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Effects of estrogen and androgen on the ultrastructure of secretory granules and intercellular junctions in regressed canine prostate.

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THE ANATOMICAL RECORD 197~111-132 (1980)
Effects of Estrogen and Androgen on the
Ultrastructure of Secretory Granules and
Intercellular Junctions In Regressed
Canine Prostate
FREDERICK B. MERK, IRWIN LEAV, PAUL W.L. KWAN, AND PETER
OFNER
Departments of Pathology, Anatomy, and Urology.Tufts University School of
Medicine, Boston, Massachusetts, 0211I ; Laboratory of Pharmacology,
Harvard School of Dental Medicine, Boston, Massachusetts, 021 15; and Steroid
Biochemistry Laboratory, Lemuel Shattuck Hospital, Boston, Massachusetts,
02130
ABSTRACT
Epithelial cells in the prostate of the castrated or hypophysectomized dog were studied by thin-section and freeze-fracture electron microscopy to
determine in vivo responses to estradiol-17P 17-cyclopentylpropionate (ECP) and
testosterone cyclopentylpropionate (TCP). Particular attention was given to
changes in specific organelles and intercellular junctions that might reflect hormone action. The few secretory granules that remain in the regressed epithelium
(vestigial granules) serve as markers of prior androgen responsiveness. Pharmacologic doses of ECP caused regressed glandular cells to acquire a novel phenotype. Characteristic features of these estrogen-modified glandular (EMG)cells are
newly formed secretory granules and tonofilament bundles that coexist with vestigial granules, thus demonstrating bipotentiality of response. Glandular cell-tight
junctions appear unaltered by the endocrine manipulations. Although EMG cells
have squamous cell features, tight junctions remain intact. Desmosomes in the
canine prostate are dimorphic and are classified 70F and l O O F according to the
width of the filaments that converge on the dense plaques. In intact dogs, l O O F
desmosomes are associated with basal-reserve cells, whereas only the 70F variety
is found between glandular cells. TCP treatment does not alter this distribution.
Following ECP administration, both 70F and l O O F desmosomes are present between EMG cells. The coexistence of newly formed secretory granules and tonofilaments of lOOF desmosomes in the same EMG cell represents estrogen-induced
bidirectional differentiation. Our findings indicate that androgens and estrogens
are individually capable of controlling the expression of secretory granules and
desmosomes. In intact animals, male and female sex hormones may act in concert
to direct epithelial cell differentiation of the prostate.
Differentiation and function of prostatic cells
have traditionally been considered to be exclusively androgen-directed processes. It is now
well documented that in a number of species
the gland responds to pharmacologic doses of
estrogens by undergoing a proliferative change
termed squamous metaplasia (Triche and
Harkin, 1971;Kroes and Teppema, 1972;Ofner
et al., 1974;Kjaerheim et al., 1974;Helpap and
Steins, 1975; Hohbach, 1977). Although unphysiologic blood levels of female hormone are
required to induce the squamous metaplasia,
the occurrence of this abnormal differentiation
of the prostatic epithelium suggests that estro-
0003-276X/80/1972-0111$03.800 1980 ALAN R. LISS, INC.
gen action may also be involved in androgendependent normal epithelial growth and in
promoting growth in benign prostatic hyperplasia and prostatic carcinoma. Estrogen receptors have been identified in the cytosols of
hyperplastic (Hawkins e t al., 1976) and
carcinomatous (Bashirelahi and Young, 1976)
human prostate.
With these considerations in mind, we focused our attention on the effects of estrogen on
prostatic epithelial cell differentiation and the
Received February 14, 1979; accepted January 4, 1980
111
112
F.B. MERK, I. LEAV, P.W.L. KWAN, AND P. OFNER
17p-hydroxysteroid pathway of testosterone
transformation, characteristic of androgen
metabolfsm by normal prostatic epithelium.
The dog was chosen as our experimental model
because it shares with man the propensity to
develop age-related benign prostatic hyperplasia (Berg, 1958) and prostatic carcinoma
(Leav and Ling, 1968).To avoid the interactive
effect of endogenous androgens and pituitary
hormones with those of female sex steroids, we
have recently utilized castrated or hypophysectomized dogs to characterize direct estrogen
actions on the prostate (Leav et al., 1978). Our
results show that the pharmacologic doses of
estradiol-17P 17-cyclopentylpropionate(ECP)
employed, not only cause basal-reserve cell proliferation and the attendant squamous metaplasia but also stimulate regressed glandular
cells, with accompanying major alterations in
prostatic androgen and estrogen metabolism.
The regressed glandular cells, which had been
responding as tall columnar epithelium to
endogenous androgen prior to castration or
hypophysectomy, undergo redirected differentiation in response to estrogen treatment.
We have designated this cell type “estrogenmodified glandular” (EMG) cells (Leav et al.,
1978).
Published reports concerning prostatic
ultrastructure have dealt primarily with
hormone-induced intracellular events. In the
present study emphasis is placed on sex
hormone-mediated alterations in cytoplasmic
and cell-surface components associated with
intercellular junctions. These structures,
which represent a significant cohesive element
in the organization and development of normal
tissue (Staehelin, 1974;McNutt, 19771, are frequently modified during pathologic processes
including neoplastic transformation (McNutt
and Weinstein, 1973; Weinstein et al., 1976).
Our objective has been to determine the effects
of hormonal manipulations on the ultrastructure of epithelial cell junctions in the canine prostate. Following the reorganization of
prostatic acini that accompanies estrogeninduced metaplasia, we find that some desmosomes between EMG cells differ from those between glandular cells, but that tight junctions
appear unchanged.
