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Histochemical characteristics of mucopolysaccharides in salivary and exorbital lacrimal glands.

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Histochemical Characteristics of Mucopolysaccharides
in Salivary and Exorbital Lacrimal Glands
S . S. SPICER AND JALE DUVENCI1
Section on Biophysical Histology, Laboratory of Experimental Pathology,
National Institute of Arthritis and Metabolic Diseases, National Institutes
of Health, Public Health Service, United States Department of Health,
Education, and Welfare, Bethesda, Maryland
ABSTRACT
Comparison of results obtained by S3504' autoradiography with those
from histochemical staining methods and evaluation of the effect on the staining
resulting from prior sialidase digestion, methylation, methylation and saponification,
or sulfation, substantiates the selectivity of some procedures for sialomucin and others
for sulfomucin. These two types of mucopolysaccharide may be differentiated by the
high iron diamine-alcian blue or aldehyde fuchsin-alcian blue sequences or comparison
between staining with the alcian blue pH 2.5-PAS sequence and staining with the
alcian blue pH 1.0-PAS method. These techniques reveal that some rodent or lagomorph salivary glands form sialomucin alone, others apparently only sulfomucin,
whereas a number produce both types of acid mucopolysaccharide either in different
mucous cells throughout the gland or in the same cell.
Differences in affinity for basic dyes, periodate reactivity, and susceptibility to
elimination of basophilia by methylation or enzymatic digestion are evident among
both the sulfo- and the sialomucins in different glands. After brief saponification the
sialomucin in rat sublingual glands becomes sialidase digestible and gains alcohol
resistant metachromasia.
Several serous or seromucous glands secrete neutral mucopolysaccharides; others
secrete polysaccharides containing sulfate or sialic acid groups whose affinity is masked
for some but not for other basic dyes.
Containing abundant mucous elements,
salivary glands and their secretions have
been analyzed in a number of biochemical
investigations for the presence of mucopolysaccharides. Tanabe ('39), for example, isolated from cattle sublingual and
submandibular glands a glycoprotein and
two polysaccharides possibly resembling
hyaluronic acid and blood type substance.
Blix et al. ('52) isolated two neutral polysaccharides from cattle submandibular
glands in addition to an acid polysaccharide rich in sialic acid. Meyer ('45) has
investigated a sialic acid containing mucosubstance in salivary glands and Landsteiner and Harte ('41) and Bray and
Stacey ('49) have found blood group reactive polysaccharides in human saliva. The
precise histologic localization of these polymers is unknown. Progress has been made
in characterization of the isolated polysaccharides. Analyses of human salivary mucoid by McCoombe et al. ('61) and of
purified bovine salivary mucin clot by
Nisizawa and Pigman ('59) revealed the
presence in these materials of sialic acid,
galactose, hexosamine, and fucose. No
ANAT. REC.,149: 333-358.
evidence has been obtained, however, for
the occurrence of sulfate esters or specific
hexuronic acids in salivary mucins.
Histologically, the secretory portion of
the salivary glands consists of mucous and
serous acini, mixed mucous acini with
serous demilunes, mucoserous acini or tubules, granular tubules and intercalated
ducts. In general, the mucous glands like
the serous form a morphologically homogeneous group. Although morphologic features at the light microscope level fail to
reveal differences in the various serous
glands, it has been assumed that chemical
differences undetected by histological
methods exist between the secretions in
the parotid, lingual and other serous glands
(Bloom and Fawcett, '62). Presumably
the same assumption applies to the preponderant mucous elements, and in this
case specific histochemical methods are
available to differentiate the secretion in
various glands through identification or
characterization of some of the constituents in the mucopolysaccharides formed.
1 Present address: Boston City Hospital, Boston,
Massachusetts.
333
334
S . S. SPICER AND JALE DUVENCI
Histochemical studies with basic dyes
have demonstrated acid polysaccharides in
salivary mucous glands and examination
with the periodic acid-Schiff (PAS) technique has revealed polysaccharides containing vicinal hydroxyls (Hoyer, 1890;
Krause, 1895; Lillie, '49; Leblond, '50;
Halmi and Davies, '53; Gomori, '54; Spicer,
'60; Bignardi et al., '60; Materazzi and
Travaglini, '60; Quintarelli et al., '61;
Shackelford and Klapper, '62a). From the
effect of sulfation on metachromasia, Bignardi ('40) obtained histochemical evidence for the presence of acid polysaccharide in some sites and polysaccharide
lacking acid groups in others. The combined basic dye - PAS methods, have
demonstrated neutral polysaccharide in
certain serous salivary glands as distinct
from acid polysaccharide in the mucous
acini (Spicer, '60; Bignardi, '61).
Autoradiography following injection of
NazS3504
permits further characterization
of the type of acid mucopolysaccharide.
This technique provides a means of localizing sulfated mucopolysaccharides histologically since glands producing sulfomucins incorporate the injected isotope
quickly and selectively with rapid turnover
of labeled mucin (Odeblad and Bostrom,
'52; Curran and Kennedy, '55; Dziewiakowski, '54; Jennings and Florey, '56). Although sulfated polysaccharides have not
been identified biochemically in salivary
mucosubstances, Bklanger ('54) has demonstrated by means of gross autoradiography with NazS3s04that the sublingual
gland of the golden hamster and palatal
glands of the rat actively incorporate the
isotope and, therefore, form sulfated mucopolysaccharide. Similar evidence has
been presented for the formation of sulfomucin by the oral molar glands of the rat
(Davies and Young, '54) and glossal mucous glands of the mouse (Spicer et d.,
'61 ). In our experience certain other histochemical methods particularly sensitive to
differences in the basophilia of tissue mucins indicate the presence in various salivary glands of acidic components different
from sialic acid and often distinctly more
acidic. Presumably these methods also
identify sulfomucins since the more
strongly acidic sulfate esters are the only
other known acid groups in polysaccharides.
On the other hand, the autoradiographic
technique revealed that the sublingual and
submandibular glands of the mouse fail to
incorporate PO4' despite the presence in
these glands of histochemically demonstrable acid mucopolysaccharide (Spicer
et al., '61). The lack of isotope incorporation in these glands and the histochemical
properties of their mucous secretion, as
well as specific assay and histochemical
enzymatic digestion, established that the
basophilia in mouse sublingual and submandibular glands is attributable entirely
to sialic acid (Spicer and Warren, '60).
The basophilia of mucin in the rat sublingual and bovine submandibular glands
has also been attributed to sialic acid by
Quintarelli et al. ('61) on the basis of
removal by acid hydrolysis, and in the
latter case sialidase digestion and chemical
assay as well. From the removal of affinity
for alcian blue or colloidal iron by means
of sialidase digestion, Quintarelli ('62)
has identified as sialomucin the secretion
in pig and monkey submandibular and dog
parotid glands. Strong alcian blue staining in the submandibular gland of the
hamster was found by Shackelford and
Klapper ('62b) to correspond with a high
level of sialic acid, and the more intense
alcianophilia in hamster females parallels
their higher sialic acid concentration. By
means of specific enzymatic digestion,
Shackelford ('63) has identified the alcianophilia of the parotid and submandibular gland of the armadillo as sialic acid
dependent. Aureli et al. ('61) have shown
by chemical assay and comparative histochemical staining that the parotid glands
of several species form sialomucin.
Because of these findings demonstrating
formation of sialomucin in some and sulfomucin in other salivary glands and the
apparent discrepancy between histochemical and biochemical data regarding the
presence of sulfated mucopolysaccharide,
further histochemical studies were undertaken to investigate the type of mucosubstance secreted by the different salivary
glands in several species. The present
study attempts identification of these two
types of mucins in various salivary glands
SALIVARY MUCOPOLY SAC CHARIDE S
and also attempts to characterize more
fully the mucosubstances elaborated by
employing additional methods which differentiate between mucopolysaccharides in
the two groups.
METHODS
Except for the tissues obtained from the
Chinese hamsters, which were kindly provided by Dr. George Yergainian, Children's
Hospital, Boston, specimens were obtained
from animals in the National Institutes of
Health breeding colony. Tissues obtained
immediately post mortem from animals
usually fasted a few hours were fixed
24 hours in 2% calcium acetate, 10%
formalin prior to routine paraffin embedding and sectioning at 5 u. Sections were
stained by a battery of methods for demonstrating and characterizing mucopolysaccharides.
The PAS stain for vicinal hydroxyls in
polysaccharides (Hotchkiss, '48) was employed as a general method for polysaccharides together with a periodic acid para
diamine variant which presumably demonstrates polysaccharide hexose (Spicer and
Jarrels, '61). Azure A was used at 0.02%
concentration in 0.1 N HC1 of approximately pH 1.0 or in pH 2.0 buffer containing 20 ml 0.1 N HC1 and 30 ml 0.1 M K
HzP04,or in pH 3.5 buffer containing 1.4
ml 0.1 M citric acid, 0.6 ml 0.2 M Naz
HPO, and 48 ml distilled water. In sections stained 30 minutes at these pH levels
and then dehydrated through graded alcohols and mounted, the strongly acidic
sulfomucins, metachromatic or orthochromatic at pH 1.0-2.0, are distinguished
from weakly acidic sulfated mucins
and nonsulfated acid mucopolysaccharides
which stain only at higher pH levels
(Spicer, '60). In a few instances where
specified, instead of alcohol resistant metachromasia, the more metachromatic color
and more sensitive staining of the undehydrated mucins were recorded as the sections were viewed wet from the staining
jar.
In accordance with Mowry's ('56) modification of Steedman's procedure, alcian
blue was employed in 1% concentration
in 3% acetic acid at pH 2.5 as a single
stain to visualize most acid mucopolysaccharides and, unless stated otherwise, was
335
used at pH 2.5 in various combinations
with other stains. Alcian blue has been
employed at controlled pH levels (Spicer,
'60; Johnson et al., '62) and in the range
pH 0.4 has been found selective for sulfated mucins. Consistent selective staining
of sulfomucins with pH 0.5 or 1.0 alcian
blue can only be accomplished by blotting
dry the sections from the staining jar prior
to rinsing in xylol and mounting (Lev and
Spicer, in press). In the pH 1 .O technique
sections were stained 30 minutes in a
solution prepared by dissolving 0.5 gm of
alcian blue in 50 ml of 0.1 N HC1. The
alcian blue-periodic acid-SchifE ( AB-PAS )
method of Mowry and Winkler ('56) was
used which distinguishes blue periodate
unreactive, or blue-purple periodate reactive, acid mucopolysaccharides from magenta neutral mucins. The latter, lacking
acid groups, have no affinity for alcian
blue and stain only with the PAS step in
this sequential precedure. In addition, alcian blue staining at pH 1.0 was followed
after blotting and drying the sections, by
the PAS method, a sequence which distinguishes blue or purple sulfomucins from
red or magenta neutral polysaccharides
and sialomucins. In this case sialic acid
groups lack alcianophilia so that the sialomucins stain like neutral polysaccharides
giving results comparable to those in sections digested with sialidase and stained
with the usual alcian blue pH 2.5-PAS
sequence. Sections were also stained with
an aldehyde fuchsin-alcian blue sequence
(Spicer and Meyer, '60) which differentiates strongly acidic purple or less acidic
blue-purple sulfomucins from blue sialomucins (or weakly acidic sulfomucins).
