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Protein secretion in the rat ventral prostate and the relation of Golgi vesicles cisternae and vacuoles as studied by electron microscope radioautography.

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Protein Secretion in the R a t Ventral Prostate and the
Relation of Golgi Vesicles, Cisternae and Vacuoles, as
Studied by Electron Microscope Radioautography '
CHARLES J. FLICKINGER
Department of Anatomy, School of Medicine, University of Virginia,
Charlottesville, Virginia 22901
ABSTRACT
Proteins, including enzymes such as acid phosphatase, are
among a variety of substances secreted into semen by the prostate gland. The
formation, intracellular transport, and discharge of protein components of prostatic secretion were studied in the rat ventral prostate following an injection of
leucine-3H. Samples were prepared for light and electron microscope radioautography at intervals ranging from four minutes to two hours after the injection.
In samples prepared four or ten minutes after administration of the precursor,
most of the silver grains overlay the rough endoplasmic reticulum. Beginning
30 minutes after the injection, while label associated with the endoplasmic reticulum was declining, the proportion of grains over the Golgi apparatus began
to increase, reaching a maximum in one-hour samples. Secretory vacuoles at
the apical ends of the cells became heavily labeled two hours after administration of the l e ~ c i n e - ~ Labeling
H.
of secretions in the lumen of the prostatic alveoli
was observed in samples taken two hours after the injection. These results indicate that secretory proteins in the prostate are synthesized in the rough endoplasmic reticulum, transported to the Golgi apparatus, and packaged into secretory vacuoles, which move to the apical ends of the cells and release their
contents to the lumen. Additional analysis of the pattern of labeling of different
elements of the Golgi apparatus suggests that some protein is transported sequentially from Golgi vesicles to stacks of cisternae and finally into Golgi vacuoles.
Radioactive secretory proteins move through prostatic cells more slowly than
through the seminal vesicle epithelium of the same animals. The main mode of
protein secretion in the prostate appears to be a merocrine type, since apical
protrusions such as have been suggested to participate in an apocrine form of
secretion were observed infrequently and did not become heavily labeled.
The prostate gland, like the seminal
vesicle, secretes a variety of substances
and contributes significantly to the volume
of semen in most mammalian species
(Price and Williams-Ashman, '61; Mann,
'64). It is of particular interest in medicine because, in contrast to the other sex
accessory organs, it is very succeptible to
disease in men, including carcinoma and
nodular hyperplasia (Robbins, '67). Ultrastructurally, the epithelial cells of the
prostate contain abundant rough endoplasmic reticulum, a large Golgi apparatus,
and prominent secretory granules, and
their development and maintenance is
highly dependent upon androgen (Brandes,
'63, '66; Brandes and Groth, '61, '63;
ANAT. REC., 180: 427448.
Brandes and Portela, '60; Brandes et al.,
'59, '62, '64; Dahl et al., '73; DiDio, '61;
Flickinger, '70, '71a, '74b; Franks, '59;
Groth and Brandes, '60; Harkin, '57a, '57b,
'61, '63; Helminen and Ericsson, '70, '71,
'72a, '72b; Helminen et al., '70; Hruban
et al.. '65; Kanai, '61; Schantz, '64;
Schrodt, '61, '63; Seaman ind Winell, '62;
Triche and Harkin, '71).
The secretion of the prostate in most
species is a slightly acidic fluid that is less
viscid and proteinaceous than that of the
seminal vesicle. It is notable for its content of citric acid and of enzymes, includReceived Jan. 30, '74. Accepted Apr. 2, '74.
1 Supported by contract N01-HD-1-2506 with the
National Institute of Child Health and Human Development.
427
428
CHARLES J. FLICKINGER
ing proteolytic enzymes and phosphatases.
Prostatic acid phosphatase is of importance
clinically because i t is found not only in
semen, but also into the blood of patients
with metastatic carcinoma of the prostate.