MATERIALS AND METHODS
Thirteen German Shepherd crossbreeds
ranging from 3 to 5 years of age were utilized in
this study. The experimental dogs were treated
according to a protocol described in detail elsewhere (Leav et al., 1978). In brief, all experi-
mental dogs were conditioned for 2&25 days
after purchase from a licensed dealer. Each of
the animals was given a complete physical examination including rectal palpation of the
prostate. In no case was the prostate abnormally enlarged.
Androgen depletion was accomplished either
by castration (Lacroix, 1959) or by hypophysectomy (Markowitz et al., 1964).Levels of circulating 17p-hydroxy-C19-steroids (testosterone plus 5a-dihydrotestosterone, not separated) and circulating estrogens (estradiol-l7p
and estrone, separately) were monitored before
and at varying intervals after surgery. The
plasma was stored a t -20°C until radioimmunoassay (RIA) could be performed (Collins
et al., 1972). Estradiol-17p 17-cyclopentylpropionate (ECP) was administered to the dogs
by intramuscular injection and testosterone
cyclopentylpropionate (TCP) was given in the
same manner. Both compoundswere purchased
from the Upjohn Co., Kalamazoo, Michigan.
We examined prostate glands taken from
seven groups of dogs: 1)two intact untreated
animals (dogs # 1and 2); 2) two animals killed
four weeks after castration (dogs #3 and 4);
3) two animals, one killed two weeks, the other
four weeks after hypophysectomy (dogs #5 and
6, respectively); 4) two castrated animals that
each received a first injection of ECP (8 mg)
four weeks after orchiectomy, the second 8 mg
dose one week later and were killed one week
after the second injection (dogs #7 and 8);
5) two castrated animals that each received one
8 mg injection of ECP two weeks after orchiectomy and were killed one week later (dogs #9
and 10); 6) two hypophysectomized animals
that each received a first injection of ECP (8
mg) two weeks after the operation, a second
8 mg dose one week later and were killed one
week after the second injection (dogs #11 and
12);7) one hypophysectomized animal that received a 600 mg dose of TCP two weeks after
the operation and was killed 56 hours later (dog
#13). Our results with the 13 animals are outlined in Table 1.
The dogs were weighed and then killed by a n
overdose of sodium pentobarbital. The prostate
glands were quickly excised and weighed and
the tissues were divided for various experimental procedures including thin-section and
freeze-fracture electron microscopy. Specimens
of sellae turcicae were taken from hypophysectomized dogs and decalcified. Sections were
prepared for histologic examination to verify
completeness of the hypophysectomies.
Tissues for thin-section electron microscopy
113
EFFECTS OF SEX STEROIDS ON CELL JUNCTIONS
TABLE 1. Concentration ofplasma 17phydroxy-Cl9-steroid and weights ofprostates from
intact and treated dogs
Dog #
Protocol’
1
2
3
4
5
6
7
8
9
10
11
12
13
Intact
Intact
Castrate
Castrate
Hypox
Hypox
Castrate-ECP
Castrate-ECP
Castrate-ECP
Castrate-ECP
Hypox-ECP
Hypox-ECP
Hypox-TCP
Pre-ablation
Post-ablation
17p-hydroxy-C,,-steroidt(ng%*)
-
110
200
70
150
135
75
70
400
130
165
135
-
20
< 10
< 10
12
12
12
< 10
< 10
< 10
15
14’
Prostate
(g)
25.0
38.0
10.5
7.0
4.0
6.0
18.0
8.0
11.4
11.0
11.0
15.0
16.5
‘Refer to Materials and Methods.
17P-hydroxy-C,,-steroidincludes testosterone and 5rr-dihydrotestosterone. The latter steroid cross-reads 56% with testosterone at 50% displacement. Cross-reactivity of other Cl.-steroidsis 3% or less.
*ng/100 ml plasma.
‘RIA value three days before TCP treatment.
t
were fixed by immersion in 2.5% glutaraldehyde-Wo paraformaldehyde solution in 0.1 M
phosphate buffer (pH 7.3) for three hours a t
room temperature. They were then rinsed in
the same buffer overnight, postfixed in 1%
osmic acid in 0.1 M phosphate buffer for one
hour at 4”C,dehydrated with a n ethanol series,
and embedded in Epon 812. Silver sections
were cut using a diamond knife and stained
with uranyl acetate followed by lead citrate. In
order to avoid biased sampling, the sections
from each prostate were obtained from not
fewer than four Epon blocks. The evaluation of
desmosomes was carried out a t a final magnification of 200,000 x . Desmosomes were not included in the data unless both dense plaques
appeared distinct. The widths of desmosomal
filaments and plasma membranes were measured on the original plates and on prints enlarged exactly 2.5 X, using a Bausch & Lomb
magnifying reticle. Illustrations of thinsectioned material were printed on highcontrast paper to enhance delineation of the
filaments.
Specimens for freeze-fracture electron microscopy were prepared according t o the
method of Moor and Miihlethaler (1963).Tissue
blocks 1 mm3 that had been fixed in 0.1M
cacodylate-buffered Wo glutaraldehyde and
stored in the same buffer were soaked overnight in 20%0 glycerol-0.1 M cacodylate. They
were quenched in Freon 22 (cooled with liquid
nitrogen) and fractured in a Balzers model
BAF’301 freeze-etch device (Balzers High Vacuum Corp., Hudson, New Hampshire). Plati-
num-carbon replicas were cast a t -115°C or
-100°C and were subsequently cleaned in a sodium hypochloriteKOH mixture. All specimens were examined in a Philips 300 electron
microscope which was calibrated with a carbon
grating having a spacing of 0.463 pm per line.
The terminology of Branton et al. (1 975) is used
in descriptions of the freeze-fracture replicas.