Three procedures were used in which
sections were stained in solutions of meta
plus para diamine or in solutions of meta
plus para diamine with added FeCL
(Spicer, '61; Spicer and Henson, '64).
These stains are referred to here for convenience as the mixed diamine method and
the low iron diamine and the high iron
diamine methods. The mixed diamine
technique stains most acid mucopolysaccharides purple selectively and differentiates periodate sensitive ( ? hexose containing) from periodate unreactive acid
mucins. Periodate reactive mucins, unlike
periodate insensitive ones, lose their stain-
336
S. S. SPICER AND JALE DUVENCI
ability by the mixed diamine method if
previously oxidized ten minutes with 1%
H5106. The method involves a 24-48 hour
exposure of deparaffinized, Feulgen hydrolyzed slides to a solution containing 30 mg
of N, N - dimethyl m - phenylenediamine
(HC1)22with an additional 5 mg of the
para isomer (monohydrochloride) and
enough 0.2 M Na2HP04(usually about 0.25
to 0.6 cm’) to bring the solution to pH 3.6
to 4.0. Inclusion of the para isomer may
be omitted with impure preparations
of meta diamine. The low iron diamine
method, which colors most sialo- and sulfomucins gray to black, involves an 8-24
hour exposure to a solution containing
30 mg of N, N - dimethyl - m - phylenediamine (HC1)2, 5 mg of the para isomer
(monohydrochloride) and 0.5 cm3 of N.F.
official FeCL solution ( 10% Fe++ ) . This
procedure, if followed by a 30 minute exposure to 1% alcian blue in 3% acetic
acid, colors most acid polysaccharides gray
to black but stains blue a few nonsulfated
mucins lacking affinity for iron diamine.
The high iron diamine technique, which is
unreactive for sialomucins but stains all
sulfomucins thus far examined, is similar
except that sections are exposed 6-18 hours
in a solution containing 120 and 20 mg of
meta and para isomer, respectively, and
1.4 cm3 of the FeCL solution. This high
iron diamine stain, if followed similarly
by alcian blue, distinguishes purple-black
mucins, which in all instances thus far
known are sulfated from blue acid mucopolysaccharides having sialic acid or possibly other unidentified acidic residues.
A one-hour blockage at 25°C with a solution of 5% phenylhydrazine hydrochloride
in distilled water followed by a five minute
rinse in running water was interposed between periodic acid oxidation and the
Schiff stain in the conventional PAS technique. This periodic acid-phenylhydrazineSchiff sequence stains periodate reactive
acid mucopolysaccharides selectively, because the cationic phenylhydrazine
condenses more readily with periodateengendered aldehydes in neutral mucopolysaccharides (such as glycogen and
gastric surface mucins), thus blocking the
PAS reactivity of these substances preferentially (Spicer, ’61). As a means of
blocking selectively the basophilia of most
-
+
nonsulfated acid mucins, sections were
subjected to a ‘‘mild” methylation procedure, involving a five-hour exposure at
37°C to methanol containing 0.1 N HCl
(Fisher and Lillie, ’54). Loss of basophilia
resulting from mild methylation was visualized in the change from blue-purple of
acid to the magenta of neutral mucins with
the alcian blue-PAS procedure. Active
methylation on the other hand involving
a 3-4 hour exposure of sections at 60°C removes sulfate esters (Spicer et al., ’61)
while esterifying carboxyls and thus blocks
nearly all acid mucopolysaccharide basophilia. Restoration of basophilia in sections from which sulfate esters were removed by active methylation was undertaken through de-esterification of carboxyls
by a 15-30 minute saponification in a solution containing 0.1% KOH in 70%
ethanol. A five minute exposure of deparaffinized sections to this alcoholic KOH
(herein designated brief saponification)
was also employed to enhance basophilia
of some secretions and to render certain
sialomucins susceptible to enzymatic digestion empirically. Sections were sulfated
15 minutes in a modified Moore and
Schoenberg (’57) procedure as employed
previously.
To identify sialomucins specificially, sections were digested with sialidase purified
from a culture of Vibrio cholerae (General
Biochemicals, Chagrin Falls, Ohio), employing the method described previously
(Spicer and Warren, ’60). The histologic
sections were incubated 8-24 hours at
39”C, except that sections which had previously been saponified by means of a five
minute exposure to a solution of 1%KOH
in 70% ethanol were digested only 4-7
hours. Untreated or briefly saponified control sections were incubated similarly with
the buffer in which the enzyme was dissolved. Following digestion, the enzymatic
removal of sialic acid residues was visualized by: (1) the loss of alcian blue affinity
in the simple alcian blue stain or the high
iron diamine-alcian blue sequence; ( 2 )
change in coloration from blue-purple of
acid to red of neutral mucopolysaccharide
in the AB-PAS sequence; (3) loss of wet
2 Available from Eastman Kodak Co., Rochester,
N.Y., Schuchardt Chemlcal Co., Munich Germany,
or Gallard-Schlesinger Chemical Corp., Girden City,
Long Island, N. Y.
TABLE 1
Histochemical properties of mucopolysaccharide comtaining secretions in salivary glands
High iron diamine-AB
m
iIistologic
site
al
.*
1
Alcian blue
pH 1.0
AFAB
pH2.5
Active
MeOH
15 min
KOH
AFAB
Low
=on
diamine
AB
After
No
pretreatment
Active
g
z
sulfation
PAS
afber
diastase
PAPS
Mixed Diamines
at pH 4.0
Without
HI04
With
H104
AB pH 2.5-PAS
After
AB
pH 1.0
PAS
KOH
[ajor
sublingual
glandmucous
acini
M
Rat
SH
CH
GP
O
0
0-3B5
4B
0-B
4B
4B
4B
4B
4B
4B
4B
4B4BP
4P
4B
2B
B
2B
2B
B
3B-P '3
B
2BP-2P
0
0-B
0-fB
0-kB
0
2B
3N
3N
4B3N
4N
4B4BN
4B
4B
4B4N
4N
4B
2B
2B
2B
2N
2B
4PN
4PN
4PN
NB-B
2 NB
3B-N13
1-2N
2N
0
1-2B
0-B
3B-2BN13
N
3N
0
0
0
0
0
0
0
4PN 11
3PN"
3PN l1
2 N
0
B
0-B
N
0
0
0
B
0-B
0
0
0
0
0
0
0
0
0
0
N
N
N
0
0
2N
N
2N
0
0
0
0
0
3P
4N
4N
4N
4N
4PN
4N
4N
4N
4N
4N
4PN
4N
1-2B
0-'-B
0-lB
0
4PN
4PN
4N
0
3PN
4PN
4PN
5 min
KOH 2
18 hr
buffer
5 min
Autoradiograph
with
s3504=
3R
3R
3R
0-fR
4R.
2R
2R
3R
0-"R
3R
2P
3P
3P
4P
3P
0-CF
0-CF
0-CF
4P
2F
3R
3R
3R-3PB'
4B
4R
4PB
4B
4BP
4B
4PB lo
3R
3R
3R-3BP
2R
1-2R
3R.
0-R
0-R
1-2R
R
0
1-3R14
0-R
0-ltR
2P
2P
2P
0
2P
0
0-P
1-2P
0-YF
2R
1-2R
3R-3P
R-BP
2B
1-2R
3BP l1
1-2P 11
3P-3BP
R-BP
2B 11
1-2R
3RP
1-2P
3R-3P
R-BP
2B
1-2R
2R
2R
3R
3R
2R
0
0-R
0
O-kR
0-R
O-kR
0
0
0
0-P
0
0
2R
0
0
0
0
2P 1s
0
2-3R
2R
2R
-~
2R
0-R
2R
4P
4P
4P
4P
4P
4P
'
4B
4R
3R
3R
4P4BP 8
0 3
0
0-+
++ S
4B
4P
~
ubmandib-
ular
glandmucous
acini
Lotid
glandserous
acini
M
Rat
SH
CH
R
GP
O
2B
0
0-2B12
0-B
2B
0
2B
1-3B
B
3B
0
M
Rat
SH
CH
R
GP"
O
0
0-kB
0
O
0
0
0
1-2B
0-B
0
0
0
B
0-B
0
0
0
0
0-B
0
0
2P
0
0-P
4B
4B
4B
4B
0-2B
4B
4P
4P
4P
4P
4P
4P
arotid
glandintercalated
ducts
M
Rat
SH
CH
R
osterior
glossal
mucous
glands
M
0
0
0
l'
O-aB
0
ltP
2 P
0-B
0
0
0
P
0
4PN 1'
2PN
3P
3NP
3PN
3N
0
2R
2R
0
0
0
0-P
0
0
P
0
CF
0
0
0
0
l5
15
3R-3P
0-B
0-R
3R
3R
3R-3P
3R
2R
0
3R
3R
3R-3BP
3R
2R
0
3R
3R
3R
3R
2R
0
3R
3R
3R
3R
2R
0
2R
2R
2BP
0-R
2R
2R
2R
2P 19
2R
2R
2R-P
2R
2R
2R
4BP
4BP
4PB
4PB
2B
4PB
4BP
4BP
4PB
4PB
3B zo
4PB
4BP
4BP
4PB
4PB
3B 20
4PB
4BP
4BP
4PB
4PB
3B 2o
4PB
0
0
0
0
"J6
2R
R
~
'
Rat
SH
CH
R
GP
3B
4B
4B
4B
1-2B
4B
2B
1-2B
B
0-B
2B
B
4R
4R
3R
3R
0-R
3R
0-CF
CF-3P
3P
3P
4P
1-3P
0
0
+++
+++
+
0
0
+
++
++++
++++
++++
+++++
mouse. SH Syrian hamster. CH Chinese hamster. R Rabbit. GP Guinea pig.