The prostate is a collection of many individual small glands and it is divisible in
most species into several lobes, which
differ in structure, histochemistry, and the
character of the secretion (Price and
Williams-Ashman, '61 ; Brandes, '65). The
prostate of the rat comprises ventral, dorsal, and lateral lobes, and the coagulating
gland. The rat ventral prostate has been
extensively studied, probably because it is
the largest of the lobes and samples are
readily obtained without extensive dissection. It contains acid and alkaline phosphatase, although in lower concentration
than in prostates of many other animals
(Mann, '64), as well as other components
of semen such as amino acids and citric
acid (Price and Williams-Ashman, '61 ;
Mann, '64). Many of the enzymes thought
to be secreted by the prostate are normally
found intracellularly and not in extracellular fluids (Mann, '64). It has been proposed that these intracellular enzymes
reach semen as the result of an apocrine
type of secretion in which protrusions of
the apical cytoplasm of prostatic epithelial
cells are shed into the lumen of the gland
(Brandes and Portela, '60; Franks and
Barton, '60; Brandes, '66). Histochemical
studies at the electron microscope level
have localized acid phosphatase within the
Golgi apparatus and in secretory vacuoles,
however, suggesting that it at least is released by exocytosis in a conventional
merocrine type of secretion (Helminen and
Ericsson, '70).
With the exception of the ultrastructural and histochemical studies cited
above, information on cellular events in
secretion in the prostate is limited. Therefore, the present study was undertaken to
furnish basic information on the site of
synthesis, intracellular pathway, and rate
of protein secretion in the rat prostate following an injection of l e ~ c i n e - ~as
H a precursor to secretory protein, as in the case
of the seminal vesicle (Flickinger, '74a).
In addition, an attempt was made to assess
the relative contributions of the proposed
apocrine and merocrine modes of secre-
tion. During the course of the study, an
opportunity was presented to analyze the
passage of secretory protein through the
Golgi apparatus, and the relation between
Golgi vesicles, cisternae, and vacuoles was
examined,
MATERIALS AND METHODS
Adult male white rats two to three
months old were killed by cervical dislocation at intervals of 4, 10 and 30 minutes,
and 1, 2, 5 , and 10 hours after each received an intraperitoneal injection of 5
mCi of le~cine-~H.
Pieces of the ventral
prostate were removed, covered with Axative, and diced into 1-2 mm cubes. Fixation was carried out in ice-cold 1% 0 ~ 0 ,
in 0.1 M phosphate buffer at pH 7.3 containing 50 mg of sucrose per ml. The tissue blocks were embedded and sectioned
according to methods described previously
(Flickinger, '74a).
Light microscope radioautographs were
prepared using Kodak NTB-2 emulsion
according to the method described by Prescott ('64). Electron microscope radioautographs were prepared using Ilford L-4
emulsion by a method modified from that
of Stevens ('66). Details of the preparation
and analysis of both light and electron microscope radioautographs are recorded in
the preceding paper (Flickinger, '74a).
RESULTS
Fine structure
The prostate is a compound tubuloalveolar gland that is lined by a cuboidal or
columnar epithelium. The nucleus of prostatic epithelial cells is round to oval and
is located in the basal part of the cell. The
rough endoplasmic reticulum occupies
approximately half of the volume of the
cell. It is the predominant organelle in the
basal and perinuclear regions (fig. 4 ) and
cisternae extend into the cytoplasm apical
to the nucleus (figs. 3, 6 ) , being found
immediately above the nucleus, lateral to
the Golgi apparatus, and in significant
amounts even in the cytoplasm at the
apical ends of the cells. The cisternal space
of the rough endoplasmic reticulum in the
prostate is distended with a fine, flocculent
material of moderate electron density.
Around the margin of the Golgi apparatus
PROTEIN SECRETION IN THE PROSTATE
there are numerous transitional elements
of endoplasmic reticulum that lack ribosomes on part of their surface facing the
Golgi apparatus (fig. 5).
