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Incorporation of 3H-fucose and the secretion of glycoproteins in the coagulating gland of the mouse.

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THE ANATOMICAL RECORD 21453-60 (1986)
Incorporation of 3H-Fucose and the Secretion of
Glycoproteins in the Coagulating Gland of
the Mouse
LYNN H. SAMUEL AND CHARLES J. FLICKINGER
Department ofdnatomy, University o f Virginia School of Medicine, Charlottesville, VA 22908
ABSTRACT
The coagulating gland of rodents, which is part of the prostatic
complex, secretes components of semen. Although possessing some ultrastructural
features of other exocrine glands, the mechanism of secretion by these cells has been
problematic. In the present study the pathway, kinetics, and mode of secretion in
the coagulating gland of the mouse were studied by light and electron microscope
autoradiography at intervals between 10 minutes and 3 hours after injection of 3Hfucose. The majority of silver grains overlay the Golgi apparatus a t the initial
interval, but in addition, more than a third of the grains were associated with
extremely distended cisternae of the rough endoplasmic reticulum. At later intervals, radioactivity of the Golgi apparatus and the endoplasmic reticulum declined,
while labeling of secretory granules increased greatly. Luminal contents became
labeled 1 hour after administration of precursor. The results indicate that the
pathway for secretion of glycoproteins proceeds through the Golgi apparatus to
secretory granules and the glandular lumen, as in many other cells. In particular,
heavy labeling of secretory granules at later intervals indicates that merocrine
secretion is the most likely mechanism in the coagulating gland. However, the
unusual observation that a significant proportion of grains overlay the rough endoplasmic reticulum at the initial interval raises the possibility that some fucose is
incorporated into glycoproteins in the endoplasmic reticulum, as has been reported
for other cell types with similarly configured endoplasmic reticulum.
The paired coagulating glands of rodents are considered part of the prostatic complex since they arise from
buds derived from the urogenital sinus (Narbaitz, 1974)
and contribute components of semen. The most widely
studied product of the coagulating gland is vesiculase,
which has a role in clotting of seminal vesicle proteins
in the formation of the copulatory plug in the vagina of
the female (Bradshaw and Wolfe, 1977; Notides and Williams-Ashman, 1967), supposedly preventing backflow
of spermatozoa following coitus (Gotterer et al., 1955).
The secretory mechanisms in the coagulating glands
have previously been little studied. Although resembling other exocrine glands in some respects, the coagulating gland epithelium has been reported to have
several unusual ultrastructural characteristics such as
extremely distended rough endoplasmic reticulum (RER)
(Brandes, 1966; Dahl et al., 19731, relatively few secretory granules, and, in some preparations, apical cytoplasmic blebs (Hawkins and Geuze, 1977). Thus, in
addition to the conventional merocrine secretion characteristic of exocrine glands, less common secretory
mechanisms have been proposed including apocrine secretion (Hawkins and Geuze, 1977) and long-term storage of products in the RER (Brandes, 1966). In addition,
the observation that monensin, which arrests transport
through the Golgi saccules in many cell types (Tartakoff,
19831, blocked secretion of only one of two major coagu0 1986 ALAN R. LISS, INC
lating gland secretory products led to the suggestion
that at least one component bypasses the Golgi apparatus (Bartlett et al., 1984).
The present electron microscope autoradiographic
study was undertaken to determine the pathway, kinetics, and mechanism of secretion in the coagulating gland,
and to attempt to relate the morphological characteristics of the secretory organelles in this gland to their
function in the secretory process. Since the luminal contents of the coagulating gland are intensely periodicacid Schiff (PAS)3positive (Brandes and Bourne, 1954;
Aumuller, 1979), H-fucose, a terminal su ar, was used
as a precursor to products of the gland; ‘H-fucose has
the additional merit of being specific for glycoproteins
because it is not metabolized to other compounds (Bekesi
and Winzler, 1967). The amino acid 3H-threonine was
also used as a precursor in a separate experiment reported elsewhere.