$, a1deh;de f&hsin. AB, alcia; blu;?;PAPS, periodic ahd~phenyl~ydra&e-Schiff.MeOH methylation.
~lors: B, blue; C,'grey; F, brown; N, black; P, purple; R, red; V. violet; Y,'yellow:
1 StaininspropeFties describe secretpry material. in
each site. istinctions between color m vanous sites
apply only for results with a given procedure.
2Prior to staining sections were exposed five minUtes to alcoholic KOH .and 18 hours to buffer of
sialidase solution or sialidase.
3 Serous demilunes show light radiosulfate incorporation.
4 4B if saponification is .omitted.
5 Some cells in distribution of those showin S3504'
autoradiographs are stained others unstaine.8.
6 Many cells blue; man oihers especially rn males,
stain blue and gre or black in'the iron diamine or
Dumle
in
aldehrvde
fuchsin-alcian blue seauence.
~ ~ _ -~~
_ _ the
.~
.
7 Many red cells, gomered and purple or b1ue:purple
in same distribution as those incorporating S3504=
and staining purple or black with aldehyde fuchsinalcian blue or iron diamine-alcian blue methods.
8 Scattered cells are blue-purple. Most are purple.
9Many cells negative- some show moderate and
others marked isotope &corporation.
10 Staining is bluer than in Syrian hamster but
more purple than in mouse.
11Adjacent granular tubules stain 1-3R with ABPAS and are unstained with sulfation - high iron
diamine-alcian blue method. They are more prominent in male than female in mouse and rabbit.
Intercalated ducts in female Syrian hamster stain
3R with AB-PAS.
1ZAt u H 1.0 male and female =lands are about
equal in- stainini intensity but
pR 2.5-male gland
has mostly PB with a few 3B cells and female mostly
3B.
1s In males clusters of large cells near intralobular
d ucts show blue fibrillar mucus. Most cells: nearly all
of them in females show aldehyde fuchsin reactive
purple or iron diamine reactive black granules mixed
vvith blue material. Grey iron diamine staining of
intercalated ducts in females is enhanced by prior
brief saponification.
14The roups of larger cells near ducts in males
stain darger.
1 s Groups of large cells in males, usually near ducts,
stain 3R. Predominant cells with more granular secretion stain 3RP-P.
16 Large cells in groups around ducts in males have
41P-BP fibrillar material. Most cells have eranular
secretion staining 3PR to P in males and 4P 50 BP in
females.
17 Goblets in interlobular ducts are 4P and 2F with
mixed diamines without and with prior periodate respectiyely, 4B with both a!dehyde fuchsin-alcian blue
and high iron diarmne-alcian blue, 4N4BN wlth low
iron diamine-alcian blue, unstained with pH 1.0 alcian
blue and 2R with the periodic acid- henylhydrazinSchiff method. Sialidase and mild met lation destroy
their azurophilia and alcianophilia. %hey stain 2B
with aldehyde fuchsin-alcian blue after 4 hour 60°C
meth lation then 15 minute saponification.
1s&ain only in mixed diamine solution in which
ca 5 cm3 N NaCl replaces ca 5 cm3 of the water.
1s 3BP if not initially exposed to KOH and buffer.
20 + R-2B if not exposed to KOH and buffer or if
not methylated.
338
S . S . SPICER AND JALE DUVENCI
metachromasia toward azure A at pH 3.5
in sections viewed directly from the staining solution; or ( 4 ) loss of alcohol resistant metachromasia. The sialic acid concentration in the several glands frozen on
dry ice immediately post mortem, was
kindly measured by Dr. L. Warren of this
Institute, employing a thia-barbituric acid
assay (Warren, '60) on acid hydrolysates.
Two mc of carrier free NadP504from
Oak Ridge National Laboratories were
injected in each of three weanling rabbits,
1.5 mc in each of three Syrian hamsters,
0.6 mc in each of three rats and 0.4 mc in
each of three pregnant mice. Three to six
hours following the intraperitoneal injection of the isotope, the animals were sacrificed and tissues were fixed in calcium acetate formalin, dehydrated, embedded and
sectioned according to routine procedures.
Sections prestained for mucopolysaccharides by the PAS, alcian blue, aldehyde
fuchsin and pH 3.5 azure A methods, and
the AB-PAS and aldehyde fuchsin-alcian
blue sequences together with duplicate unstained sections were prepared promptly
for autoradiography with Kodak AR-10
stripping film using conventional techniques. Following a 6-12 day exposure,
autoradiographs were developed, air dried,
dehydrated through alcohol and xylene and
mounted in permount.
RESULTS
The acini terminal to the granular tubules or intercalated ducts in the submandibular glands of all the species examined
here are referred to as mucous acini. This
designation is employed, rather than the
term seromucous (Shackelford and Klapper, '62a), because of the conspicuous acid
mucopolysaccharides demonstrable by several histochemical methods in all of these
acini (except those of the guinea pig) as
in the mucous acini of the sublingual and
glossal glands. However, the designation
mucoserous might be preferable since these
glands contain considerable ribonucleic
acid and thus form protein rich secretion.
In general, the sublingual and glossal mucous acini of this study show the most
abundant and strongly basophilic mucopolysaccharide and little chromidial substance. The submandibular mucous acini
display intermediate abundance of both;
and the parotid, lingual and exorbital lacrimal serous acini reveal the most abundant
ribonucleic acid and little to moderately
abundant polysaccharide with weak or no
basophilia (table 1, 2).
Mouse submandibular and sublingual
glands. The secretions in the mucous
acini of the submandibular and major
sublingual glands of the laboratory mouse
(NIH general purpose mixed breeding
colony) reveal histochemical properties indicative of a nonsulfated acid mucopolysaccharide. This is evident in their blue
staining with the aldehyde fuchsin-alcian
blue or high iron diamine-alcian blue sequence and failure to incorporate S3504'
(fig. l A , table 1). It has been shown that
the mucous elements in these glands secrete sialomucins uniformly (Spicer and
Warren, '60). The secretion in the submandibular differs from that in the sublingual gland, however, in showing alcohol
resistant orthochromasia with azure A at
pH 3, weak affinity for the low iron diamine method and marked resistance to
sialidase digestion. Both glands reveal
periodic acid-phenylhydrazine-Schiff staining indicative of periodate reactive acid
polysaccharide, but the staining in the submandibular gland is relatively weak (table
1 ) as is the PAS reactivity. Since the submandibular gland retains part of its reactivity in the mixed diamine procedure following oxidation with periodate, it appears
that this secretion has intermediate periodate reactivity compared with secretions the
staining of which totally resist or succumb
to periodate.
Secretions in the granular tubules of the
mouse submandibular gland and in granular cells in intralobular ducts of male
mouse sublingual glands contain neutral
polysaccharide as judged by the (diastase
resistant) red staining with the AB-PAS
method and failure to incorporate S3'00,'.
Small serous demilunes with granules
which stain red in the AB-PAS sequence
cap the mucous alveoli in the sublingual
gland. Although thus lacking in alcianophilia and unreactive toward azure A, aldehyde fuchsin and the iron diamine stains,
these granules appear to be the site of weak
F504'uptake (fig. 1A) and presumably
produce lightly sulfated mucopolysaccharide with masked or inapparent basophilia.
SH
CH
M, Rat
SH, CH}
Parotid
intercalated
ducts
Posterior glossal
mucous glands
4PR
0
0
4PR
PR
0
3PR
1-2PR
3PR4
PR
34PR
3PR
4PR
4PR
4PR
2%;
4PR
PR
3PR
1-2PR
1-2PR
0-PR
0
0
0-2PRa
4PR
0
0
0
3PR
1-2PR
1-2PR
0
0--tPR
3PR
0-3PRZ
4.hr
siahdase
5KOH
min
4V4P
4P
4RP
0-B
B
’
3V-4P
0
0
0
0
0
0-B
0-B
0-B
0
0
0
B
B
2-3P
kB
3P
2B
4P
4VP
4P
0
0
0
4VP
0
0
0
0
Azure A
PH 1.5 Untreated
1
S3504=
0
B
0-B
2PB
4P
0
B
0-B
0-PB
0
0-2P
0
0
a
5 min
KOH
4hr
sialidase
isotope label.
2-3RP
2-3P
4P
KOH
4 hr
buffer
1
5 min
Azure A pH 3.5
2
4V-4P
0
B
0-B
B-PB
0
0
%3P
0-3P
%
$
:
4.hr
siahdase
Alcohol resistant staining of secretion
Similar results were obtained with exposure to buffer then KOH.
a Most cells unstained. Scattered metachromatic cells are distributed like those in autoradiographs showing heavy
8 Applies only to the female.
‘Male glands show 1-2PR staining in most cells with scattered groups of larger 3PR cells often near ducts.
5 Male glands B-2BP.
6 Not examined in rat.
Abbreviations as in table 1.
R
M, Rat
GP and R
SH, CH
Parotid
serous
alveoli
-cB
PR
3PR
1-2PR
3PR *
-t- PR
0
M
Rat
SH
CH
GP
Submandibular
gland mucous
alveoli
0
0
34PR
3PR
0-2PR2 4PR
4PR
4PR
0
4PR
0
0
A~~~~ A
pH 1’5
M
Rat
SH
CH
GP
Species
Azure A pH 3.5
5min
5min
Untreated
4hr
buffer 1 sialidase
Staining- of secretion in sections viewed wet
Major sublingual
gland mucous
alveoli
Histologic
site
TABLE 2
Effect o f sialidase digestion and o f sulfation on azurophilia of salivary glands
lfEh
-cB
*B
4P
3P
3B
4B
2vB
2VB
2VP
VB
2VB
2-3B
0
3P
3P
2P
4BP
3BP
0
Chromidial
substance
mth
azure A
pH 2.0
2VB
2VB
2VB
2VB
1-2-
4P
4P
4P
4PB
4P
sulfation
Azure A
pH 0.5
340
S. S . SPICER AND JALE DUVENCI
Rat submandibular and sublingual
glands. Secretions in mucous acini of rat
submandibular and sublingual glands have
histochemical properties resembling in
some respects those of the mouse. Like the
latter, they color blue with the aldehyde
fuchsin-alcian blue or high iron diaminealcian blue method and fail to incorporate
S3'04' (fig. 1B) and accordingly contain
non sulfated acid polysaccharide. Unlike
the other sublingual glands of this study
which are metachromatic toward azure A
in dehydrated sections, the rat gland is
orthochromatic (fig. 3A). After brief exposure to KOH, however, rat sublingual
gland mucin shows alcohol resistant metachromasia (fig. 3B). Although containing
sialic acid at such levels that this residue
presumably accounts for their basophila,
the mucous secretions in these rat salivary
glands resist destruction of basophilia by
sialidase digestion (fig. 2A) (Spicer and
Warren, '60). Quintarelli et al. ('61) have
provided evidence that the basophilia of
the rat sublingual gland is attributable to
sialic acid because it succumbs to mild acid
hydrolysis. Preliminary brief exposure to
alcoholic KOH renders the basophilia in
the sublingual but not the submandibular
gland of the rat not only metachromatic
but also susceptible to sialidase (figs. 2B,
3 C ) (table 2). The sublingual gland forms
periodate reactive acid polysaccharide
judging from the strong periodic acid-phenylhydrazine-Schiff staining and loss of
mixed diamine staining after periodate.