The Golgi apparatus is found i n the cytoplasm apical to the nucleus. It consists of
three kinds of elements: vesicles, stacks
of cisternae, and vacuoles (figs. 5, 7 ) . The
vesicles are predominantly the smoothsurfaced variety and are about 400 A in
diameter, but some coated vesicles slightly
larger than this are also present. The
stacks are composed of two to five highly
flattened cisternae that usually exhibit a
small degree of curvature. The cisternae
within a stack often are of unequal length,
so that elements of one stack extend from
it and associate i n a n unpredictable fashion with other Golgi cisternae and vesicles.
Golgi vacuoles have a spherical or irregular shape and are scattered about within
the Golgi region i n association with one
pole of the stacks. The vesicles are distributed around the margins of the Golgi
region and in its interior between the
stacks. Protrusions approximately the
diameter of Golgi vesicles project from the
surfaces of transitional elements of endoplasmic reticulum, Golgi cisternae, and
Golgi vacuoles, as if the images represented vesicles fixed i n the process of
budding from or fusing with the other
membranous elements. The vesicles and
cisternae of the Golgi apparatus have a
content of moderate electron opacity. Golgi
vacuoles have a variable content of increased density compared to the other
Golgi elements.
I n the cytoplasm between the Golgi
apparatus and the apical plasma membrane are numerous membrane bounded
secretory vacuoles (figs, 3, 6, 8, 9). There
is a gradient i n the density of their contents. Those found deep in the cytoplasm
have a moderate electron opacity similar
to that of the denser Golgi vacuoles.
Smaller secretory vacuoles found i n rows
at the extreme apical ends of the cells have
a much greater electron opacity. The secretory vacuoles are usually spherical and
have a smooth contour, but in some instances a vacuole is surrounded by a rim
of small vesicles, and protrustions from
the vacuole membrane similar in size to
the vesicles are visible (fig. 9 ) .
429
Mitochondria are located predominantly
in the cytoplasm between anastomosing
cisternae of rough endoplasmic reticulum.
Membrane bounded structures, morphologically identified a s lysosomes on the
basis of their polymorphous content, are
present particularly in the supranuclear
region. The apical surface contains short
microvilli (figs. 3, 6). The lumen of the
prostate is electron-lucent, usually containing a fine flocculent material (figs, 3, 8).
Dome-like protrusions of the apical surface, smooth due to a scarcity of microvilli, are sometimes present. These “blebs”
usually lack most organelles and contain
only cytoplasmic matrix, free ribosomes,
and some endoplasmic reticulum. Spherical masses of cytoplasm with a similar
morphology are observed in the lumen, but
the occurrence of the blebs and spheres
is very variable from one specimen to
another.
The height of the epithelium varies
from one alveolus to another. In alveoli
lined by a tall columnar epithelium, the
rough endoplasmic reticulum is more abundant, the Golgi apparatus larger, and secretory vacuoles more numerous than in
alveoli lined by the shorter cuboidal
epithelium.
Light microscope radioautography
I n all the light microscope radioautographs of ventral prostate, silver grains
were distributed throughout the epithelial
cells, although there was a moderate concentration of label a t the apical ends of
the cells one and two hours after injection.
Material in the lumen of the prostatic acini
became labeled two hours after administration of the precursor. Silver grains were
more numerous over the alveoli lined by a
columnar epithelium than over those lined
by a cuboidal epithelium, and alveoli lined
by the taller epithelial cells were selected
for thin sectioning and electron microscope radioautography.
Electron microscope radioautography
In electron microscope radioautographs
of prostates from animals killed four or
ten minutes after the injection of leu~ i n e - ~ (figs.