RESULTS
Support o f experimental data
No pituitary tissue was observed in sections
of sellae turcicae from the hypophysectomized
dogs. As a consequence of castration or hypophysectomy, levels of circulating 17phydroxy-C19-steroids declined sharply and in
many instances approached the sensitivity
limits (10 ng%) of our RIA (Table 1).No significant differences in the reduced levels of male
sex steroids were apparent when the two ablative procedures were compared. The RIA values
were obtained immediately prior to ablation,
just before hormone treatment and a t sacrifice.
Levels of plasma estradiol-17p ?nd estrone
were also monitored. Following either ablative
procedure, titers of these hormones decreased
about 3&50%0.After ECP administration, they
were increased 200-500% above preablation
levels assuring us that our data represent a
bona fide estrogen effect.
Intercellular junctions in canznp prostate
A terminal bar or “junctional complex” (Farquhar and Palade, 1963) is located between
prostatic glandular cells a t the lirrninal poles
114
F.B. MERK, I. LEAV, P.W.L. KWAN, AND P. OFNER
(Figs. 1-8,14,15, and 18).The elements of each
complex generally appear in the following
order: a distally located tight junction (zonula
occludens), an intermediate junction (zonula
adherens), and a proximally oriented desmosome (macula adherens).
Two types of desmosomesjoin epithelial cells
in the canine prostate. Both types are present
either as a component of thejunctional complex
or as independent structures along the plasma
membrane. We classify them “100F and “ 7 0 F
according to the criteria outlined by McNutt
and Weinstein (1973).These classifications depend on the width of the filaments (100F or
70F) associated with the dense plaques
(McNutt and Weinstein, 1973). We find that
lOOF desmosomes, which frequently have a
long profile (Fig. 131, are associated with distinct 9-10 nm wide tonofilaments (Fig. 16).The
tonofilaments often extend deep into the cytoplasm, giving the desmosome a “shaggy” appearance (Fig. 16). However, 70F desmosomes
have short, fine filaments that are 5-7 nm in
width (Fig. 8) or are indistinct (Fig. 14). The
70F desmosomes are generally smaller than
the lOOF variety and they usually have a
“square” appearance because the total length of
both sets of filaments, extending in opposite
directions into adjacent cells, is about equal to
the width of the junctional profile (Figs. 8 and
14). Although the type of desmosome cannot be
positively identified at low magnification, the
characteristic square or shaggy appearance is
often apparent (Figs. 1-3).
(RER) and lack secretory granules (Fig. 1).The
other population consists of well-differentiated
columnar (glandular) cells. They contain
abundant RER and Golgi membranes, but the
most prominent feature in the cytoplasm is
many membrane-bound secretory granules
(Fig. 1).The average diameter of the largest
profiles (presumably granules that have been
sectioned through the equator) is 1.05 i 0.17
Pm.
Following either castration or hypophysectomy there is atrophy of glandular epithelium in the prostate (Fig. 2). Because morphological responses to both forms of ablation
are essentially the same, they are described
together. After ablation, no change is detected
in the architecture of basal-reserve cells. However, glandular cells have increased nuclear to
cytoplasmic ratios because of loss of cytoplasmic volume and the nuclei appear pleomorphic. There is a n increase in the number of
autophagic vacuoles and lytic-dense bodies.
Marked reductions in the amounts of RER,
Golgi membranes, and secretory granules are
observed. The few secretory granules that remain (vestigial granules) retain their characteristic appearance but some have undergone
extraction artifact. The extracted material is
presumably lipid, which is a major constituent
of canine prostatic secretory granules
(Gouvelis et al., 1971). Vestigial granules are
found either sequestered in autophagic vacuoles or free in the cytoplasm (Fig. 2).
Ultrastructural comparison of normal and
Intercellular junctions in acini of intact
regressed glandular cells
and ablated dogs
Two populations of epithelial cells are found
Typical junctional complexes (Farquhar and
in acini of intact-untreated dogs (Fig. 1).One of Palade, 1963) are found between glandular
them is the undifferentiated basal-reserve cell cells of the intact dog prostate (Figs. 1and 14).
(Timms et al., 1976) that is usually oriented Freeze-fracture replicas of normal tissue reveal
with it long axis parallel to the basement that the tight junctions are complete fibrillar
lamina. Basal-reserve cells have scant networks, consisting of 5-8 intramembranous
amounts of rough endoplasmic reticulum strands. Type A-1 gap junctions (Staehelin,
Fig. 1. Portion of a prostatic acinus from a normal intact dog (#l). Basal-reserve cells (B), which are located at the periphery
of the acinus, appear inactive. Secretory (glandular)cells extend from the basal lamina (BL) to the lumen (L). They contain
moderate amounts of rough endoplasmic reticulum (RER) and numerous secretory granules. A junctional camplex (JC) is
present at the apical-lateral surfaces of two cells and its proximal component is a small “square”desmosome (arrow).
x 13,700.
Fig. 2. Acinus from a 4-week castrate(control) dog (#3). Involution ofthe glandular cellsis accompanied by an appearance
of secondary lysosomes and by reduction in the number of cytoplasmic organelles including secretory granules. A small
numberofvestigial granules remainand some of them are incorporatedinto developing-autophagicvacuoles (AV).Although a
few vestigial granules have undergone extractionartifacts (longarrows), they retain the appearance of the secretory granules
found in intact animals. Considerable cytoplasmic debris is present in the lumen. Junctional complexes, which include small
“square” desmosomes, are similar to those of intact untreated dogs (short arrows). x 7,000.
EFFECTS OF SEX STEROIDS ON CELL JUNCTIONS
115
116
F.B. MERK, I. LEAV, P.W.L. KWAN, AND P. OFNER
EFFECTS OF SEX STEROIDS ON CELL JUNCTIONS
1974) appear on the P-fracture face as relatively small aggregates of subunits (30-50 particles). Similar observations have been made in
prostates of other species (Sinha and Bentley,
1975; Sinha et al., 1977).