Since the opposite is the case for the submandibular gland, this secretion apparently contains periodate unreactive acid
polysaccharide (table 1) .
The granular tubules in the submandibuIar gland form a histochemically neutral
polysaccharide which as in the mouse lacks
autoradiographic evidence of S3504' incorporation and stains red in diastase digested slides stained with the AB-PAS
method. The granular secretion in the serous demilunes of the rat sublingual gland
also contain diastase resistant neutral polysaccharide which stains red with the ABPAS method and lacks any basophilia or
evidence of S3504'uptake.
Submandibular gland of the Syrian
hamster. The mucous acini in the submandibular gland of the Syrian hamster (Meso-
cricetus auratus) reveal a granular grayblack secretory substance with admixed
blue coloration throughout the cytoplasm
when stained by the high iron diaminealcian blue method (fig. 5). The aldehyde
fuchsin-alcian blue sequence also distinguishes two types of secretion in the mucous acini, a blue-purple granular material
distinct from a blue stained mucosubstance.
In the female hamster some variability is
evident with these two methods; most cells
reveal blue and gray-black or purple staining, but the proportion of blue and of grayblack or purple differs among the individual cells. The same variability exists in the
male, but the gray-black or purple staining
is much more prominent. In addition there
are groups of larger, mostly blue cells with
a granular cytoplasm collected usually adjacent to intralobular ducts (fig. 5).
The cells of the mucous alveoli in the
female show fairly uniform staining
throughout with the PAS, pH 2.5 alcian
blue (fig. 4), or combined AB pH 2.5-PAS
(fig. 10) methods, but those of the male
vary in alcianophilia, there being groups of
somewhat larger and more alcianophilic
cells usually collected at or around intralobular ducts.
Alcian blue at pH 1.0 imparts to most of
the mucous alveoli of the male and female
submandibular glands comparably a moderate blue which is considerably less intense
than that with alcian blue at pH 2.5. The
gray coloration with the high iron diaminealcian blue and purple with the aldehyde
fuchsin-alcian blue methods, and the residual staining with alcian blue when the
pH of the solution is lowered to 1.0 (table
1), all indicative of sulfated mucopolysaccharide in the granular secretion, parallel
the strong autoradiograph obtained with
S3504'as shown in figures 4 and 8.
Sialidase digestion removes the diffuse
alcian blue coloration predominant in the
larger blue periductal cells of the male and
present together with gray coloration in
most cells of both sexes, leaving the gray
diamine staining unaltered in sections
stained with the high iron diamine-alcian
blue method (fig. 6). Lack of any alcian
blue staining here after sialidase digestion
demonstrates that sialic acid accounts for
the alcianophilia in this combination procedure and shows further that the prior ex-
SALIVARY MUCOPOLYSACCHARIDES
posure to iron diamine blocks subsequent
alcian blue staining of sulfate esters in the
secretion. Following sialidase digestion
most of the acini in the male and nearly all
of those in the female retain some alcianophilia changing from the normal purpleblue to purple coloration with the AB pH
2.5-PAS method (fig. 11). However, the
large cells around ducts in the male lose
alcianophilia after digestion and then color
predominantly red with this sequence so
that these cells contain mainly sialomucin.
Staining with the AB pH 2 5-PAS technique
after sialidase resembles that with the AB
pH 1.0-PAS sequence in undigested glands
as would be expected from the accumulated evidence that sialomucins lack alcianophilia at pH 1.0. Persistence of decreased alcianophilia after sialidase or on
lowering the pH of staining to 1.0 provides
evidence that part of this staining at pH
2.5 is accounted for by sulfate and part by
sialic acid. Since sialidase digestion decreases but does not eliminate azurophilia
part of this staining, particularly with regard to wet metachromasia, is also attributable to sialic acid and part to sulfate esters
(table 2).
From the strong periodic acid-phenylhydrazine-Schiff staining and the susceptibility of mixed diamine reactivity to prior
periodate oxidation, it appears that this
submandibular secretion contains periodate reactive acid polysaccharide. Following
removal of sulfate esters by active methylation and subsequent deesterification of
carboxyls by saponification, the mucous
alveoli fail to stain or in a few areas show
faint blue coloration with the aldehyde
fuchsin or high iron diamine-alcian blue
sequences. Most of the sublingual glands
in this study stain bright blue with the
basic dye sequences after methylationsaponification, and it is not clear whether
this lack of staining in the Syrian hamster,
as well as in the mouse and rat submandibular mucous acini, reflects qualitative difference between submandibular and sublingual sialomucins. However, it seems
possible that the difference is quantitative
and results from hydrolysis of sialic acid
during methyIation and saponification so
that submandibular glands having less
sialomucin are more susceptible to destruction of alcianophilia.
341
As observed originally by Shackelford
and Klapper (’62b), the mucous (or seromucous) acini of the female gland stain
more heavily with alcian blue at pH 2.5
than do those of the male, a difference correlated with the higher level of sialic acid
in the female. From the present study it
appears that azurophilia at pH 3.5 (table 2)
is also clearly more intense in the female.
Whereas the PAS reactivity is slightly if at
all greater in the female gland, the periodic
acid-phenylhydrazine-Schiff staining is
considerably stronger in this sex. This result, together with the observation that the
large periductal cells in the male stain
more strongly than the mucous acini with
the periodic acid-phenylhydrazine-Schiff
method, suggest that sialomucin mainly accounts for this staining in the submandibular gland of the Syrian hamster. From the
various staining, digestion and blockage
procedures, it may be concluded that most
acini in this gland form both sulfo- and
sialomucin but a few cells, especially those
in the male with periductal distribution,
produce mainly sialomucin. In general the
gland of the female stains more strongly
for sialomucin, that of the male for sulfomucin.
The granular tubules distal to intralobular ducts in the Syrian hamster submandibular gland, lacking evidence of S”O,=
incorporation and staining red in the ABPAS sequence (fig. lo), secrete (diastase
insensitive) polysaccharide apparently devoid of acid groups. On the other hand, the
terminal branches of the granular tubules
form a granular secretion which, although
stained red with the AB-PAS method, colors
gray with the high iron diamine-alcian
blue and faint purple with the aldehyde
fuchsin-alcian blue techniques. Prior five
minute saponification intensifies this diamine staining. These intercalated ducts,
which it should be noted parenthetically
are more conspicuous in femaIe glands,
apparently form sulfomucin with masked
alcianophilia. It has not been possible to
demonstrate either presence or absence of
isotope uptake in these ducts because of
the technical difficulties from the heavy
surrounding label in the mucous acini
and the relatively poor resolution obtainable with S350,=autoradiographs.
342
S. S . SPICER AND JALE DUVENCI
Sublingual gland of the Syrian hamster.
Mucous acini of the Syrian hamster sublingual gland stain a uniform red with the
PAS procedure (fig. 7A), a uniform blue
with pH 2.5 alcian blue (fig. 7 B ) and a
uniform blue-purple with the AB-PAS combination (fig. 10). However, the apparent
uniformity of the secretion throughout this
gland indicated by these procedures is refuted by methods which differentiate between types of acid mucopolysaccharides.
Autoradiographs following injection of
S 3 s 0 0 , z ,developed on unstained sections or
sections prestained with pH 2.5 alcian
blue, reveal little or no uptake of isotope by
many of the cells but moderate to heavy
labeling of one or several cells in numerous
acini (fig. 7B ). The isotope incorporation
here, confirming that observed in gross
autoradiographs by Bklanger ('54), appears
markedly variable from cell to cell when
examined with the higher resolution stripping film technique. The aldehyde fuchsinalcian blue sequence, which in general
stains sulfated mucins purple to blue-purple and nonsulfated acid mucins blue,
colors many of the cells of the hamster
sublingual gland blue but shows one or several blue-purple cells in a number of acini.
These purple cells correspond with those
in which the overlying autoradiograph
demonstrates isotope incorporation (fig. 8).
Many of the cells are unreactive with the
low or high iron diamine method; a large
number stain lightly and some heavily. Accordingly, with the high iron diaminealcian blue sequence, many cells stain
blue, some blue and gray, and some black;
and the pattern of distribution of the gray
to black cells resembles that of cells showing isotope incorporation (fig. 12). Alcian
blue at low pH is considered selective for
sulfated mucins and at pH 1.0 fails to stain
most of the cells but reveals one or several
blue cells in a number of acini with the
same pattern of distribution as that of the
blue-purple or black cells in the previously
mentioned techniques (fig. 9A). Alcian
blue at pH 1.0 followed by PAS staining
shows some cells with dark purple-blue,
others with red and others with both types
of staining. The periodic acid-para diamine procedure shows brown staining presumably indicative of hexose also differing
in various cells (fig. 9B).