H
3, 4 ) , by far the greatest
number of silver grains was over the rough
endoplasmic reticulum. Aside from a small
430
CHARLES J. FLICKINGER
amount of label associated with the Golgi
apparatus, very few grains overlay the
other cellular organelles. By 30 minutes
after injection of the radioactive precursor,
grains became associated with vesicles and
stacks of Golgi cisternae in greater numbers than previously (fig. 5 ) . In samples
prepared one hour after injection (fig. 6 ) ,
the endoplasmic reticulum and Golgi vesicles and cisternae remained labeled, but
radioactive protein had progressed also to
vacuoles in the Golgi region, Finally, in
prostates fixed two hours after injection,
grains were found overlying Golgi vacuoles
(fig. 7 ) , secretory granules at the apical
ends of the cells (figs. 8, 9), and material
in the lumen (fig. 8), while the amount
of label associated with the rough endoplasmic reticulum appeared to be less than
at earlier intervals,
A clearer idea of the pattern of labeling
of the prostatic epithelium can be gained
by considering the percent of silver grains
associated with different structures at intervals following the injection, as shown
in table 1 and figure 1. At the earliest interval, four minutes following injection,
nearly three-fourths of the silver grains
were located over the rough endoplasmic
reticulum. The proportion of grains associated with the rough endoplasmic reticulum
showed a progressive decline in all subsequent samples, but in the next sample,
taken ten minutes after the injection, the
rough endoplasmic reticulum still contained about two-thirds of the grains, Beginning 30 minutes after the injection,
while the proportion of grains over the
endoplasmic reticulum continued to decline, the percent of grains over the Golgi
apparatus began to rise, reaching a peak
of almost 30% of the grains 60 minutes
after injection. Secretory vacuoles began
to exhibit a small amount of labeling one
hours following the injection, and this
labeling rose more rapidly by two hours
after administration of radioactive leucine.
The percent of silver grains over mitochondria, nuclei, cytoplasmic matrix and
other structures fluctuated but remained
relatively low. A small fraction of the
grains was observed over the lumen two
hours after injection.
Relative grain concentrations were calculated by dividing the percent of grains
(table 1 ) by the percent of volume (table
2) for each structure, The highest concentration of radioactive protein was initially ( 4 minutes) associated with the
rough endoplasmic reticulum, and this
concentration declined slowly over the next
two hours. On the other hand, the relative
concentration of radioactivity in the Golgi
apparatus was initially low but it began to
rise rapidly at ten minutes and reached a
maximum at 30 to 60 minutes, declining
thereafter. Although the labeling of secretory vacuoles was unimpressive when expressed as percent of the total, the relative
gain concentration over secretory vacuoles
rose sharply beginning in the one hour
sample and reached a value in the twohour sample that was several times that
attained by any other cell organelle. This
emphasizes that fact that, although the
secretory vacuoles occupied only a few percent of the cell volume, they contained a
high concentration of radioactive protein
in these samples.
Labeling of the Golgi apparatus
Since the radioactivity associated with
the Golgi apparatus in the prostate rose
during the time required for two samples
and then declined in a third sample, it
TABLE 1
Distribution o f silver grains overlying the epithelium o f the prostate
after injection of leucine-SH
% Total grains
Min
4
10
30
60
120
RER
Golgi
73
66
56
49
43
13
Secretory
vacuoles
21
0
1
1
29
22
4
14
12
Lumen
Nuclei
Mitochondria
Matrix,
other
Total
grains
0
2
6
3
4
4
6
3
7
5
4
6
11
11
5
8
200
395
685
687
813
1
1
4
5
PROTEIN SECRETION IN THE PROSTATE
431
\
60
In
40
.E
!
i
8
/-
20
Secret0 ry
*I.*
Lumen
wwmmmwmmwwmwmm~wm.wmmwwmm~*
4
10
30
60
I
120
minutes
4 io
30
60
minutes
Fig. 1 The percent of silver grains overlying parts of the prostate at intervals after
injection of l e ~ c i n e - ~ H
Top,
. endoplasmic reticulum, Golgi apparatus, secretory vacuoles and
lumen; bottom, mitochondria, nuclei, cytoplasmic matrix and other structures.