MATERIALS AND METHODS
A preliminary study was conducted to determine the
amount of radioactive precursor to be administered.
Either 1.25 mCi or 2.5 mCi of 3H-fucose (Amersham
Corp., Arlington Heights, L,specific activity 27 Ci/
Received March 29, 1985; accepted July 26, 1985.
54
L.H. SAMUEL AND C.J. FLICKINGER
mmole) was injected into the jugular vein of adult male randomly selected areas of the tissue with care taken to
mice. After 30 minutes the coagulating glands were ensure equal representation of apical and basal portions
removed and fixed for 2 hours in half-strength Karnov- of the cells.
The distribution of silver grains was analyzed using
sky’s fixative (Karnovsky, 1965), followed by a 1-hour
postfixation in cacodylate-buffered 1%osmium tetrox- the probability circle method (Salpeter and McHenry,
ide. After fixation the tissue was rinsed, dehydrated in 1973). The radius of a circle a t 8,000 x magnification
a graded series of alcohols and propylene oxide, and with 50% probability of containing the site of origin of
embedded in Araldite. Sections 1pm thick were cut with the disintegration producing a silver grain was calcuglass knives and mounted on glass slides for light micro- lated. The 1.2-cm circle was drawn on a piece of clear
scope autoradiography. The slides were dipped in Kodak plastic and superimposed over each grain on a microNTB-2 emulsion diluted 1:l with distilled water (Pres- graph with the center of the circle placed over the center
cott, 1964). Upon drying, they were stored a t 4°C in of the grain. If a single cellular organelle was included
light-tight boxes for 2-4 weeks. The autoradiographs in the area defined by the circle, that organelle was
were developed with Dektol, fixed, and stained with allotted a single exclusive grain count. If several organAzure 11. The slides were examined with a light micro- elles occupied the space within the circle, each received
scope to evaluate whether the density of label would be an equal fraction of the shared grain. Approximately
sufficient for electron microscope autoradiography. Since 500 grains were analyzed for each animal (yielding about
labeling was light even with the 2.5-mCi sample, a 1,000 grainskime interval). The percentage of grains
higher dose of 3.75 mCi was used in the main over each organelle was determined for each time
experiment.
interval.
In the main autoradiography experiments, adult male
Relative grain concentrations (% grains/% volume)
SW mice were anesthetized with Penthrane and a n in- (Feeney and Wissig, 1972) were also calculated for each
cision was made in the neck to expose the jugular vein. organelle. The percent of the total cell volume occupied
An aqueous solution containing 3.75 mCi of 3H-fucose, by the organelles was determined directly from the miconcentrated to 0.2 ml by evaporation with dry nitrogen, crographs counted for each interval using a point hit
was injected directly into the vein. The skin incision was method (Flickinger, 1974).
closed with autoclips and the animals were returned to
RESULTS
cages. At intervals of 10 minutes, 30 minutes, 1hour, 2
Fine
Structure
hours, and 3 hours following injection two animals were
The low, evenly contoured pseudostratified columnar
killed. The coagulating glands were removed, fixed in
half-strength Karnovsky’s fixative (Karnovsky, 1965)for epithelium of the coagulating gland was characterized
2 hours, and prepared for light microscope autoradiog- by greatly distended cisternae of rough endoplasmic reticulum, which were particularly abundant in the basal
raphy as described in the pilot experiment.
The light microscope autoradiographs were used to half of the columnar cells (Fig. 1).This extensive endoevaluate the intensity of label and quality of fixation of plasmic reticulum was filled with a fine, homogeneous,
the tissue. In addition, the distribution of grains over granular material of moderate electron density. The
the lumen and the epithelium was determined a t each other cytoplasmic constituents, including small mitointerval. Grains were counted over equal areas (5,000 chondria and dense lysosomes, were primarily confined
pm2) for epithelium and lumen on one slide for each to narrow strands of cytoplasm between the cisternae of
animal, using a Zeiss light microscope equipped with a n rough endoplasmic reticulum. The heterochromatic, cenocular grid. Areas were selected for the presence of lu- trally located nucleus was irregular in shape. Although
minal secretory material, which was readily visible in the endoplasmic reticulum was distended, the perinuclear cisterna had a narrow profile as in other cells. The
stained slides, and good tissue preservation.