Both 70F and lOOF desmosomes are present
in thin-sectioned prostatic epithelium obtained
from intact dogs. From six blocks we examined
thin sections that included profiles of more
than 25 acini, and found that basal-reserve
cells are joined to glandular cells almost exclusively by l O O F desmosomes (Fig. 13). In contrast, an examination of 105 desmosomes between glandular cells provided no clear indications that any are the 100F variety. The fine
filaments, associated with desmosomes of the
junctional complexes,usually appear indistinct
and form a filamentous mat that appears similar to the mats associated with intermediate
junctions (Fig. 14). Serial sections of the 70F
desmosomes fail to reveal any contact with
10-nm filaments. Compact fascicles of tonofilaments, which are very electron-dense, occasionally appear in the cytoplasm of glandular
cells but they are not found in contact with 70F
desmosomes. We have found that the only
desmosomes with distinct tonofilaments in
glandular cell cytoplasm are those also joined
to basal-reserve cells.
Based on thin-section and freeze-fracture
data, the ultrastructure of tight and gap junctions within regressed acini appears essentially the same as in intact untreated animals.
Tight junctions with the full complement of
junctional strands and small gap junctions persist between the regressed glandular cells. Similar observations have been made in other
species (Tice et al., 1975; Sinha and Bently,
1975). Following ablation-induced regression,
100F desmosomes join basal-reserve cells and
glandular cells. Although the 70F desmosome
continues to be the predominating form between glandular cells (Fig. 151, a few lOOF
desmosomes now appear (18out of 112 counted,
or 16%).
Response of regressed prostatic acini
to estrogen stimulation
No cytological differences have been observed between the ECP-stimulated epithelia
of castrated or hypophysectomized dogs. Following estrogen administration, squamous
metaplasia is evident in most of the acini. In
addition, the regressed glandular cells are also
stimulated. However, the extent of response in
both types of cells is not uniform and varies
from acinus to acinus.
117
Three types of epithelial cells are present in
the acini (Leav et al., 1978).The basal-reserve
cells are somewhat enlarged, but their cytoplasmic organelles do not appear modified by
the female hormone. Squamous cells, which
appear elongated, are fairly uniform in appearance (Fig. 3). They are generally located in the
periphery of the acini and, as squamous metaplasia becomes advanced, these cells become
stratified. Squamous cells are characterized by
large perinuclear bundles of tonofilaments but
secretory-, keratohyalin-, and membranecoating granules are absent. The basal-reserve
cells and their squamous-cell progeny are
joined to one another almost exclusively by
l O O F desmosomes. The third type is the pleomorphic estrogen-modified glandular (EMG)
cells (Fig. 31, which form a single layer. They
combine features of secretory and squamous
cells. The EMG-cell contains numerous tonofilaments that are scattered or arranged in
bundles. Coexisting with the tonofilaments are
abundant RER, Golgi membranes, and large
numbers of secretory granules (Fig. 4). Two
distinct populations of membrane-bound
granules are frequently found in the same
EMG cell (Figs. 5 and 6). One type is easily
recognized as the vestigial granules. They are
relatively sparse and are encountered in various stages of lysosome-induced degradation
(Fig. 5). These granules, which have the same
texture and dimensions as those found in glandular cells prior to ablation, can be distinguished from lipid droplets by virtue of their
limiting membrane (Figs. 5 and 6). They are
consequently considered to be structures that
were largely formed under the influence of androgen. The other type is very numerous. They
are usually about % the size, less “electrondense,” and more peripherally located than the
vestigial granules (Figs. 5 and 6). Since the
small granules were never found in regressed
acini, their development must be considered to
be an estrogen-mediated phenomenon.
Intercellular junctions between the modified
glandular cells of estrogen-treated dogs
The EMG cells, laden with secretory
granules, a r e easily recognized in freezefracture replicas (Fig. 91, and the tight junctions associated with these cells (Fig. 10)
closely resemble those in intact and ablated
animals. We never observe focal attenuations
or discontinuities in the network of intramembranous strands. Gap junctions are not readily
apparent in EMG cell membranes. However,
unusual junctional combinations (Orwin et al.
118
F.B. MERK, I. LEAV, P.W.L. KWAN, AND P. OFNER
EFFECTS OF SEX STEROIDS ON CELL JUNCTIONS
1973; Alroy and Weinstein 1976; Elias and
Friend 1976) consisting of granulofibrillar
areas encircled by tight-junctional strands are
occasionally seen (Fig. 11).
Following estrogen treatment, l O O F desmosomes are the only type observed between
squamous and EMG cells. They are also found
between EMG cells (54 out of 127 counted, or
43%).The 100F desmosome is present on EMG
cell surfaces at many locations, including the
proximal position of junctional complexes
(Figs. 4 and 7). The tonofilaments of these junctions tend to be long and straight (Figs. 7 and
16) and they often intermingle with cytoplasmic bundles of tonofilaments (Fig. 16).
However, 70F desmosomes are also found between EMG cells. Many of them appear unaffected by the hormone treatment (compare
Figs. 7 and 8). They are associated with short
filaments that are thready (Fig. 8) or indistinct
(Fig. 5).Serial sections indicate that the dense
plaques are usually not in contact with 10-nm
filaments (Figs. 5a and b). However, some
small desmosomes are associated with a few
tonofilaments (lower part of Fig. 7). These junctions probably represent a spectrum that spans
the two prototypes described above.
Response of regressed acini
to androgen stimulation
In this preliminary experiment we found
that, during the brief period (56 hours) following androgen administration, full restoration
of the secretory epithelium has occurred. The
basal-reserve cells appear unchanged, but the
glandular cell cytoplasm is packed with secretory granules (Fig. 18) that have the same
“electron density” as their counterparts in intact dogs. Freeze-fractured tight junctions (Fig.