120
432
CHARLES J. FLICKINGER
TABLE 2
Relative volumes of cell components i n radioautographs of prostatic epithelium
% Cell volume
Min
RER
Golgi
50
55
49
50
50
18
10
13
4
10
30
60
120
17
17
Secretory
vacuoles
Lumen
Nuclei
1
1
2
2
2
5
8
4
4
9
7
Mitochondria
6
7
9
6
6
6
5
8
4
Matrix,
other
13
13
18
13
12
Intersections
counted
965
908
844
869
879
TABLE 3
Relative concentration of silver grains overlying components of the prostate after
%. grains/% cell volume
the injection o f l e ~ c i n e - ~ H
Grain concentration
Min
RER
4
1.30
1.20
1.14
0.98
0.84
10
30
60
120
Golgi
0.79
1.20
1.61
1.70
1.35
Secretory
vacuoles
0.50
1.00
0.50
2.00
7.00
Lumen
0
0.13
0.25
1 .oo
0.50
Nuclei
1.20
1.oo
0.60
0.50
1.oo
Mitochondria
Matrix,
other
0.86
0.79
0.85
0.61
0.38
0.66
0.43
0.78
0.83
0.66
TABLE 4
seemed of interest to try to determine
Distribution
of
silver
grains overlying components
whether the label was preferentially associof the Golgi apparatus in the prostate after
ated with different parts of the Golgi appainjection o f leucine-3H
ratus in any of these samples. Therefore,
% Grains
the silver grains overlying the Golgi appaMin
Cistemal
Grains
ratus at intervals between ten minutes and
Vesicles
stacks
Vacuoles counted
two hours after injection of l e ~ c i n e - ~ H
10
70
20
10
30
were assigned to either Golgi vesicles,
71
30
61
31
8
stacks of cisternae, or vacuoles. Individual
60
53
30
17
100
vesicles are
400 A in diameter and are
120
43
21
36
47
beneath the resolution of electron microscope radioautography, but in the prostate
they occur in groups (figs. 5, 7), and percent of grains over the vacuoles exmany grains could readily be related to ceeded that in the stacks.
these groups of vesicles. The results, exDISCUSSION
pressed as percent of total grains over the
In the earliest sample, taken four minGolgi apparatus, are shown in table 4 and
figure 2. Ten minutes after administration utes after the injection of leucine-sH,more
of the precursor, 70 percent of the grains than 70 percent of the silver grains were
overlay groups of vesicles. Although the located over the rough endoplasmic reticuvesicles retained the highest proportion of lum. This indicates that incorporation of
grains, the fraction associated with the l e ~ c i n e - ~into
H protein took place in the
vesicles declined progressively while that rough endoplasmic reticulum, in accord
over the stacks rose to a maximum in 30- with observations on other protein secretminute and one-hour samples and then ing gland cells (e.g., Siekevitz and Palade,
declined. In contrast, the percent of grains '58; Car0 and Palade, '64; Jamieson and
over the Golgi vacuoles was initially low, Palade, '67a, '71; Whur et al., '69; Nadler
but it rose in the one-and two-hour samples et al., '70; Nakagami et al., '71; Castle et
when the percent in the stacks and vesicles al., '72; Feeney and Wissig, '72). At later
was declining. In the two-hour sample, the intervals, while the percent of grains over
-
PROTEIN SECRETION IN THE PROSTATE
433
6C
In
5
E 4c
[J,
ae
2c
I
10
do
I
30
4
120
minutes
Fig. 2 The percent of silver grains overlying components of the Golgi apparatus at intervals after injection of leucine-SH. The results are expressed as the percent of the total
number of grains that overlay the Golgi apparatus at each interval.
the rough endoplasmic reticulum declined,
the proportion of grains associated with
the Golgi apparatus and secretory vacuoles
increased in turn, suggesting that the
labeled protein was transported to the
Golgi apparatus where i t was condensed
and packaged into secretory vacuoles.
Concentration of the protein in secretory vacuoles apparently occurred, since
labeled secretory vacuoles at the apical
ends of the cell had a denser content than
those close to the Golgi region. Release of
secretory protein began about two hours
after the injection of radioactive leucine.
Thus, the steps in the pathway of secretory protein in the prostate resembled
those in the seminal vesicle (Flickinger,
’74a), but the transport of secretory protein occurred more slowly in the prostate
since labeled secretions were present in the
lumen of the seminal vesicle within 30
minutes but did not reach the lumen of
the prostate until two hours after the
administration of Ieu~ine-~H.Similarly,
proteins labeled with cy~tine-~H
are cleared
more rapidly from the mouse seminal vesicle epithelium than from the coagulating
gland (Morehead, ’69).