Enzymatic digestion demonstrates that
sialomucin is located in the isotopically unlabeled cells which lack affinity for aldehyde fuchsin (fig. 8 ) or the high iron diamine reagent (fig. 12) and therefore stain
blue with sequences of either of these dyes
followed by alcian blue. Thus sialidase digestion removes the blue coloration in sections stained by the high iron diaminealcian blue method without affecting the
gray to black staining indicative of sulfomucin in the scattered mucous cells as
well as adjacent serous demilunes and mast
cells (fig. 12). Whereas all cells stain uniformly blue-purple with the AB pH 2.5-PAS
technique (fig. 10) in undigested sections,
many stain red with this procedure following exposure to sialidase (fig. 1 1 ) . This
red staining results from loss of the acid
groups responsible for alcianophilia without which the polymer to which the sialic
acid is attached stains red like a neutral
polysaccharide. However, one or more cells
in a number of acini resist digestion, retaining their capacity to stain completely
blue-purple or partly blue-purple and partly
red with the AB-PAS method. The distribution of the sialidase resistant cells resembles that of cells staining purple with the
aldehyde fuchsin-alcian blue, gray to black
with the iron diamine-alcian blue and bluepurple with the AB pH 1.0- PAS procedures. The distribution of enzyme resistant
cells is also similar to that of the isotope
labeled cells so that sublingual glands from
S3504= treated hamsters give autoradiographs in sections previously digested with
sialidase and stained with the AB-PAS procedure which reveal isotope label selectively over the sialidase resistant blue-purple cells (fig. 11).
Methylation 2-4 hours at 60°C, which
removes sulfate esters by methanolysis but
blocks carboxyls by esterification, occludes
basophilia toward high iron diaminealdehyde fuchsin or alcian blue in this
gland. Subsequent saponification to deesterify the carboxyls restores uniform
bright blue staining with the high iron
diamine-alcian blue sequence or the aldehyde fuchsin-alcian blue method. This uniform blue staining resembles that seen in
the mouse, rat and guinea pig sublingual
glands after methylation-saponification
(table l ) , a result which confirms that
SALIVARY MUCOPOLY SACCHARIDES
sulfate esters combine with the iron diamine or aldehyde fuchsin and sialic acid
residues with alcian blue in these sequences. It may be concluded from staining, digestion and blockage procedures
that many of the cells in the mucous acini
of the Syrian hamster sublingual gland
form sialomucin exclusively, some form
sulfated mucopolysaccharide predominantly at least, and a large number produce
both types of secretion. In the six animals
of each sex examined here, the sublingual
glands of the male contained on the average many more sulfomucin-laden cells
than did those of the female.
The sialomucin in this gland differs from
most in lacking affinity for the low iron
diamine reagent and resisting complete
blockage of basophilia in the AB-PAS
method by "mild methylation.
The secretions in all the mucous cells of
the sublingual like those in the submandibular glands of the Syrian hamster show
strong periodic acid-phenylhydrazine-Schiff
reactivity uniformly and complete loss of
staining by the mixed diamine technique
in sections previously oxidized with periodic acid (table l). These results, interpreted as indicative of the presence of vic
glycol groups in close proximity to acid
residues, are consistent with the possibility
that sialic acid residues or sulfate esters
may be linked to hexose and conceivably
in a given organ or cell are bound interchangeably to the same polymer. Sialic
acid assay kindly carried out by Dr. L.
Warren provides further evidence that the
hamster salivary glands secrete sialomucin,
there being 4.4 WM of acid hydrolyzable
sialic acid per gram of wet tissue in several pooled submandibular glands and
16.2 VMin pooled sublingual glands.
Granule-filled serous demilunes cap the
mucous alveoli in the sublingual glands of
the Syrian hamster. These demilunes although stained red like neutral mucopolysaccharide with the AB-PAS method (fig.
10) have moderate iron diamine affinity.
A five minute exposure to KOH accentuates
this staining so that after such brief saponification the demilunes color gray with the
low iron diamine technique (fig. 12) as
distinct from the purple-black of neighboring mast cells and mucous cells. The demilunes also stain black or light purple
343
with the high iron diamine-alcian blue or
aldehyde fuchsin-alcian blue methods, respectively. It appears then that the hamster demilunes (like those in the mouse)
form sulfated mucopolysaccharide with
masked alcianophilia; but, because of the
acinar labeling adjacent to the hamster demilunes, isotope incorporation cannot be
clearly demonstrated in them autoradiographically.
Submandibular and sublingual glands of
t h e Chinese hamster. The studies of
Chinese hamster (Cricetulus griseus) tissues, not including autoradiography with
S3'04', are restricted to histochemical staining methods. The mucous alveoli in the
submandibular gland of this species stain
gray uniformly with the iron diaminealcian blue methods and light blue with
alcian blue at pH 1.0 and 2.5 and appear
to form acid mucopolysaccharide with a
low level of sulfation. They have weak
PAS reactivity unaltered by active methylation.
Uniform metachromasia of all mucous
alveoli is seen in the Chinese hamster sublingual gland stained with azure A at pH
1.0 (fig. 13); uniform purple coloration is
evident with the aldehyde fuchsin-alcian
blue sequence (fig. 14) and uniform black
staining with the high iron diamine-alcian
blue technique. Thus, it appears reasonably certain that all the mucous cells of
the sublingual gland in the Chinese hamster form a strongly acidic sulfomucin uniformly. The sulfate esters in this mucopolysaccharide differ from all others yet
examined, except those in mast cells and
in the colon of the Chinese hamster, in
resisting complete hydrolysis by four hour
methylation at 60°C. After such treatment
they stain gray-black in the high iron diamine-alcian blue procedure either with
saponification after the methylation as
shown in table 1 or without such exposure
to alkali.
The mucous acini in the Chinese hamster sublingual gland are PAS unreactive
and stain blue (rather than blue-purple) in
the AB-PAS method (fig. 15) and purple in
the pH 3.6 mixed diamine method with
and without prior periodate oxidation.
From these properties it is clear that a
periodate unreactive acid mucopolysaccharide is formed. This sulfomucin has the
344
S. S. SI'ICER AND JALE DUVENCI
unusual property - shared with that in
rabbit glossal and laryngotracheal mucous
glands and colonic goblets - of being essentially PAS unreactive (fig, 16) but gaining strong PAS reactivity after removal of
sulfate esters by active methylation (fig.
17). Apparently this mucosubstance contains sulfate esters on vie glycols which
until removal by methylation occlude PAS
reactivity. It may be noted in this connection that full sulfation of tissue sections
in nitro blocks the PAS reactivity of all
polysaccharides and subsequent methylation restores the staining (Spicer, '60).
Guinea pig submandibular and sublingual glands. Judging from the histochemical staining reactions, the guinea pig submandibular gland secretes only a neutral
polysaccharide (table 1 ) ( Shackelford and
Klapper, '62a).
Mucous secretion in the sublingual gland
of the guinea pig closely resembles that of
the mouse with a few exceptions, particularly the darker and more purple alcohol
resistant metachromasia which presumably reflects the higher level of sialic acid
present (Spicer and Warren, '60). Correspondmgly, the periodic acid-para-diamine procedure (Spicer and Jarrels, '61)
shows stronger orange-brown staining following enzymatic removal of sialic acid.
The reactions in general are those of a
periodate reactive (hexose rich?) sialomucin formed uniformly throughout the
gland. This sialomucin and that in the
Syrian hamster sublingual gland are different from those in the mouse and rat sublingual glands in that they have little or
no affinityfor the low iron-diamine reagent
and relatively high resistance to blockage
of basophilia in the AB-PAS stain by mild
methylation (table 1 ) . Although previously reported as susceptible to mild methylation relative to sulfomucins this secretion
appears, from the present study, less susceptible than that of most sialomucins,
possibly on a quantitative rather than a
qualitative basis.
Rabbit ssbmandibular gland. The mucous alveoli of the rabbit submandibular
gland produce acid mucopolysaccharide
which lacks alcohol resistant azurophilia
at or below pH 3.5 despite staining with
alcian blue at pH 2.5 and 1.0 and showing
purple-gray coloration with the high iron
diamine-alcian blue or blue-purple with
the aldehyde fuchsin-alcian blue sequences
(figs. 18-20) (tables 1 and 2). The latter
histochemical evidence for sulfation of the
polysaccharide agrees with the active
S3504' incorporation demonstrable autoradiographically (figs. 18 and 19). Biochemical assay shows a low level of' sialic
acid in this gland (2.8 pM/gm of wet tis,sue), a fact which concurs with the predominant staining of sulfomucin in this
site. The lack of PAS reactivity here corresponds with the persistent staining by
the pH 3.6 mixed diamine solution after
periodate oxidation (table 1 ). The various
properties are those of periodate unreactive
sulfomucin. This lack of azurophilia is
unique among the periodate unreactive
acid mucopolysaccharidesin that the latter
are usually the most strongly acidic toward
azure A, staining with Y metachromasia at
relatively low pH.
The intralobular ducts of the rabbit submandibular gland lack autoradiographic
evidence of S3504"incorporation except
over clearly labeled secretion in the lumen.
To determine whether a relatively low level
of radiosulf ate incorporation occurs in the
granular tubules of this gland is difficult
because of the relatively poor resolution
with PO4' and the fact that label here
might be obscured by the adjacent strong
autoradiograph of the mucous acini. Granules in these tubules stain red with the
AB-PAS method and also lack affinity for
the other basic stains. The proportion of
the gland occupied by granular tubules and
by mucous alveoli in the few animals studied appears to differ in the sexes. The
granular tubules are relatively more abundant in the male and the mucous acini in
the female rabbit, as in mice, but the differences are less marked.
Parotid glands. Despite lacking affinity
for the basic dyes and having staining
properties indicative of a neutral mucopolysaccharide, the secretion in the mouse
parotid shows moderate S350,=uptake in
autoradiographs (figs. 21 and 22) (table 1).
Possibly this label reflects presence of
otherwise undetected sulfate esters in the
mucopolysaccharide since the autoradiographic method is undoubtedly more sensitive than staining procedures and is not
subject to the stearic restrictions to stain-
SALIVARY MUCOPOLYSACCHARIDES
ing imposed by polymer structure or masking copolymers.
The secretion in the serous alveoli of the
rat parotid also stains like a neutral polysaccharide histochemically but in this
instance shows autoradiographic labeling
with P O 4 ' not discernibly above background (fig. 23).
The serous alveoli in the parotid of the
Syrian hamster display moderate but variable blue coloration with the AB-PAS, high
iron diamine-alcian blue and aldehyde
fuchsin-alcian blue methods (figs. 24-26)
(table 1). These latter two properties,
indicating nonsulfated acid polysaccharide
concur with the marked decrease in alcianophilia after sialidase digestion (table 1).
