confirm specificity, normal rabbit serum or PBS was different infish fromthe seaandthe river; the
was substituted for anti-N24 serum. antigenicity ofN24was more intense in the mater-
nal river fish sample (Fig. 4).
Theolfactoryrosette ofmature kokanee salmon
RESULTS
stained with Nissl solution showed olfactory
Soluble extracts of the olfactory system and epithelium and several branches of the olfactory
;
290 M. Shimizu, H. Kudo et al.
Fig. 1. SDS-PAGE(A)andWesternblotting(B)ofsolubleextractsoftheolfactorysystem(1,olfactoryepithelium
2,olfactorynerve;3,olfactorybulb),otherpartsofthebrain(4,telencephalon;5,optictectum;6,cerebellum;7.
hypothalamus) andhypophysis(8)fromkokaneesalmon. ArrowheadinAidentifiesanolfactorysystem-specific
protein ofmolecularweight 24kDa (N24). Anti-N24 serum (1:2500) recognizes a band ofmolecularweight 24
kDa in the olfactory system but not in other parts ofthe brain. Numbers at left are Mr standards XlO3.
Chum Sockeye Masu Chum Sockeye Masu
Salmon Salmon Salmon Salmon Salmon Salmon
Fig. 2. SDS-PAGE (A)andWestern blotting(B)ofsolubleextractsoftheolfactorysystem(1.4.8,olfactorynerve:
2, 5, 9, olfactory bulb; 7, olfactoryepithelium) and telencephalon (3, 6. 10) from chum salmon, sockeyesalmon
and masusalmon. Anti-N24serum (1 :2500) recognizesa band ofmolecularweight 24kDaonlyintheolfactory
system. Numbers at left are Mr standards X 10\
Olfactory System-Specific Protein in Salmonids 291
94-
fUl
67- 1
1 1
1
43-
30-
EH
20-
14-
12 12 12 12
3 4 3 4 3 4 3 4
Parr Smolt Parr Smolt
Fig. 3. SDS-PAGE (A) and Western blotting (B) of soluble extracts of the olfactory nerve system (1, olfactory
epithelium; 2, olfactory nerve; 3, olfactory bulb) and telencephalon (4) in masu salmon during parr-smolt
transformation. The immunoreactivity of anti-N24 serum (1:2500) is more intense in parr than in smolt.
Numbers at left are Mr standards X103.
94-|
9
67-
43-
30-
20-
P
14- '"" Hi
A
12 12 12
3 3 3
On Ob T On Ob T
Fig.4. SDS-PAGE (A) and Western blotting (B) ofsoluble extracts ofthe olfactory system (On, olfactory nerve;
Ob, olfactory bulb) and telencephalon (T) in chum salmon during spawning migration (1, coastal sea; 2,
pre-spawningfishinthematernalriver;3,spawningfishinthematernalriver). Theimmunoreactivityofanti-N24
ismoreintenseinfishfromthe maternalriverthaninfishfromthecoastalsea. NumbersatleftareMrstandards
XlO3
.
292 M. Shimizu, H. Kudo et al.
B
Fig. 5. Adjacent 10-,«mthicksectionsofthe olfactoryrosette ofmature kokaneesalmonstainedwithNisslsolution
(A), sABCmethodwith anti-N24 at a dilution of1:2500 (B), andsABCmethodwith normal rabbitserum at a
dilutionof1 :2500 (C). N24immunoreactivityoccurs in someolfactoryneuroepithelialcellsandin branchesof
the olfactory nerve. MP, melanophore; OE, olfactory epithelium; ON, olfactory nerve. X250.
nerve (Fig. 5A). ThesABCmethoddemonstrated salmon and chum salmon. However, no cross-
thatN24immunoreactivitywaslocalizedexclusive- reactivity of anti-N24 serum was observed in the
ly in almost all branches ofolfactory nerve and in olfactory system of carp (unpublished data).
some olfactory epithelial cells, presumably Therefore, all salmonids examined to date possess
neuroepithelial cells (Fig. 5B). The immunoreac- a 24kDa protein (N24) specific to the olfactory
tivity ofapical portions ofthe olfactory epithelium system.