It was possible to follow the passage of
a “front” of label through the prostatic
cells, but once a cell organelle became
labeled the intensity of labeling did not
decline as rapidly as in the case of the
seminal vesicles of the same animals
434
CHARLES J. FLICKINGER
(Flickinger, ’74a). For example, by two percent of label associated with the vesihours after the injection the percent of cles was steadily declining, the percent of
grains associated with the rough endo- grains over the stacks of cisternae rose and
plasmic reticulum of the prostate declined then fell. Finally, the proportion of grains
to about half of its initial value, but still over Golgi vacuoles increased. This behavcomprised 43 percent of the total. This ior suggests that the stacks received labeled
suggests that in the prostate newly syn- protein from the vesicles and passed it on
thesized secretory protein may be retained to the Golgi vacuoles. Although the percent
or stored within the cavity of the rough of grains associated with vesicles declined
endoplasmic reticulum to a greater degree throughout the experiment, it remained
than in the seminal vesicle. More detailed higher than that of either stacks or vacuinformation on the availability of the pre- oles, possibly due to the relatively large
cursor at different times throughout the volume occupied by groups of vesicles.
These results suggest that at least some
experiment would be necessary to study
precisely the emptying of the prostatic secretory proteins are transported sequenendoplasmic reticulum. However, since the tially through Golgi vesicles, stacks, and
data on the seminal vesicle and the pro- vacuoles. They do not provide information
state were obtained from the same ani- on the behavior of Golgi membranes durmals, the availability of precursor to both ing the process, but the data are certainly
glands was probably very similar or even consistent with the hypothesis that Golgi
identical, making ,this comparison of the cisternae are formed by fusion of vesicles
two glands reasonable. Furthermore, con- derived from the endoplasmic reticulum at
sideration of the fine structure of the endo- a “forming” face of the Golgi apparatus
plasmic reticulum lends some support to and pass in a train through the stacks,
the idea that more protein is contained undergoing maturation in the meantime to
within the cisternae in the prostate, since emerge at a “mature” face as the memthe rough endoplasmic reticulum cisternae brane surrounding a secretory vacuole
in the seminal vesicle have a narrow (Mollenhauer and Whaley, ’63; Grove et
lumen, but those in the prostate are wider al., ’68; Yunghans et al., ’70; Morr6 et al.,
and are more distended with a flocculent ’ 7 1 ) - It may be noted also that the present
material which probably represents secre- study has not determined whether it is
obligatory for secretory proteins to pass
tory protein.
The mechanism of transport of secre- through the stacks. Electron microscope
tory protein from the rough endoplasmic images suggested fusion (or fission) of
reticulum to the Golgi apparatus in the vesicles with both Golgi stacks and Golgi
prostate is presumed to be by means of vacuoles. Thus, the possibility remains that
small smooth vesicles such as those in- some proteins might by-pass the stacks and
volved in this activity in other cells (Jamie- enter Golgi vacuoles directly as reported
son and Palade, ’67a, ’67b). These vesicles for the unstimulated guinea pig pancreas
are numerous in the Golgi region of the (Jamieson and Palade, ’67b, ‘71b). In
prostate and images suggesting that they addition, the presence of images (fig. 9 )
detach from transitional elements of endo- suggesting that vesicles (of the same or
plasmic reticulum and fuse with Golgi ele- different type) may fuse with secretory
ments are present. The analysis of silver vacuoles (Helminen and Ericsson, ’70)
grains overlying different parts of the Golgi raises the possibility of further addition or
apparatus at different times following in- removal of components of the secretory
jection supports this contention and gives vacuoles before their release from the cells.