Although isotope incorporation cannot be
demonstrated, a low level of uptake might
be obscured by the adjacent strong autoradiograph. It is of interest in this connection that Aureli et al. ('61) have found
abundant sialic acid in the parotid glands
of a number of species. The low concentration of sialic acid, 2.6 pM/gm of wet
tissue, found here on chemical assay probably accounts for the weak alcianophilia
evident in most of the serous alveoli.
The prominent intercalated ducts of the
Syrian hamster parotid present contradictory results staining red like neutral polysaccharide with the AB-PAS technique
(fig. 24) but showing purple coloration
with the aldehyde fuchsin-alcian blue
(fig. 25) or gray-black with the high iron
diamine-alcian blue method (fig. 26). A
preliminary five minute saponification accentuates to gray and black the staining
with the low and high iron diamine, respectively. The affinity for high iron diamine and aldehyde fuchsin, indicating
sulfated polysaccharide, parallel the autoradiographic evidence for the presence of
sulfated polysaccharide in the intercalated
ducts (fig. 25). These ducts then apparently secrete a sulfated mucopolysaccharide with affinity for aldehyde fuchsin
and the high iron diamine but not for the
other basic dyes employed.
The serous alveoli of the Chinese hamster parotid reveal alcian blue affinity too
weak to be apparent in the AB-PAS stain
(fig. 27) but visible with the iron diaminealcian blue sequence in some alveoli (fig.
28). Intercalated ducts in the parotid of
345
the Chinese hamster like those in the
Syrian hamster, although lacking alcian
blue affinity, color gray-black with the
high iron diamine-alcian blue sequence
(fig. 28). Unlike those in the Syrian hamster these ducts reveal light metachromasia
when viewed wet from the pH 3 azure A
solution. Apparently these ducts in the
Chinese hamster also secrete sulfated
mucopolysaccharide which differs from the
comparable secretion of the Syrian hamster in lacking affinity for aldehyde fuchsin and having azurophilia and (periodate
susceptible) affinity for the mixed diamine
reagent (table 2).
Unlike any other parotid serous alveoli
examined here, those of the guinea pig
afford no indication of the presence of
either neutral or acid polysaccharide. The
interlobular ducts, however, contain numerous goblets (Shackelford and Mapper,
'62a) with a mucin showing histochemical
characteristics of a sulfate free, periodate
reactive sialomucin (table 1) which like
that in the guinea pig sublingual gland
largely lacks affinity for the low iron diamine reagent.
The rabbit parotid serous alveoli lack
basophilia (fig. 29) for all the stains employed except for light gray staining with
the low iron diamine-alcian blue sequence
(table 2). The latter result corresponds
with the moderate isotope incorporation
evident in PO4' autoradiographs (fig. 29).
Intercalated ducts in this gland form an
acid polysaccharide which presumably is
sulfated since they display affinity for high
iron diamine as well as alcian blue in the
A 3 pH 1.0-PAS stain (table 1).
Posterior glossal mucous glands. The
mucous glands of the posterior tongue in
all five species uniformly secrete mucosubstances with histochemical properties
of strongly acidic, sulfated mucopolysaccharide and give, in the four species investigated, correspondingly strong P O 4 ' autoradiographs (figs. 30 to 34) (tables 1 and
2 ) . These secretions differ in the several
species; in general the susceptibility of the
mixed diamine staining to periodate parallels the intensity of the PAS or the periodic
acid-phenylhydrazine-SchifFreactivity except in the Syrian hamster and to a lesser
extent the guinea pig glands. The latter
have periodic acid-phenylhydrazine-Schiff
346
S . S. SPICER AND JALE DUVENCI
reactivity, but show nearly as strong mixed
diamine staining with as without prior
periodate oxidation. On the other hand,
these two properties correlate inversely
with the degree of alcohol resistant metachromasia and the pH extinction value for
azurophilia (table 2). It is noteworthy
that of all the glossal mucous glands
studied, those in the rabbit are the most
periodate unreactive and show the strongest metachromasia and lowest extinction
value for azurophilia (below pH 0.5) but
paradoxically have the least affinity for
alcian blue (table 1). The histochemical
reaction of these glands differs significantly in another respect. Some, such as
those in the mouse, display moderate blue
staining with the aldehyde fuchsin-alcian
blue and high iron diamine-alcian blue
sequences following active methylation and
saponification. Possibly this staining denotes a relatively low level of sialic acid
in sites containing predominantly sulfomucin.
Miscellaneous glands. The posterior
glossal serous glands were examined in the
mouse and Syrian hamster. From their
lack of basophilia and negative P O 4 = autoradiographs, these glands apparently secrete (diastase resistant) neutral polysaccharide in the mouse (fig. 3 5 ) . The glossal
serous glands of the Syrian hamster,
although lacking affinity for aldehyde
fuchsin, azure A or alcian blue show
moderately active S”04’ uptake in autoradiographs (figs. 36 and 3 7 ) and pre-
sumably form a sulfated mucopolysaccharide with masked basophilia.
Exorbital lacrimal glands in the mouse
(table 3) secrete a mucopolysaccharide
with weak alcian blue affinity (fig. 38A)
and PAS reactivity and no azurophilia but,
from autoradiographic evidence, a high
level of sulfation (fig. 38B). The isotope
incorporation concurs with the light purple
and purple-gray staining by the aldehyde
fuchsin-alcian blue and high iron diaminealcian blue sequences, respectively. Brief
saponification enhances the latter.
The secretion of the exorbital lacrimal
gland in the rat (table 3 ) stains blue in
both the AB-PAS (fig. 39A) and aldehyde
fuchsin-alcian blue (fig. 39B) sequences.
This secretion does not combine with alcian blue at pH 1.0, however. The similar
staining by the mixed diamine technique
with and without prior periodate oxidation
corresponds with the lack of PAS reactivity
of this basophilic component. These results taken together with the negative
S3504= autoradiographs (fig. 39B) imply
the presence of a periodate unreactive nonsulfated acid mucopolysaccharide. The
only other secretion with such properties
thus far encountered is that in the submandibular gland in the rat, that of the submandibular gland in the mouse being
roughly similar but with somewhat greater
periodate reactivity. These two secretions
of the rat presumably provide examples
heretofore unencountered of periodate unreactive sialomucin unless some acid group
as yet unknown is present,
TABLE 3
Histochemical properties of secretions in acini of exorbital lacrimal glands
Mouse
0-B
Alcian blue pH 1.O
Alcian blue pH 2.5
0-B
Alcian blue pH 2.5-PAS
2R-P
Diastase-PAS
1-2R
0
Diastase-PAPS
Azure A pH 2.0
0
Azure A pH 3.5
Aldehyde fuchsin-alcian blue
2P
Mixed diamine pH 4.0
2P
CP
Mixed diamine pH 4.0 after periodate
Low iron diamine-alcian blue
%N
High iron diamine-alcian blue
2PN
0-P
Sulfation - high iron diamine-alcian blue
S3504’ incorporation
Abbreviation for methods and colors as in table 1.
1 Chromidial substance at base of cell i
n each species is 2VB.
+++ +
Rat
Hamster
0
B
B
0-R
0
0
0
B
2P
1-2P
-cB
B
CB
0-PN
0
-cB
4P
B
4RP
4R
3R
0
0-CP
F
0-B
0
347
SALIVARY MUCOPOLYSACCHARIDES
The exorbital lacrimal gland in the Syrian hamster (table 3 ) forms an intensely
PAS-reactivemucopolysaccharide with very
little or no basophilia except for faint blue
staining by the aldehyde fuchsin-alcian
blue procedure (fig. 40) (table 3 ) . The
strong periodic acid-phenylhydrazine-Schiff
reactivity of this mucosubstance affords
the only histochemical evidence for acid
polysaccharide and suggests the presence
of acid groups proximal to uic glycols.
This gland shows little if any isotope uptake in S3'00,' autoradiographs which probably are within background level (fig. 40B),
but specific assay reveals a high concentration of sialic acid, comparable to that
conveying strong alcian blue affinity to the
major salivary glands in this species. Thus,
a value of 17 UM of bound sialic acid per
gm of wet tissue was found for the pooled
exorbital lacrimal glands of several Syrian
hamsters. Masking of sialic acid basophilia
in this organ compares with the occluded
basophilia of sulfate esters in the mouse
exorbital lacrimal gland.
In the mixed glands of the pharynx and
palate as well as the larynx and trachea
of the mouse, the AB pH 2.5-PAS method
visualizes red serous demilunes containing
neutral diastase resistant polysaccharide,
together with uniformly blue-purple mucous acini (fig. 41). Whereas sialidase
digestion (fig. 42) removes such alcianophilia from most of the cells of these
mucous acini, the alcian blue staining of
some of the cells resists this treatment.
Either the pH 1.0 AB-PAS technique or a
mild methylation-AB-PAS sequence reveals
the same pattern of blue cells scattered
among a majority of red cells in the mucous acini as is seen with the sialidase-ABPAS sequences. Cells with the same distribution as the sialidase resistant cells
show both selective S3504=incorporation in
autoradiographs and selective purple or
black staining with the aldehyde fuchsinalcian blue or high iron diamine-alcian
blue techniques. Moreover, isotope label
is localized over the purple cells in autoradiographs of aldehyde fuchsin-alcian
blue stained sections (fig. 43). The cells
with the distribution pattern of the sialidase susceptible elements stain blue in the
latter two techniques. Thus, the mucous
forming component of these mixed glands,
like the mucous alveoli in the Syrian hamster major salivary glands, are composed
of a mixture of sulfo- and sialomucin producing cells. These mucous glands contain
acid polysaccharide with similar periodate
reactivity in the sialomucin and the sulfomucin producing cells, since these glands
containing both cell types stain strongly
and uniformly with the PAS and the periodic acid-phenylhydrazine-Schiffmethods
and lose completely their mixed diamine
reactivity following periodate oxidation. It
is to be noted then from the present results
that the mucous glands in the mouse trachea and larynx produce sialomucin predominantly but not exclusively as reported
previously (Spicer and Warren, '60).
Human salivary glands. On the basis
of the experience in animals it appears
possible with histochemical staining, blockage and digestion methods to differentiate
several types of acid mucopolysaccharides
in human salivary glands despite lack of
positive identification of sulfomucins with
radiosulfate. In the human as in the Syrian hamster sublingual gland, sialo- and
sulfomucin forming mucous cells lie intimately intermingled as shown particularly
by staining with the high iron diaminealcian blue and aldehyde fuchsin-alcian
blue sequences or the pH 1.5 azure A
method and by effect on alcian blue pH
2.5-PAS staining of sialidase digestion or
mild methylation. There is marked variation in the proportion of these two secretions and in the acidity and periodate reactivity of the sulfomucin in the different
lobes; in general, cells secreting sulfated
mucopolysaccharides predominate. The
submandibular gland shows, in addition to
sulfated mucopolysaccharide in granules of
mucoserous alveoli, mucous acini predominantly forming sialomucin interspersed in
roughly equal proportions with mucous
acini containing mainly sulfomucin.