12) are indistinguishable from those in intact
or ablated dogs, and large gap junctions (Fig.
17) are present. Desmosomes of the junctional
complexes and elsewhere between glandular
cells are predominantly (89 of 100 counted) the
70F variety (Fig. 18). Compact fascicles of tonofilaments, similar to those described in intact
119
dogs, are sometimes present in the cytoplasm
but they are usually not in contact with the
desmosomes.
DISCUSSION
When the prostate of the androgen-depleted
dog is exposed to pharmacological levels of circulating estrogen, two populations of epithelial
cells are affected simultaneously. One population is the basal-reserve cells, which proliferate
(Helpap and Steins, 1975;Hohbach, 1977;Leav
et al., 1978), and whose progeny differentiate
into squamous cells, giving rise to squamous
metaplasia. The other population is the regressed glandular cells. When stimulated with
estrogen, they undergo cytoplasmic transformation from one adult cell type into another
and therefore are also participants in the metaplastic process. However, unlike the basal-reserve cells, they probably do not divide. Autoradiographic analysis of 3H-thymidine incorporation in prostatic explants, exposed to
estrogen in vivo, indicates a high labeling
index over the basal(squamous cell) regions of
the acini and a low index over the superficial
(EMG cell) regions (Helpap and Steins, 1975;
Leav et al., 1978).
The regressed prostatic glandular cell in our
canine model is a unique tool for monitoring
ultrastructural responses to individual sex
steroid hormones in vivo. Following estrogen
stimulation, these cells undergo pronounced
changes in appearance. However, their original
identity is not lost because the vestigial
granules, a distinctive cytoplasmicmarker, are
retained. Their presence indicates that these
cells were responding to androgen prior to ablation. The small secretory granules that appear
followingECP treatment were never present in
the regressed epithelium. Consequently we
consider them to be an indicator of estrogen
action. Coexistence of the small estrogeninduced secretory granules andlor tonofilament bundles with vestigial granules is a distinguishing feature of EMG cells and is indicative of bipotential hormone responsiveness
Fig. 4. Detailed view of EMG cells from a castrated ECP-treated dog (#7). Small membrane-boundsecretory granules are
situated near the plasma membrane and they coexist in the cytoplasm with wide bundles of tonofilaments (TF),Golgi
apparatuses(G),and rough endoplasmic reticulum (ER),and lytic-densebodies (DB)are common.The EMGcells arejoinedby
tightjunctions (TJ) and lOOF desmosomes (D). A junctional complex from the same specimen is illustrated in the inset. The
desmosomal(1OOF)component is tangentially sectioned and distinct tonofilaments are seen converging on the dense plaques.
x 41,000; inset x 45,000.
120
F.B. MERK, I. LEAV, P.W.L. KWAN, AND P. OFNER
EFFECTS OF SEX STEROIDS ON CELL JUNCTIONS
121
Fig. 5. Estrogen-modifiedglandular cells from a hypophysectomized, ECP-treated dog (#12). Two populations of secretory
granules coexist in the same cell. Small granules, which appear in large numbers, are arranged subjacent to the luminal
membrane. A second population of granules, which is larger and more electron-dense than the first, is illustrated in various
stages of lysosome-induced degeneration (LYS). These are vestigial granules. At the far left is a junctional complex that
includes a “square” 70F desmosome (arrow).A high magnificationview (inset a) and a non-consecutive serial section (inset b)
show that this tangentially sectioned desmosome is associated with fine as well as indistinct filaments and that it has no
apparent contact with tonofilaments. Inset c is a high magnification view of the area within the rectangle (from a serial
section). The outer membrane is that of the lysosome, whereas the inner membrane is part of the vestigial granule. Arrows
indicate regions where the “unit”character of the membranes is evident. x 14,500;insets a and b x 63,000;inset c x 95,000.
Fig. 6. Estrogen-modifiedglandular cells from the same specimen as the preceding figure (dog # 12).Ajunctional complex
that joins two EMG cells includes a lOOF desmosome (D). Tonofilaments of the lOOF desmosome (see Fig. 7) and small
luminally oriented secretory granules coexist in the same cell with a vestigial granule (V).Vestigial granules, including the
one in the inset (alsofrom dog #12), have amorphous centers, whereas lytic-densebodies (DB)have heterogeneous interiors. A
rim of unextracted material, which includes the limiting membrane of the vestigial granule, is seen in the inset (arrow).The
outer membrane is probably that of a lysosome whose body is entirely out of the plane of section (compare with Fig. 5).
x 14,500 inset x 44,000.
122
F.B. MERK, I. LEAV, P.W.L. KWAN,AND P. OFNER
EFFECTS OF SEX STEROIDS ON CELL JUNCTIONS
Fig. 7. High magnification of the EMG-EMG cell desmosomes illustrated in Figure 6. The coarse filaments
(long arrows) associated with the lOOF desmosome near the lumen are long and they have the rigid appearance
characteristic of tonofilaments. The dense plaques of the other desmosome are associated with a fine network of
cytoplasmic filaments (short arrows)that stain less intensely than tonofilaments. However, a few coarse (%10nm)
filaments (long arrows) also converge onto the dense plaque. This junction may represent a transition stage
between 70F and lOOF desmosomes. x 65,000.
Fig. 8. Detailed view of a n EMG-cell junctional complex that includes a “square” 70F desmosome (dog #12).
Structural differences between the filaments associated with 70F and IOOF desmosomes will be appreciated by
comparing Figures 7 and 8, both printed a t the same magnification. The short rippled microfilaments that
converge on the dense plaque of this 70F desmosome are 5-7 nm wide and do not exceed the width of transversely
sectioned plasma membrane (at asterisk). x 65,000.