Although the general features of the
some indication of the pathway of protein
through the Golgi apparatus. At the earliest pathway of secretory proteins through
interval at which Golgi label was studied, gland cells are now understood, the subthe majority (70% ) of the grains overlay ject of membrane synthesis and turnover
vesicles, suggesting that the vesicles were during secretion and the relation between
the first components of the Golgi region to the membranes of the organelles that parreceive radioactive protein from the endo- ticipate in this transport is controversial.
plasmic reticulum. Subsequently, while the Some investigators propose that mem-
PROTEIN SECRETION IN THE PROSTATE
branes accompany secretory proteins
through the cells, being transferred successively from endoplasmic reticulum to
Golgi apparatus and ultimately to the
plasma membrane (Cheetham et al., '70;
Yunghans et al., '70). This view is supported by studies indicating that in the
parotid gland the synthesis of microsomal
membrane protein occurs concomitantly
with the synthesis of secretory proteins
(Amsterdam et al., '71). In contrast, others
maintain that the different cytoplasmic
membranes (endoplasmic reticulum, Golgi
apparatus, secretory vacuoles, etc.) are
separate and distinct types of membranes
that relate functionally but do not mix
with one another (Meldolesi et al., '71;
Meldolesi and Cova, '71, '72; Bergeron
et al., '73). Thus transport of secretory proteins from endoplasmic reticulum to Golgi
apparatus is visualized as occurring by
means of a shuttle system of vesicles
which return to the endoplasmic reticulum
after depositing their contents in the Golgi
apparatus, and a similar shuttle system of
vacuoles is thought to operate in transport
between the Golgi apparatus and the
plasma membrane. Support is lent to this
view by experiments indicating that stimulation of pancreatic secretion does not
result in a corresponding increase in synthesis of microsomal membrane proteins
(Meldolesi and Cova, '71), and the observation that membrane proteins of the endoplasmic reticulum, Golgi apparatus, and
zymogen granules turn over much more
slowly than do secretory proteins (Meldolesi, '74), suggesting that membranes are
indeed reutilized. While cell fractionation
studies have been useful, radioautographic
investigations such as the present study
have not contributed much to solving this
problem, in part because the resolution of
electron microscope radioautography is not
sufficient to distinguish between the labeling of membranes and that of their contents in endoplasmic reticulum, Golgi apparatus, and secretory vacuoles. On the other
hand, accumulation of radioactivity in the
plasma membrane as the result of incorporation of many labeled secretory vacuole
membranes might be detectable but was
not observed, possibly because, as pointed
out by numerous authors, there must be a
mechanism for removal of the excess
435
plasma membrane contributed by the addition of many secretory vacuole membranes (Palade, '59; Fawcett, '62; Amsterdam et al., '71; Jamieson and Palade, '71a,
'71b; Meldolesi and Cova, '71; Holtzman
et al., '73; Geuze and Poort, '73). The
nature of this mechanism is not definitely
known, but the excess plasma membrane
could either be degraded (Fawcett, ' 6 2 ) by
digestion in lysosomes following endocytosis (Holtzman et al., '73; Geuze and
Poort, '73) or intact patches of membrane
could be returned to the Golgi apparatus
for reutilization (Palade, '59; Amsterdam
et al., '69; Jamieson and Palade, '71b).
It has been suggested that the formation
of apical "blebs" or protrusions on some
prostatic epithelial cells might precede the
detachment of the apical ends of the cells
in a form of apocrine secretion (Brandes
and Portela, '60; Franks and Barton, '60;
Brandes, '66). Protrusions were uncommon
in our samples, and silver grains did not
appear to be concentrated over them at
any time. Although protrusions were not
observed with sufficient regularity to permit an analysis of the relative grain
concentration, this qualitative evaluation
coupled with the extremely high relative
concentration of radioactivity that was
present in membrane-bound secretory
granules suggests that the principal mode
of protein secretion in the prostate is the
merocrine variety. The proposed apocrine
mode, if it operates a t all, seemingly
accounts for a relatively small proportion
of prostatic protein secretion. Histochemical studies (Helminen and Ericsson, '70)
have led to a similar conclusion with respect to the secretion of acid phosphatase.
ACKNOWLEDGMENTS
The author is indebted to Miss Sharon
Odum for technical assistance, and to
Dr. James Simmons for assistance in performing injections of radioactive materials.