DISCUSSION
The frequent PAS reactivity and nearly
constant affinity for basic dyes with high
selectivity for acid polysaccharides encountered in all sites showing strong autoradiographs here, substantiates the already well
established observation that injected S3504=
is incorporated only as sulfate esters in
polysaccharides during the first six hours.
348
S. S. SPICER AND JALE DUVENCI
Conversely, where autoradiographic evidence is available, isotope incorporation
has been demonstrated invariably in sites
with high iron diamine reactivity or strong
aldehyde fuchsin affinity. Of the several
histologic structures which give weak autoradiographs with radiosulfate only two the mouse parotid and Syrian hamster
lingual serous acini - lack definite histochemical staining for sulfomucin.
The secretions formed in the histologic
sites investigated vary so widely when examined by a battery of methods, that each
mucosubstance has a profile of characteristics different in some respects from any
other. This is not surprising considering
the number of different residues known
to occur in mucopolysaccharides and the
variety possible in polymers of these units.
Part of the value of the histochemical approach aside from localizing known substances lies in detecting differences not
otherwise evident and providing evidence
for presence of uncharacterized polymers.
Histochemical methods, although limited
in achieving detailed knowledge of the
various polymers, in some instances permit
specific identification of the components
responsible for the differences observed.
The incorporation of sulfate esters in polysaccharides of numerous mucous or serous
secretions has not been recognized by other
methods but is established by autoradiography and confirmatory specific histochemical staining methods. Comparison of
results by staining methods with those of
the definitive autoradiographic and enzymatic digestion procedures provides a
basis for interpretation of the specificity of
the staining methods, and the present results confirm and extend earlier experience
indicating differential staining of sialo- and
sulfomucins in particular. Thus, in the
high iron diamine-alcian blue or aldehyde
fuchsin-alcian blue sequences, the correlation of black or purple staining with radiosulfate incorporation and of blue staining
with sialidase digestibility establishes the
specificity of these procedures. Further
evidence is provided by the removal of
black or purple but not of blue staining by
the active methylation-saponification sequence which hydrolyzes sulfate esters and
leaves some at least of the sialic acid
groups unaltered. Sulfation of the tissue
sections introducing sulfate esters into all
the tissue polysaccharides affords supportive evidence in that following sulfation
neutral mucopolysaccharides as well as
sialo- and sulfomucins all show exclusive
black (table 1) or purple high iron diamine
reactivity or purple aldehyde fuchsin reactivity (Spicer and Meyer, '60) with these
basic stain sequences.
With available methods it is apparent
that some glands form sialomucin uniformly and others sulfomucin uniformly
whereas some glands produce a mixture of
sulfomucin and sialomucin either in different or the same cells. In the latter group,
however, the histochemical methods do
not distinguish whet her sialic acid residues
and sulfate esters are present in varying
proportions on the same macromolecule or
whether some polymer molecules contain
only sialic acid whereas other neighboring
molecules of the same or a different type
of polysaccharide contain only sulfate. This
is certainly true of the mucous cells in
the submandibular but is perhaps less true
of those in the sublingual gland of the
Syrian hamster. With the high iron diamine-alcian blue technique this sublingual
gland (like mouse pharyngeal mucous
glands) shows all gradations of staining of
a morphologically similar material from
pure blue to blue-black to black in different
cells and even in a single cell, indicating
that the same polymer may have sulfate
and sialic acid in differing proportions.
The uniform periodic acid-phenylhydrazine-Schiff staining and sensitivity of
mixed diamine staining to prior periodate
oxidation in these glands are consistent
with this possibility. The submandibular
gland of the Syrian hamster reveals morphologically different blue and gray particles in the same cell, suggesting the
presence of two different acid mucopolysaccharides here. Since the high iron
diamine or aldehyde fuchsin are darker
stains and may overwhelm the alcian blue
subsequently applied, the combined sequences may not detect a small proportion
of sialic acid in a polymer predominantly
sulfated or a small proportion of sialomucin in a cell forming mainly sulfomucin.
However, evidence for the occurrence of
such a situation is provided by the blue
staining observed with these sequences in
SALIVARY MUCOPOLYSACCHARIDES
certain but not all glossal glands after removal of sulfate with four hour methylation and subsequent deesterification of carboxyls with saponification (table 1 ) .
Histochemical methods also differentiate
mucosubstances on the basis possibly of
the proximal spatial relationship between
anionic charges and periodate oxidizable
vicinal hydroxyls in certain acid polysaccharides. Mucosubstances, presumed from
the periodic acid-phenylhydrazine-Schiff
reactivity and the susceptibility of mixed
diamine staining to prior periodate oxidation, to contain hexose with closely associated acid groups and vic hydroxyls, are
found in both the sialo- and the sulfomucins. From the present results it appears
there are also examples of periodate unreactive acid polysaccharides in both these
categories. The possibility merits consideration that this difference in susceptibility
of mixed diamine staining to prior periodate oxidation reflects attachment of the
acidic groups to hexose in the periodate
sensitive category and hexosamine in the
others.
There is no apparent explanation for the
relative resistance of basophilia to mild
methylation and weak affinity for the low
iron diamine reagent encountered in Syrian hamster and guinea pig sublingual
sialomucin. Also unexplained is the enhancement of alcohol resistant metachromasia, high iron diamine staining or sialidase digestibility observed in the secretions
in certain sites after brief saponification.
Possibly such exposure to alkali decreases
the hypothesized masking effect on basophilia of a cationic copolymer, It is of
interest in this connection that Quintarelli
('62) has demonstrated enhanced affinity
for alcian blue and colloidal iron in several
sites following digestion with pepsin for
the purpose of removing associated masking protein.
Sulfomucins and sialomucins in mucous
glands show certain general differences
from those in serous acini and intercalated
ducts. The latter as a rule have weak
basophilia, staining lightly with the high
iron diamine or aldehyde fuchsin procedures but little if at all with azure A and
alcian blue. Steric hindrance to the dye,
possibly a result of a masking by an associated polycation, seems a more likely
349
explanation of this observation than does
relative sensitivity of these methods, since
in sites containing sialomucin alcian blue
imparts stronger coloration than does aldehyde fuchsin, and the one serous site producing a sialic acid rich polymer - the
Syrian hamster exorbital lacrimal gland displays feeble alcian blue affinity. Notably
these glands contain abundant ribonucleic
acid and protein rich secretion (table 2).
Certainly in a gland which gives one of
the strongest S3'04' autoradiographs observed in the mouse, i.e. the exorbital
lacrimal gland (Spicer et al., '61), masking
accounts for the lack of basophilia rather
than quantity of sulfate groups present.
By coincidence two ribonucleic acid rich
exorbital lacrimal glands display little or
no basophilia, although one, the mouse,
shows a high level of radiosulfate incorporation, and the other, the Syrian hamster, a high concentration of sialic acid.
Quintarelli ('63) has found evidence of
masking of sialic acid carboxyls by basic
proteins.
ACKNOWLEDGMENTS
The author is grateful to Mrs. Jacqueline
G. Henson and Mrs. Susan D. Hash for
valued technical assistance and to Mr. Don
R. Tyson for photomicrographs.
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PLATES
PLATE 1
EXPLANATION O F FIGURES
1A
Mouse submandibular (left) and sublingual glands showing blue staining indicative of nonsulfated acid mucopolysaccharide. In both glands there is failure to
incorporate S3504' except for the sublingual serous demilunes ( t ) the secretion
of which is unstained here but colors red like neutral polysaccharide in sections
stained with the AB-PAS method. Aldehyde fuchsin-alcian blue sequence followed
by S 3 5 0 4 ' autoradiograph. X 630.
1B Like figure 1 A except rat glands. The mucous secretions in both glands stain blue
indicating nonsulfated acid mucouoplysaccharide. Incorporation of radioactive
sulfate is not evident. X 630.
352
2A
Rat submandibular (left) and sublingual glands showing alcian blue affinity of
mucous secretions similar to that of untreated section despite exposure to enzyme.
Four hour sialidase digestion then AB-PAS stain. X 205.
2B
Like figure 2A except section was exposed five minutes to alcoholic KOH prior to
digestion. Following such saponification sialidase removes acid groups in the rat
sublingual (but not submandibular) gland which bind alcian blue so that the
mucous secretion stains red like a neutral instead of blue like acid mucopolysaccharide. X 435.
3A
Rat sublingual gland showing weak orthochromatic basophilia of mucous alveoli
in untreated section. Azure A pH 3.5. X 345.
3B
Like figure 3A except that as a result of a five minute saponification prior to
staining the mucous secretion shows alcohol resistant metachromasia. X 205.
3c
Rat sublingual gland demonstrating loss of azurophilia following digestion of
saponified section. Five minute saponification-four hour sialidase-azure A pH 3.5.
x 325.
4
Submandibular gland of female Syrian hamster showing uniform alcianophilia
and S3504' incorporation throughout the mucous alveoli. Granular tubules (e.g.
upper left) lack basophilia and autoradiographic label. Alcian blue pH 2.5-S3504'
autoradiograph. X 415.
5
Male Syrian hamster submandibular gland. Note (periductal) cells ( T ) which
stain blue in contrast to preponderant cells with gray-purple granules in addition
to a variable degree of diffuse blue staining. The cells showing only affinity for
alcian blue are infrequent to absent in females. Mast cells (bottom center) stain
purple. High iron diamine-alcian blue sequence. x 350.
6
Section adjacent that in figure 5 showing removal by sialidase of alcianophilia
but not high iron diamine affinity. Blue coloration is absent from cells which in
untreated sections (fig. 5 ) stain blue exclusively ( 1' ) principally (lower right)
or inconspicuously. The residual gray-purple staining indicative of sulfomucin
varies in different cells. Five minute saponification - four hour sialidase - high
iron diamine-alcian blue sequence. x 350.
x 210.
7A
Syrian hamster sublingual gland showing uniform staining. PAS.