123
124
F.B. MERK, I. LEAV, P.W.L. KWAN, AND P. OFNER
Fig. 9. Freeze-fracture replica from the prostate of a hypophysectomized ECP-treated dog (#ll).The EMG cells are
characterized in replicas by the presence of secretory granules near the luminal membranes (compare with Figs. 4-61, Except
for a small region of PF face luminal membrane (P) and nuclear envelope tN), the cells have been cross-fractured, clearly
revealing the proximity of the secretory granules to the lumen (L).The granulemembrane relationship facilitates identification of EMG cell membranes for junctional analysis. X 43,000.
EFFECTS OF SEX STEROIDS ON CELL JUNCTIONS
125
Fig. 10. Freeze-fractured tight junction (zonula occludens) from a hypophysectomized ECP-treated dog (#ll).This
EMGcell junction consists of an uninterrupted fibrillar network that is 5-8 strands in depth. The luminal membrane with
cross-fractured microvilli (MV) is oriented above the junction and the lateral membrane is below. x 46,500.
Fig. 11. Unusual combinationof a tight junction and desmosomeswithin an EMG-cell plasma membrane (dog # 11). Some
PF-face strands of the tight junctions encircle granulofibrillar areas of the membrane that are freeze-fractured desmosomes
(D). It is not possible to determine whether these desmosomes are 70F or 100F. x 54,000.
Fig. 12. Freeze-fracture replica of a tightjunction from a hypophysectomized TCP-treated dog (#13). The tight junctional
network, ranging from 6 to 9 strands in depth, forms a continuous belt within the membrane of an androgen-stimulated
glandular cell. Thisjunction closely resemblesthe zonulae occludentes found in prostatesof intact-untreated dogs. x 40,000.
126
F.B. MERK, I. LEAV, P.W.L. KWAN, AND P. OFNER
EFFECTS OF SEX STEROIDS ON CELL JUNCTIONS
127
Fig. 13. lOOF desmosome from a prostatic acinus of an intact normal dog (#l).This junction, which has a long profile, is
located between a basal-reserve cell and a glandular cell. x 82,000.
Fig. 14. Junctional complex between prostatic glandular cells of a n intact normal dog (#U. Two epithelial cells arejoinednear
the luminal surface (L)by ajunctional complex which includes a tightjunction (TJ),intermediatejunction (IJ), and “square” 70F
desmosome (DES). Although unit membranes and dense plaques of the desmosome are distinct, the associated filaments are not
individually resolved, They form a filamentous mat that resembles the one adjacent to the intermediate junction. X 82,000.
Fig. 15. Junctional complex between regressed epithelial cells of acastrated dog (#3). A large 70F desmosome is the proximal
component ofthisjunctional complex. A smaller 70F desmosome is also present. Adjacent to the dense plaques, fine filaments are
seen in longitudinal section (long arrows) and in cross section (short arrows). The width of the 5-6 nm filaments is less than
transversely sectioned plasmalemma (asterisk) and unit membrane within the desmosome itself. X 114,000.
Fig. 16. Two “shaggy” lOOF desmosomes in prostatic epitheliumofa castrated ECP-treateddog (#7). The desmosomesjoin a n
EMG cell (below)with a squamous cell (above).Tonofilaments associated with the desmosomesextend deeply into the cytoplasm
and mingle with bundles of tonofilaments. The tonofilaments (between arrows) have a width of 9-10 nm. Most of the plasma
membranes between the cells appear thickened because of tangential plane of section. A short portion of transversely sectioned
plasmalemma (asterisk) is narrower than the tonofilaments. x 82,000.
Fig. 17. Freeze-fractured gap junction from the glandular epithelium of a hypophysectomized TCP-treated dog (#13). A large
aggregation of 7-9 nm particles is present on the P fracture face. x 90,000.
Fig. 18. Detailed view of a junctional complex in the glandular epithelium of a hypophysectomized TCP-treated dog (#13).
Fifty-six hours after androgen administration abundant secretory granules are present in the cytoplasm. Large and small
“square” 70F desmosomes are similar to those that join glandular cells in intact-untreated animals. x 65,000.
128
F.B. MERK, I. LEAV, P.W.L. KWAN, AND P. OFNER
(Fig. 19). In addition, the EMG cells also exhibit bidirectional (divergent) differentiation.
This phenomenon is expressed in the cytoplasm
as development of secretory (small granules)
and squamous (tonofilaments) components
within the same cell (Fig. 19). Thus estrogen
appears to be capable of imposing both secretory and squamous cell features onto a
framework that previously exhibited only secretory cell characteristics.
In the normal prostatic acinus, a continuous
layer of glandular epithelium forms a lining
between the lumen and the basement lamina.
Tight junctions (zonulae occludentes) between
the glandular cells are the paracellular permeability barrier to diffusion (Claude and Goodenough, 1973; Staehelin, 1974;McNutt, 1977).
When the glandular cells acquire squamous
characteristics, as the result of estrogen treatment, no modifications of the tight-junctional
networks are observed. This observation may
be significant in view of the nature of tightjunctional development in other epithelia.
squamous cells in mammalian tissues such as
epidermis and vagina are joined by focal (discontinuous) tight junctions (Elias et al., 1978).
However, when a squamous epithelium is exposed to retinoic acid (vitamin A) in organ culture, tonofilaments and other squamous features are progressively replaced by glycogen
and mucous granules (Elias and Friend, 1976).