LITERATURE CITED
Amsterdam, A., I. Ohad and M. Schramm 1969
Dynamic changes in the ultrastructure of the
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~.
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PLATE I
EXPLANATION O F FIGURES
3-4
438
In samples of ventral prostate prepared for radioautography four or
ten minutes after the injection of l e ~ c i n e - ~ H
the
, great majority of
silver grains are located overlying the rough endoplasmic reticulum
(ER). There are a few scattered silver grains over the Golgi apparatus (G) and mitochondria, but secretory vacuoles ( S ) and the
lumenal secretions ( L ) are not labeled at this time. N, nucleus.
Figure 3 illustrates the apical end of prostatic epithelial cells, while
figure 4 depicts the basal region. Both figures are from a sample
fixed four minutes after injection. Figure 3, x 19,000. Figure 4,
X 16,000.
PROTEIN SECRETION I N THE PROSTATE
Charles J. Flickinger
PLATE 1
439
PLATE 2
EXPLANATION O F FIGURE
5
440
In specimens prepared 30 minutes after the administration of the
precursor, label remains associated with the rough endoplasmic reticulum (ER), but the number of silver grains overlying the Golgi apparatus has increased greatly. Note that most of the silver grains overlying the Golgi elements are associated with groups of small vesicles
( A ) or stacks of cisternae (B), and few Golgi vacuoles ( C ) are
labeled (cf. fig. 7). x 21,000.
PROTEIN SECRETION IN THE PROSTATE
Charles 5. Flickinger
PLATE 2
441
PLATE 3
EXPLANATION OF FIGURE
6 In samples of prostatic epithelium prepared one hour after administration of radioactive leucine, many silver grains overly the Golgi
apparatus ( G ) , as well as the endoplasmic reticulum (ER). Grain
counts show that the maximum labeling of the Golgi apparatus is
attained at this stage. Secretory vacuoles ( S ) and the lumenal secretions (L) have not yet become significantly labeled. x 19,000.
442
PROTEIN SECRETION IN THE PROSTATE
Charles J. Flickinger
PLATE 3
443
PLATE 4
EXPLANATION OF FIGURE
7
444
The Golgi apparatus remains labeled two hours after injection,
although grain counts show that the proportion of grains associated
with it has declined. The type of Golgi element labeled differs from
earlier intervals, however. Labeled vesicles and stacks of cisternae
predominate at earlier stages and Golgi vacuoles are lightly labeled,
(fig. 5 ) , but i n the two hour samples the number of grains associated with Golgi vacuoles has increased noticeably. A, groups of vesicles; B, stacks of cisternae; C, vacuoles. Note the differences in density among Golgi vacuoles, probably reflecting differing degrees of
concentration of secretory product. x 25,000.
PROTEIN SECRETION IN THE PROSTATE
Charles J. Flickinger
PLATE 4
445
PLATE 5
EXPLANATION OF FIGURES
446
8
Secretory vacuoles ( S ) in the apical parts of prostatic epithelial cells
become labeled two hours after injection of l e ~ c i n e - ~ HIn. addition
some silver grains overly the lumen of the gland (L). The intensity
of labeling of the cells, especially of secretory vacuoles, remains high
in the two hour sample, indicating that release of secretory proteins
has just begun. x 18,000.
9
Labeled secretory vacuoles (S) are present in the apical region of
prostatic epithelial cells two hours after injection. The secretory
vacuoles have a dense content and are surrounded by small vesicles
that appear to be in the process of fusing with or detaching from
the vacuoles, suggesting that the content of the vacuoles is modified
after the vacuoles leave the Golgi apparatus. A protrusion from the
surface of a piece of rough endoplasmic reticulum (arrow) suggests
that some of these vesicles might arise from the endoplasmic reticulum in the apical parts of the cells. L, lumen; ER, endoplasmic
reticulum. x 27,000.
PROTEIN SECRETION I N THE PROSTATE
PLATE 5
Charles J. Flickinger
447
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