7B
Syrian hamster sublingual gland. Note uniform staining of gland but selective
and variable incorporation of isotope in some of the cells. Alcian blue pH 2.5
stain then P504' autoradiograph. X 250.
a
Syrian hamster submandibular (lower left) and sublingual glands showing bluepurple staining of submandibular gland alveoli and blue-purple staining of some
and blue of other cells in the sublingual gland. Isotope incorporation demonstrated by the overlying silver grains corresponds in intensity with blue-purple
staining. Mast cells stain purple. Aldehyde fuchsin-alcian blue stain then S3504'
autoradiograph. X 210.
9A
Syrian hamster sublingual gland. Scattered cells stained blue selectively show a
similar distribution to cells incorporating isotope in figure 7B. Mast cells are red.
Alcian blue pH 1.0-safranin pH 1.5 sequence. X 350.
9B
Syrian hamster sublingual gland showing selective brown staining of scattered
cells in a distribution comparable to that of cells secreting sulfomucin (figs. 7B,
8 , 9A, 11, 12). Mast cells stain black. Fortyeight hour periodic acid para
diamine. x 155.
SALIVARY MUCOPOLYSACCHARIDES
PLATE 1
S . S . Spicer and Jale Duvenci
353
PLATE 2
E X P L A N A T I O N OF FIGURES
10
11
12
13
14
15
16
17
18
19
20
Female Syrian hamster submandibular (left) and sublingual glands showing uniform staining. Silver grains overlie submandibular mucous cells uniformly and
scattered sublingual cells. The latter are obscured by the dark stain. Secretions
in granular tubules of submandibular gland (lower left) and demilunes of sublingual stain red like neutral polysaccharide. AB-PAS stain then S3'04' autoradiograph. X 210.
Syrian hamster submandibular (lower left) and sublingual glands showing sialidase susceptibility of the blue basophilia in most cells of the sublingual gland.
Much of the basophilia of secretion in the submandibular gland and part or all
of that in scattered sublingual cells resists sialidase. Autoradiographic label demonstrative of sulfated mucopolysaccharide seen at a higher focus level over this
section is restricted to basophilic areas. The blue areas here correspond in distribution to those with aldehyde fuchsin, pH 1.0 alcian blue and iron diamine affinity
in figures 8, 9A, 12. Sequence of 24 hour sialidase digestion, AB-PAS stain then
s3504' autoradiograph. x 285.
Syrian hamster sublingual gland at the border of enzyme digested area showing
removal of alcian blue affinity but not of iron diamine reactivity i n the digested
area below. As in figure 11 some (blue) cells appear to secrete only sialomucin,
some (black) cells exclusively sulfomucin and others both types of acid mucopolysaccharide. Mast cells between, and serous demilunes capping mucous cells
stain gray to black. Sialidase-high iron diamine-alcian blue. X 200.
Submandibular (above) and sublingual glands of Chinese hamster. Note strongly
acidic metachromatic mucous secretion i n the sublingual gland indicative of
sulfomucin and absence of staining i n submandibular gland. Azure A pH 1.0.
x 345.
Section adjacent to that in figure 13 showing purple stained secretion in sublingual gland. Aldehyde fuchsin-alcian blue. x 223.
Section adjacent to figure 14 showing blue staining in sublingual gland indicative
of acid mucopolysaccharide lacking periodate reactivity. The submandibular gland
secretion stains weakly. AB-PAS. x 340.
Section adjacent to figure 15. Note esentially negative reaction of sublingual
mucin and weak to negative reaction of submandibular secretion. PAS. x 250.
Section adjacent that i n figure 16. Note strong reactivity induced in sublingual
(but not submandibular) gland indicative of sulfate esters on vicinal hydroxyls.
Methylation four hours at 60°C-PAS sequence. x 205.
Male rabbit submandibular gland showing autoradiographic label demonstrative
of sulfomucin over alveoli which stain blue for acid mucopolysaccharide. Granular tubules (e.g. upper right) color red presumably because of content of neutral
mucopolysaccharide. AB-PAS-S3504' autoradiograph. x 630.
Section adjacent that in figure 18. The acid mucopolysaccharide i n mucous alveoli
incorporates isotope as in figure 18 but lacks azurophilia. Chromidial substance at
base of cell and nuclei stain blue. Azure A-pH 3.5-S3504' autoradiograph. X 680.
Female rabbit submandibular gland showing purple-gray staining indicative of
sulfomucin in mucous alveoli and lack of staining (of the neutral mucopolysaccharide) i n the granular tubule at upper right. High iron diamine-alcian blue.
x 270.
,.
Mouse parotid. Note light autoradiographic label from radiosulfate incorporation
despite lack of basophilia. Secretion i n the serous alveoli stains red like neutral
polysaccharide. AB-PAS-S3504' autoradiograph. x 325.
Mouse parotid showing moderate autoradiographic label over serous alveoli but as
in figure 21 lack of basophilia. Aldehyde fuchsin-alcian blue stain S3Q'
autoradiograph. X 325.
Like figure 22 except r a t parotid. Note lack of basophilia of serous alveoli and
occasional overlying silver grains probably not exceeding background level. x 325.
Syrian hamster parotid. Note red staining indicative of neutral polysaccharide i n
intercalated duct (lower left) and light purple basophilic staining of serous alveoli
indicating acid mucopolysaccharide. AB-PAS. X 325.
Section adjacent that i n figure 24. Note that intercalated ducts although lacking
affinity for alcian blue (fig. 24) show affinity for aldehyde fuchsin and correspondingly active S35O4' incorporation. Secretion in serous alveoli which stains
weakly and selectively with alcian blue shows little if any isotope incorporation.
Aldehyde fuchsin-alcian blue-S3'04' autoradiograph. X 375.
Section adjacent that in figure 25 showing high iron diamine reactivity (of
sulfated mucopolysaccharide) in intercalated ducts and weak alcian blue affinity
(of nonsulfated acid mucopolysaccharide) i n serous alveoli. High iron diaminealcian blue. X 325.
Chinese hamster parotid showing absence of alcian blue affinity throughout the
gland and a more magenta coloration of serous alveoli than of intercalated duct
( T ). AB-PAS. X 320.
~
21
22
23
24
25
26
27
354
~
SALIVARY MUCOPOLYSACCHARIDES
S. S. Spicer and Jale Duvenci
PLATE 2
355
PLATE 3
EXPLANATION OF F I G U R E S
28
Section adjacent that in figure 27. Note that as in the Syrian hamster the intercalated ducts stain with high iron diamine although lacking alcianophilia
(fig. 27). Occasional serous alveoli show weak alcian blue affinity. High iron
diamine-alcian blue. X 600.
29
Rabbit parotid showing red staining indicative of neutral mucopolysaccharide
but nevertheless light autoradiographic label over serous secretion. AB-PASS3504'
autoradiograph. x 590.
30
Mouse posterior glossal mucous glands showing purple staining characteristic
of sulfomucin and confirmatory heavy overlying autoradiographic label. Aldehyde fuchsin-alcian b l ~ e - S ~ ~autoradiograph.
O~'
X 630.
31
Like figure 30 except rat tissue. X 630.
32
Like figure 30 except Syrian hamster tissue and stained only with alcian blue at
pH 1.5. x 600.
33
Chinese hamster posterior lingual mucous glands showing blue-purple staining
characteristic of sulfomucin. Aldehyde fuchsin-alcian blue. X 125.
Rabbit posterior lingual mucous glands showing strong metachromasia of the
mucous secretion and heavy overlying silver deposit demonstrative of sulfomucin.
Azure A pH 1.0-S3504' autoradiograph. X 580.
Mouse posterior glossal serous gland. Note red staining of serous secretion and
35
lack of isotope incorporation indicating presence of neutral polysaccharide.
AB-PAS ~tain-S~~O4'
autoradiograph. X 800.
Syrian
hamster
posterior
glossal serous gland. Note red staining of serous secre36
tion indicative of neutral polysaccharide. AB-PAS. X 600.
Section adjacent that in figure 36. Note strong autoradiographic label despite
37
lack of basophilia here or in figure 36. Alcian blue pH 1.5-S3504' autoradiograph. X 600.
38A Mouse exorbital lacrimal gland showing weak basophilia and moderate PAS reactivity. AB-PAS. X 265.
38B Section adjacent that in figure 38A. The secretion normally reveals light purple
coloration with this sequence characteristic of sulfated mucopolysaccharide, but
the heavy overlying silver deposit of the autoradiograph obscures the staining.
Aldehyde fuchsin-alcian blue-S3504' autoradiograph. X 325.
34
39A
39B
40A
Syrian hamster exorbital lacrimal gland. Note intense magenta coloration indicative of neutral polysaccharide in apparent contradiction to the high level of
bound sialic acid demonstrable biochemically. AB-PAS. x 600.
40B
Section adjacent that in figure 40A showing weak affinity for either basic
dye and lack of isotope incorporation except for scattered silver grains not above
background level. Aldehyde fuchsin-alcian blue stain -s"04' autoradiograph.
x 600.
Mouse pharyngeal mixed mucous-serous glands. Note uniform blue coloration of
acidic secretion in mucous glands contrasting with red coloration of neutral
polysaccharide in serous demilunes ( t ). AB-PAS. x 630.
41
42
43
356
Rat exorbital lacrimal gland showing blue coloration indicative of acid mucopolysaccharide lacking periodate reactivity. AB-PAS. x 265.
Section adjacent that in figure 39A. Blue coloration indicative of nonsulfated
acid mucopolysaccharide corresponds with the lack of autoradiographic label.
Some of the purple stained mast cells like this one (left) show light labeling
five hours after isotope injection. Aldehyde fuchsin-alcian blue-Ss504= autoradiograph. X 600.
Section adjacent that in figure 41. Note red staining in some of the mucous cells
resulting from enzymatic removal of the acid groups but retained blue coloration in some cells indicative of sialidase resistant acid residues. Eighteen hour
sialidase digestion-AB-PAS. x 630.
Section adjacent that in figure 42. Blue coloration, preponderant among the
mucous cells and indicative of nonsulfated acid mucopolysaccharide, corresponds in distribution to sialidase susceptible area in figure 41. The purple
staining characteristic of sulfomucin corresponds with overlying autoradiographic label. Aldehyde fuchsin-alcian bl~e-S~~O4'
autoradiograph. x 630.
SALIVARY MUCOPOLYSACCHARIDES
S. S. Spicer and Jale Duvenci
PLATE 3
357
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