Concurrently, complete (continuous) tight
junctions are assembled at the surfaces of cells
undergoing the vitamin A-induced mucous
metaplasia (Elias and Friend, 1976). Our experimental protocol produces an opposite effect
on epithelia. The glandular cells acquire tonofilaments and other squamous features, yet the
completeness of the tight junctions is not affected. Attenuation and breakdown of tightjunctional elements can take place during
rapid cell proliferation (Staehelin, 19741, amphibian neurulation (Decker and Friend,
19741, and neoplastic transformation (Merk et
al., 1977; Sinha et al., 1977). Our failure t o
observe junctional breakdown may be due to
insufficient time for disassembly to occur. A
more likely explanation is that although EMG
cells have acquired some squamous cell features they continue to perform glandular cell
functions, including provision of a permeability
barrier for the underlying cells.
Desmosomes serve as anchorage sites for
intercellular adhesion. These cell-surface
specializations are associated with cytoplasmic
filaments that protect the plasma membrane
by distributing mechanical stress over a large
volume (McNutt and Weinstein, 1973; Staehelin, 1974). The present study demonstrates
that two morphologically distinct forms of
desmosomes are present in the canine prostate.
The 70F and lOOF desmosomes have been described previously (McNutt and Weinstein,
19731,but this is the first report that the phenotypic expression of desmosomes is under hormonal control in a target tissue. Our finding is
Fig. 19. Schematic representation of responses that regressed prostatic cells have to male and female sex
steroids. Atrophic glandular cells (GI a t the bottom of the figure are joined to one another by 70F desrnosomes (1).
Basal-reserve cells (B) form lOOF desmosornes (2) with their glandular cell neighbors. The cytoplasm of the
regressed glandular cells contains numerous lysosornes including lytic-dense bodies (3).Also present are vestigial
secretory granules (41,which frequently appear partially extracted.
When regressed glandular cells are exposed to TCP, they rapidly acquire a n appearance similar to that of the
glandular cells found in intact animals (upper left). Desrnosomes joining glandular cells, either as components of
junctional complexes or individually, are of the 70F variety (1).The lOOF desmosomes are present only between
glandular and basal cells (2). Under the influence of androgen, the glandular cells have the characteristics of a
secretory epithelium. They are laden with abundant, densely stained secretory granules. Broad bundles of
tonofilaments are lacking.
When regressed glandular cells are exposed to pharmacological levels of ECP (illustrated a t center right) they
acquire a phenotype that is not observed in normal canine prostate. Although estrogen-modified glandular cells
(EMG) continue to exhibit 70F desmosomes (1) and vestigial granules (41,new structures have appeared in
response to the female hormone. They are a population of small secretory granules, broad bundles of tonofilaments, and lOOF desmosornes (2) that join the EMG cells. Basal-reserve cells (B) also respond to estrogen
stimulation. They proliferate and their squamous cell progeny (S), characterized by lOOF desmosomes and
tonofilament bundles, are located between some EMG cells and the basement lamina.
The coexistence of vestigial granules (a marker of previous androgen responsiveness) and the small secretory
granules (a marker of estrogen responsiveness) in EMG cells indicates that a potential to respond to either sex
hormone exists in the regressed glandular cells, i.e., a bipotentiality of response. A collateral phenomenon is
observed in the EMG cells. Tonofilament bundles and lOOF desmosomes (characteristics of squamous-cell differentiation) are formed concurrently with secretory granules (characteristic of secretory-cell differentiation) under
the influences of estrogen. Coexistence of these structures in the EMG cells is an indication of bidirectional
differentiation.
129
EFFECTS OF SEX STEROIDS ON CELL JUNCTIONS
-
Androgen Induced
Secretory Granules
INTACTor TCP-TREATED
Estrogen-Induced
Secretory Granules
Bidirect ionaI
Differentiation
c3/
HYPOX.or CASTRATE
130
F.B. MERK, I. LEAV, P.W.L. KWAN. AND P. OFNER
not unexpected, because previous studies have
shown that another cell-surface structure, the
gap junction, also responds to hormones (Merk
et al., 1972; Bjersing and Cajander, 1974;
Decker, 1976; Dahl and Berger, 1978).
The predominance of 70F desmosomes in
glandular epithelium of the canine prostate
appears to be a species-specific phenomenon.
We have frequently seen l O O F desmosomes between glandular cells of normal prostate in
other species including man (Merk, Leav,
Kwan, and Ofner, unpublished observations).
The 70F desmosomes observed between glandular cells of normal canine prostate resemble
the desmosomes found during embryonic development by Lentz and Trinkaus (1971). The
W3 nm filaments of these junctions frequently
mingle with morphologically similar filaments
in the ectoplasm. Ishikawa et al. (1969) have
shown that heavy meromyosin (HMM)is bound
to the thin filaments of epithelial cells in the
same “arrowhead’ manner that it is bound to
known samples of fibrous actin. However,
thicker 10 nm filaments, including those associated with squamous-cell desmosomes (i.e.,
lOOF), are not decorated with HMM. The data
of Ishikawa et al. (1969) indicate that fine filaments in the ectoplasm of epithelial cells are
actin or actin-like protein and presumably
have contractile properties, in contrast to the
thicker filaments that are probably non-motile
and subserve a cytoskeletal function. McNutt
and Weinstein (1973) have suggested that the
thin filaments of intermediate junctions
(zonulae adherentes) and 70F desmosomes are
also F-actin because they have a close association and morphological similarity with HMMdecorated filaments. Recent evidence supports
the concept that F-actin is associated with components of the junctional complex. The protein
a-actinin, which is considered to be a membrane anchor for actin, has been localized by
indirect immunofluorescent microscopy in the
junctional complexes of intestinal epithelia
(Craig and Pardo, 1979). Our inability to resolve consistently the filaments associated
with 70F desmosomes and intermediate junctions in our osmium-fixed tissues also supports
the F-actin concept. Purified actin filaments,
prefixed with glutaraldehyde, are readily
fragmented by the action of osmic acid solutions (Maupin-Szamier and Pollard, 19781,
whereas the 10 nm tonofilaments appear stable. If the proposal of McNutt and Weinstein
(1973) is correct, the different properties that
motile actin filaments and non-motile tonofilaments confer on 70F and lOOF desmosomes,
respectively, may have functional significance.