Blocks meeting the criteria of good fixation and suffi- supranuclear Golgi apparatus (Figs. 1, 2) consisted of
ciently intense labeling were prepared for electron mi- curved, stacked saccules and a n assortment of vesicles
croscope autoradiography. Since luminal labeling was and vacuoles located in the concavity of the stacks. The
heavy a t 1 hour in the light autoradiographs, the elec- contents of the vacuoles ranged from light to moderate
tron microscope study of the secretory pathway was not in electron density. Secretory granules were located in
extended to the 3-hour samples. Thin sections (silver to the apical cytoplasm (Figs. 2,5).
gold interference colors) were cut with a diamond knife
and mounted on copper grids. The grids were dipped in
a 0.05% solution of sodium dodecyl sulfate (SDS),
Fig. 1. Perinuclear region of coagulating gland epithelium. Cisternae
mounted on glass pegs with rubber cement, and coated
the rough endoplasmic reticulum (ER), which are distended with a
with a monolayer of Ilford L-4 emulsion using the loop of
moderately dense material, are the most prominent components of the
method (Stevens, 1966). Undeveloped sample grids were cells. A portion of the Golgi apparatus (GI, including cisternae and
checked during the coating process to ensure that a n vacuoles, is also visible. N, nucleus. x 20,000.
even monolayer was obtained. Coated grids were stored
Fig. 2. Apical part of the epithelium of the coagulating gland. The
in light-tight boxes at 4°C for 9 months and then devel- rough
endoplasmic reticulum (ER) extends into this region, although
oped in Microdol-X for 5 minutes and fixed in Rapid Fix. the cisternae are not as dilated in the basal direction (Fig. 1). Secretory
Background was less than 0.1 silver graid100 pm2 and granules (S) are present near the apical plasma membrane and lumen
was considered negligible. After development, grids were (L). In this and other micrographs, the contrast is low because of the
densities of cytoplasmic matrix, content of the endoplasmic
stained with uranyl acetate followed by lead citrate similar
reticulum, and secretory granules; probably this is the result of the
(Reynolds, 1963). The grids were examined in a n elec- effective retention of proteinaceous components by the glutaraldehydetron microscope, and micrographs were obtained from formaldehyde fixative. G , Golgi apparatus. x 24,000.
56
L.H. SAMUEL AND C.J. FLICKINGER
tight Microscope Autoradiography
At all intervals following injection of 3H-fucose, silver
grains were distributed over the glandular epithelium
in the light autoradiographs. At 10 and 30 minutes
grains were concentrated over the supranuclear region
and at 30 minutes the apical parts of the cells were
heavily labeled. Grains were dense over the lumen and
the apical ends of the cells at 1 hour, while by 3 hours
both cells and lumen were diffusely labeled.
When the numbers of silver grains overlying equal
areas of epithelium and lumen were determined, the
proportion of luminal grains rose from 5.8% and 3.8% a t
10 and 30 minutes, respectively, to 62.2% a t 1hour. This
indicates that secretion of labeled material occurred between 30 minutes and 1 hour after incorporation of the
precursor, but it should be noted that these counts simply compare equal-sized areas. Since they are not adjusted to reflect the volume occupied by the lumen, they
do not furnish a quantitative estimate of the amount of
material secreted.