was non-specific because the same portions were The olfactory sensory cells (olfactory neuro-
also stained in control sections, in which anti-N24 epithelial cells) are bipolar neurons, whose den-
serumwasreplacedwithnormalrabbitserum (Fig. drites project to the external environment and
5C). Specific immunoreactivity was never observ- axons to the olfactory bulb, which form the olfac-
ed in other nervous tissues studied. tory nerve. Preliminary immunocytochemical
study showed that positive N24 immunoreactivity
occurred exclusively in the olfactory nerve and in
DISCUSSION
some olfactory neuroepithelial cells. The specific
Electrophoretic comparison of proteins re- immunoreactivity was also found in the olfactory
stricted to the olfactory system with those in other bulb where the olfactory nerve was penetrated
partsofthebrainin kokaneesalmon demonstrated (unpublished data). Wecould notidentifytypesof
the existence of several olfactory system-specific neuroepithelial cells at the light microscopic level.
proteins. Among them, the most clearly visible These observations suggest that N24 has possible
protein designated as N24 based on its 24 kDa roles in neurotransduction from neuroepithelial
molecular weight was used to raise a specific cells to olfactory bulb; however, further detailed
antibody in a rabbit. Western boltting analysis immunocytochemical studies at the electron mi-
revealed that the antiserum recognized only this24 croscopic level are needed to clarify this point.
kDa band in the olfactory system, which was A mammalian olfactory marker protein of
absent from other parts of the brain not only in molecular weight 20 kDa is localized in olfactory
kokanee salmon but also in sockeye salmon, masu sensory neuron cell bodies and axons; its appear-
Olfactory System-Specific Protein in Salmonids 293
ance coincides with the establishment of sensory salmonid species and describes some of its im-
synapses in the olfactory bulb [7]. Although it munological properties. N24 may prove to be
seemsunlikelythatthemammalianolfactroymark- good molecular marker for the study of salmonid
er protein is identical to N24 because of the olfactory functions. Detailed biochemical and
difference in their molecular weight, further im- cytological analyses of N24 as well as the other
munological approaches are necessary. Carnosine olfactory system-specific proteins are now in prog-
is the predominant dipeptide present in the olfac- ress in our laboratory.
tory neurons and nerve in mammals, birds, repti-
lians andamphibians, but has not been reported in ACKNOWLEDGMENTS
fishes [3]. Olfactomedin, a57kDa glycoprotein, is
localized exclusively in frog olfactory neuroepithe- We thank Professor Howard A. Bern, University of
lium [23]. These olfactory-specific proteins may cCrailtiifcoalrnirae,adfionrghiosf itnhteeremsatnuisncrtihpet.preWseentasltsoudythaanndk fDorr
play important roles in chemoreception, however, Osamu Hiroi, Dr Masahide Kaeriyama, Hokkaido Sal-
their precise functions remain uncertain. mon Hatchery, and Mr Hiroshi Kawamura, Hokkaido
Another biochemical approach in higher verte- Fish Hatchery, for providing the chum salmon, the
brates concerns the involvement of olfactory re- sockeye salmon and the masu salmon, respectively, and
Mr Hiroyuki Haruna and Mrs Rirako Yamada, Toya
ceptor proteins which activate adenylate cyclase
Lake Station for Environmental Biology, for their tech-
[21, 22] via a GTP-binding protein [2] leading to nical assistance. Thisstudywassupported in part bythe
theopeningofcyclicnucleotide-gatedcationchan- Special Grant-in-Aid for Promotion of Education and
nels [5, 18]. A multigene family of GTP-binding ScienceinHokkaidoUniversityProvidedbytheMinistry
protein-coupled receptors expressed uniquely in of Education, Science and Culture of Japan, and by a
olfactory tissues has recently been reported [4]. reseach grant from the Akiyama Foundation to H.U.
Immunocytochemical studies using monoclonal
antibodies against rabbit olfactory bulb have de-
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