During the early stages of desmosomal
morphogenesis in regenerating epidermis
(Krawczyk and Wilgram, 19731, tonofilaments
are attached shortly after the dense plaques are
formed. This finding indicates that the lOOF
desmosome is formed de novo and consequently
the 70F variety, under ordinary circumstances,
is not an immature form of the l O O F but is in
itself a fully differentiated structure. Previous
reports about morphological changes in mature
desmosomes of epithelial cells have been limited to multilayered tissues where the alteration has been shown to be dependent on the
location of the cells as they progress from one
position to another (Petry et al., 1961; Farquhar and Palade, 1965). Our observations
of 70F and lOOF desmosomes between EMG
cells have been made on a single-layered
epithelium. The presence of transition-stage
desmosomes,which combine 70F and lOOF features (lower part, Fig. 71, suggests that some
70F desmosomes may be transformed into lOOF
when the phenotype of glandular cells is modified by androgen depletion and estrogen administration.
100F desmosomes are part of the squamouscell phenotype that is expressed during squamous metaplasia irrespective of the causative
agent. Consequently we do not interpret their
appearance as a hormone-specific response.
However 100F desmosomes are not normally
part of the glandular-cell phenotype of the intact canine prostate. Therefore the appearance
of these junctions between EMG cells must
either by directly attributable to androgen depletion and estrogen administration or be a
nonspecific phenomenon resulting from a rapid
cellular response t o hormone stimulation. To
test the latter possibility, we examined the
glandular epithelium of a hypophysectomized
dog killed just 56 hours after androgen treatment, and we infrequently observed lOOF
desmosomes between glandular cells. Our observation that lOOF desmosomes are more
commonly found between EMG cells than between fully restored glandular cells suggests
that their formation is not the result of a generalized rapid response to sex hormone stimulation but represents a specific effect due to
androgen depletion and estrogen administration.
It is likely that androgen also actively regulates the phenotypic expression of desmosomes
in the canine prostate. We have noticed that,
when the ratio of circulating androgen to estrogen is high, 70F desmosomes predominate be-
EFFECTS OF SEX STEROIDS ON CELL JUNCTIONS
tween glandular cells. However, following
ablation, the decline in levels of circulating
endogenous androgens is much greater than
that of estrogen (Leav et al, 1978). This decrease in the androgedestrogen ratio is accompanied by a n increased incidence of lOOF
desmosomes between glandular cells. Similarly, lOOF desmosomes also predominate in
the undifferentiated prostatic epithelium of
sexually immature 6-week-old dogs (Merk,
Leav, and Cavazos, unpublished results). Our
observations suggest that the phenotypic expression of the two types of desmosome is regulated by the androgen-estrogen ratio. Whether
androgen exerts its influence by promoting
formation of 70F desmosomes or by inhibiting
the lOOF variety cannot be determined a t
present.
Prostatic glandular cells, which have developed in an environment favoring androgen, express an aberrant phenotype when exposed to
pharmacological levels of estrogen. Under
these conditions the dimorphic nature of
EMG-cell desmosomes is evidence of bidirectional differentiation a t the cell surface.
Coexistence of 70F and l O O F desmosomes on
the surfaces of the metaplastic EMG cells can
be compared with a similar phenomenon found
on unusual cells present in canine mammary
adenoacanthomas. Glandular and squamous
cells in these tumors are joined by 70F and
lOOF desmosomes, respectively. However, a
third population, the “intermediate” cell, includes both the glandular and squamous cell
types of desmosomes and therefore exhibits
bidirectional differentiation of the plasma
membrane (Alroy and Weinstein, 1976).
Freeze-fracture replicas of epithelial cells,
which combine squamous and glandular cell
characteristics, sometimes display unusual
combinations of tight junctions and granulofibrillar areas (desmosomes).The combinations
are present on “intermediate” cell surfaces
(Alroyand Weinstein, 1976)and are also found
between cultured peridermal cells of the chick
embryo (Elias and Friend, 1976) and between
keratinizing Henle’s cells of the wool follicle
(Orwin et al., 1973).These combinations, which
are not ordinarily encountered between normal
adult glandular cells, are found in the glandular epithelium of canine prostate following estrogen administration (Fig. 11).
We conclude that glandular cells of the canine prostate possess cellular mechanisms that
render them responsive t o either androgen or
estrogen. During reorganization of the acini,
which follows endocrine manipulations, cyto-
131
plasmic and cell-surface modifications reflect
the changes occurring in the hormonal environment. When glandular cells are exposed to
the abnormal stimulus of pharmacological
doses of estrogen, the hormone regulates the
expression of many types of structures that had
previously responded to androgen, including
RER, secretory granules, and desmosomes.
Under physiological conditions, estrogeninduced morphological responses may be modified or masked by androgen. The differentiation of glandular cells in the canine prostate
may be brought about by androgens and estrogens working in concert to establish the characteristic morphology found in intact animals.
ACKNOWLEDGMENTS
The authors are grateful to Bennett M. Stein,
M.D., who performed the hypophysectomies,
and to Fred W. Quimby, D.V.M., for assistance
and advice in animal care. We also wish to
thank Mr. Gary Goodrich for the steroid RIAs,
Mr. Steven Halpern for photographic assistance, Mr. Dan Casper for the schematic illustration, and Ms. Lela Silverstein and Ms. Arlene Kronman for help in preparing the manuscript.
This research was supported by grants CA
15776 and CA 16377 from the National Cancer
Institute, U.S. Department of Health, Education, and Welfare.
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