Electron Microscope Autoradiography
At the initial interval, 10 minutes after injection of
3H-fucose (Figs. 3, 41, almost all the silver grains were
associated with the Golgi apparatus and the RER, nearly
half (49.2%)being attributed to the Golgi apparatus and
38.3% localized over the reticulum (Fig. 6, Table 1).By
30 minutes, however, a large increase occurred in the
number of grains assigned to the secretory granules
(Fig. 6), and label associated with the apical plasma
membrane showed a small rise. The percentage of grains
attributed to the Golgi apparatus was relatively unchanged at 30 minutes, while that associated with the
RER declined. At 1 hour after administration of precursor (when radioactive material was present in the gland
lumen in light autoradiographs) 48.6% of the grains
were associated with the secretory granules (Figs. 5 , 6)
and label attributed to the apical plasma membrane had
increased to 12.5%, while the proportion of grains over
the Golgi apparatus declined to 19.6%. The percentage
of grains attributed to mitochondria was highest a t the
initial interval and decreased with time (Table 1). A
small proportion of grains was located over lysosomes,
basolateral plasma membrane, and the nucleus a t all
intervals. Relative grain concentrations (% grains/% volume) (Table 21, which reflect the relative concentration
of radioactivity in the various compartments, showed
trends similar to those described for the percentages of
grains.
In summary, the Golgi apparatus was most heavily
labeled initially, a substantial proportion of grains also
being associated with the RER. Labeling of the endoplasmic reticulum decreased between 10 and 30 minutes, and a substantial decline in number of grains over
the Golgi apparatus was observed a t 1hour. In contrast,
the percentage of grains and relative grain concentration increased steadily in the secretory granules a t 30
minutes and 1hour.
DISCUSSION
3H-Fucoseas a Precursor
Fucose is considered a n excellent precursor in studies
of glycoprotein synthesis because it is not metabolized
to amino acids or other sugars (Coffey et al., 1964; Bocci
~- un.
.
.
.
and Winzler. 1969: Schachter., 1974). In addition., if
~
~I
~
~
~
~
~~~~~
incorporated, it is readily soluble and thus should be
washed out of the tissue during preparation for electron
microscopy, since it is not bound by glutaraldehyde fixation (Bennett, 1978; Pelletier and Puviani, 1973). Fucose is incorporated primarily into glycoproteins, and to
a lesser extent into glycolipids (Bosmann et al., 1969).
Since glycolipids are usually dissolved by the solvents
used in dehydration of tissue prior to embedding in
plastic, sites of radioactivity within the cell indicate the
presence of glycoprotein products (Bennett, 1978). Studies of labeled fucose as an in vivo pulse indicate that
radioactivity in the bloodstream declines to less than
10% of the initial dose within 10-20 minutes after injection (Coffey et al., 1964; Bocci and Winzler, 1969). The
pulse is not as sharply defined as with amino acid precursors but is usually adequate to produce a definite
wave of radioactivity through the cells, as was observed
in the present study.
Fucose is a terminal sugar in glycoproteins and for
this reason is typically incorporated in the Golgi apparatus (Bennett, 1978; Leblond and Bennett, 1977).From
the Golgi complex, three destinations of 3H-fucose-labeled compounds have been shown in a number of cell
types: secretory products, plasma membrane, and lysosomes (Bennett, 1978; Bennett et al., 1974; Haddad et
al., 1977).
Sites of 3H-FucoseIncorporation
Fucose labeling in the coagulating gland followed a
somewhat different pattern of incorporation from that
usually seen with this precursor. At 10 minutes the
largest number of silver grains, almost 50%, was associated with the Golgi apparatus, as anticipated from
studies on other exocrine glands. However, almost 40%
of the grains lay over the RER. Of these, approximately
75% were “exclusive” grains, for which the endoplasmic
reticulum was the only organelle within the probability
circle. This makes it unlikely that the grains attributed
to the RER were the result of scatter of radioactivity
from other labeled organelles nearby. Thus the results
imply that incorporation of 3H-fucoseinto glycoproteins
occurred in both the Golgi apparatus and the RER. Although this is the pattern expected with a sugar precursor such as glucosamine which is involved in both core
and terminal glycosylation (Schachter, 19741, it is rare
with fucose, which is a specific label for terminal oligosaccharides. Although the finding could represent metabolic changes in the 3H-fucose label, this has been
demonstrated to involve a negligible amount of the fucose administered (Coffey et al., 1964; Bocci and Winzler, 1969).
Incorporation of 3H-fucoseinto components within both
the rough endoplasmic reticulum and the Golgi apparatus has been observed in the hyperstimulated “thyroidectomy” and “gonadectomy” cells seen in the anterior
Figs. 3-5. Electron microscope autoradiographs a t intervals after
administration of 3H-fucose.
Fig. 3. The Golgi apparatus is heavily labeled 10 minutes after
administration of the precursor. Grains also overlie the rough endoplasmic reticulum (ER). x 24,000.
Fig. 4. At 10 minutes after injection of 3H-fucose, silver grains
overlie distended cisternae of rough endoplasmic reticulum. x 26,000.
L.H. SAMUEL AND C.J. FLICKINGER
58
Fig. 5. Many silver grains overlie secretory granules (S) at the 1-hour interval. A small portion of the
lumen (L)is represented. G, CMgi apparatus. X 26,000.
.
50 -
....
....
...
40
30
z
v,
0
&
20
RER
-------- ------
10
I
lO'YlN
3o'MlN
Fig, 6. The percentage of grains assigned to selected organelles of
coagulating gland cells at intervals after injection of 3H-fucose. RER,
rough endoplasmic reticulum; GA, Gold apparatus; SG, secretory
1' HR
granules; APM, apical plasma membrane. Values for all compartments
are shown in Table 1.
59
SECRETION IN THE COAGULATING GLAND
TABLE 1. Percent of silver grains associated with parts of coagulating gland cells at intervals after
administration of 3H-fucose*
Time (minutes)
Oreanelle
RER
Golgi
SG
APM
Lysosomes
B&L PM
Mitochondria
Nucleus
Cytoplasm
Total No. grains
10
30
60
38.3 f 0.05
49.2 k 1.3
1.6 $- 0.8
1.0 0.2
0.5 k 0.05
0.7 $- 0.1
6.2 k 0.6
2.8 & 0.1
0.4 k 0.4
1,347
19.9* f 3.3
47.4 9.9
25.0 k 6.8
4.3* k 0.7
0.2 k 0.2
0.6 f 0.4
1.6* k 0.1
0.8** 2 0.04
0.1 5 0.1
901
16.1** k 1.3
19.6* f 3.0
48.6** f 3.0
12.5* k 2.3
0.2 f 0.2
0.3 k 0.3
1.4* k 0.3
1.3 _+ 0.7
0.0
1,015
*
*
'RER, rough endoplasmic reticulum; SG, secretory granules; APM, apical plasma membrane; B&L PM, basal and lateral
plasma membrane. The means f S.E.M. are shown. The significance of the difference between a given value and that for the
initial (10minute) interval, as determined by a t test, is designated as follows: *P < .05; **P < .01.
TABLE 2. Relative grain concentrations (% grains/%volume) at intervals after injection
of 'H-fucose'
Time (minutes)
Organelle
RER
Golgi
SG
APM
Lysosomes
B&L PM
Mitochondria
Nucleus
CvtoDlasm
10
1.45 f 0.10
3.10 f 0.45
1.24 k 0.10
0.93 & 0.27
0.52 f 0.19
0.57 k 0.03
1.14 + 0.04
0.32 f 0.01
0.01 + 0.01
30
0.68*
2.66
7.55*
2.79
0.33
0.59
0.39**
0.15*
f 0.05
k 0.60
f 0.85
k 0.54
0.33
0.41
k 0.04
k 0.02
0
60
0.70* f 0.04
1.39* f 0.01
14.76* k 2.17
10.39** k 0.19
0.20 k 0.20
0.23 i 0.23
0.26** f 0.06
0.19 0.12
*
0
'Means f S.E.M. are shown. See footnote to table 1 for abbreviations.
pituitary gland after removal of the thyroid or gonads,
respectively (Pelletier, 1974; Pelletier and Puviani,
19731, even though incorporation of 3H-fucose label into
the normal unstimulated somatotrophs and gonadotrophs was limited to the Golgi apparatus (Pelletier,
1974). It has been suggested that label associated with
the RER in the stimulated pituitary cells may be the
result of fucosyltransferases becoming activated in the
endoplasmic reticulum where their synthesis probably
occurs (Pelletier, 1974). It is striking that the extremely
distended RER of thyroidectomy and gonadectomy cells
closely resembles that of the coagulating gland. In the
thyroidectomy and gonadectomy cells, however, a constant level of radioactivity remained in the endoplasmic
reticulum even a t the longest intervals (1 and 4 hours),
either as a result of storage or of slow transport, while
that in the Golgi apparatus evidently progressed to the
secretory granules. In the coagulating gland, label associated with the RER, as well as the Golgi apparatus,
declined with time. This difference may be due to continuous secretion into the lumen in the coagulating gland
of products labeled in both organelles, whereas in the
anterior pituitary secretion is prevented until the cells
are stimulated.
In biochemical assays of fucosyltransferases in rat liver
subfractions, the highest specific activity was associated
with the Golgi fraction but a small amount was also
associated with the rough microsomes (Munro et al.,
1975; Schachter and Roseman, 1980). It should be noted
that the fucosyltransferase acitivity associated with the
mitochondria1 subfraction was even lower than that of
the rough microsomes, yet isolated mitochondria have
been shown to be capable of incorporating monosaccharides into glycoproteins (Bosmann and Hemsworth,
1970). In the present study, a small amount of radioactivity was associated with the mitochondria of the coagulating gland at all intervals, which may be due to
independent glycoprotein synthesis by these organelles.
However, the glycosyl transferases of the male sex accessory glands may, of course, differ from those of liver
since biochemical studies have shown some substances
to occur in these glands in concentrations very different
from any other location in the body (Mann, 1964; Price
and Williams-Ashmann, 1961; Gerhardt et al., 1983).
The Secretory Pathway
The pattern of labeling seen in the coagulating gland
following initial incorporation of 3H-fucose is consistent
with progression of products from the Golgi apparatus
to secretory granules, with subsequent release to the
glandular lumen, as demonstrated in a variety of cell
types (Bennett et al., 1974). The delay seen in decline of
grains associated with the Golgi complex until the 1hour interval may be attributable to labeled material
60
L.H. SAMUEL AND C.J. FLICKINGER
reaching this organelle from the RER between the 10and 30-minute intervals. Alternatively, labeled products
from both the Golgi apparatus and the RER could proceed directly to the secretory granules, with those from
the endoplasmic reticulum reaching the granules more
quickly. However, all the varieties of secretory granules
were concentrated a t the concave pole of the Golgi stacks
and thus appeared to be formed there, while there were
no morphological indications that secretory vacuoles
budded from apical regions of the RER.
At the later intervals after 3H-fucose labeling of the
coagulating gland, grains were attributed to the apical
plasma membrane. Since the surface coats of all cells
contain glycoproteins (Rambourg, 1971; Hughes, 1973;
Luft, 1976), this labeling probably reflects the presence
of fucosylated membrane components. However, some of
these grains could also be due to scatter of radioactivity
from the nearby lumen and apical secretory granules.
The autoradiographic evidence suggests that the primary mode of secretion in the mouse coagulating gland
is merocrine since the secretory granules became very
heavily labeled. Nearly half the silver grains were attributed to secretory granules 1 hour after administration of the precursor, and the relative grain concentration attained a n extremely high value (over 14) at
that time. Conversely, morphological indications of apocrine secretion were few in the present study, since
apical blebs were uncommon and almost all of the profiles of the apical ends of coagulating gland cells displayed secretory granules.
ACKNOWLEDGMENTS
This research was supported by a grant (DCB-8309364)
from the National Science Foundation.
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coagulation, gland, mouse, secretion, glycoprotein, incorporation, fucose
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