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Lysosomes in uterine involutionDistribution of acid hydrolases in luminal epithelium.

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Lysosomes in Uterine Involution : Distribution of
Acid Hydrolases in Luminal Epithelium '
E. ANTON, D. BRANDES,e AND S. BARNARD
Department of Pathology, Johns HOpkim University School of Medicine,
and Baltimme City Hospitals, Baltimme, Maryland 21205
ABSTRACT
The distribution of acid hydrolytic activities in rat uterine epithelial
cells during post-partum involution was examined by light and electron microscopic
cytochemistry. Acid phosphatase, j3-glucuronidase, N-acetyl-j3-glucosaminidase and
EBoo-resistant
esterase (cathepsin) increased during the period of involution and the
electron cytochemical preparations revealed the lysosomal nature of the acid hydrolytic positive particles visualized by light microscopy.
The newly formed particles included primary lysosomes, in the form of Golgi
vesicles, vacuoles, and secondary lysosomes such as dense bodies and autophagic
vacuoles. This apparent increase in lysosomal activity during uterine involution is in
agreement with similar patterns observed in the course of tissue regression in general.
Activation of lysosomal enzymes represents a well-defined biochemical pattern
related to tissue regression in general (de
Duve, '63), which includes several examples of hormonally induced involution
of sex accessory organs (Slater, Greenbaum and Wang, '63; Brandes, '66).
Changes in distribution of lysosomal hydrolases during post-partum involution of the
uterus, which conforms to this pattern,
have been described in light microscopic
histochemistry (Lobe1 and Deane, '62) and
biochemical studies (Woessner, '65).
Since the term lysosome covers a variety
of particles with differences in their subcellular morphology and functional properties (de Duve and Wattiaux, '66), it
seemed of interest to characterize these
bodies at the ultrastructural level by cytochemical techniques.
This paper reports h e structural
changes in the distribution of lysosomes
in the surface epithelium of the rat uterus
during post-partum involution.
MATERIALS AND METHODS
Holtzman pregnant female rats were
used in this study, and groups of three
animals were sacrificed at the following
time points: on the seventeenth day of
gestation; immediately after parturiton;
eight hours after parturition, and at 1, 2,
3, 4, 6 and 17 days after parturition. In
these groups, litters of nine or more young
sucklings were left with their mothers.
ANAT.REC., 164: 231-252.
A portion of the uterus, about 2 mm
thick was excised from the area approximately 3 mm below the separation of the
uterine horns. This area did not include
any of the placental scars and is part of
the body of the uterus. In all instances,
the tissues were fixed for two hours in 6%
glutaraldehyde in 0.1 M cacodylate buffer
pH 7.4 containing 0.33 M sucrose (Sabatini, Bensch and Bmnett, '63). Tissues
were rinsed and stored at 4°C in the same
buffer. For conventional electron microscopy, portions of the tissues were post-fixed
in 1% buffered OsOd and embedded in
Epon 812. Ultra-thin sections were cut
with a Porter-Rlum microtome and the
grids stained with uranyl acetate and lead
citrate (Reynolds, '63). Electron micrographs were taken with the RCA EMU-3F
electron microscope.
Histochemistry
Glutaraldehyde fixed frozen sections
were cut at 8 CI and 36 p. The 8 I.I sections
were mounted on slides and incubated for
the demonstration of:
( a ) Acid Phosphatase : lead salt method
(Gomori, '52); 45 minutes at 37°C and
-
Received Nov. 25, '68. Accepted Feb. 11, '69.
1This investigation was supported by grants HD
00042, National Institute of Child Health and Human
Development and CA 08518 from the National Cancer
Institute, Nitional Institutes of Health, U.S. Public
Health. Service.
2 Recipient of a Career Development Award, K3-CA21,756-04. National Cancer Institute, US. Public
Health Sernce.
231
232
E. ANTON, D. BRANDES AND S. BARNARD
counterstained with methyl green. Controls: no substrate; prolonged fixation in
20% formalin; sodium fluoride 0.01 M.
(b) p-glucuronidase, simultaneous coupling azo dye technique (Hayashi, Shirahama and Cohen, '68) for 30 minutes at
37°C and counterstained with methyl
green. Controls: specific inhibitor 0.25
mM glucosaccharo- 1:4 lactone; absence
of substrate.
(c) N-acetyl-p-glucosaminidase: simultaneous coupling azo dye method (Hayashi,
'65) 40 minutes at 37°C and counterstained with methyl green. Controls: no
substrate; inhibitor- N-acetyl-glucosaminolactone 5 mM.
resistant esterase: indoxyl ace(d) EEo0
tate method (Pearse, '61) for one hour
and counterstained with Nuclear Fast Red.
Controls : no substrate.
The 36 1.1 sections were floated in 0.33
M sucrose, and incubated in Gomori's lead
salt mixture for acid phosphatase for 20
minutes at 37"C, post-fixed in buffered
OsOc and embedded in Epon 812. Subsequent preparative procedures were identical to those of the conventional preparations.
Control sections were processed for observation when comparing the various histochemical stains, but were not included
in the figures because of the negative reactions. A description of the preparation
of control sections is included above. Grids
which were not counterstained with either
lead or uranyl acetate, were also observed
in order to rule out the possibility that
lead deposits in the lysosomes were due to
inspecific deposition during lead counterstaining.
RESULTS
Light microscopy
On the seventeenth day of pregnancy
(fig. 1) and immediately following parturition (0 hour post-partum), the surface
epithelial cells contained very few demonstrable lysosomes. At eight hours after
parturition, a slight acid phosphatase reaction (fig. 2), restricted to minute particles, was observed in the luminal epithelial
cells.
There was a progressive increase in the
size of the acid phosphatase-positive particles, and in the intensity of reactions,
through six days post-partum (figs. 3, 4).
Three other lysosomal enzymes, p-glucuronidase (fig. 3), N-acetyl-p-glucosaminidase (fig. 6), and E"'-resistant esterase
(fig. 7) showed a parallel increase during
the involution stage.
On the seventeenth day post-partum
(fig. 8), the concentration of lysosomes in
the luminal epithelial cells was markedly
decreased and similar in appearance to
that seen at 0 hour post-partum and on
the seventeenth day of gestation.
Electron microscopy
On the seventeenth day of gestation
(fig. 9), no lysosomes could be detected in
most luminal epithelial cells of the uterus,
and in most of these cells, the Golgi complex was poorly developed and showed no
acid phosphatase activity (inset to fig. 9).
The same description applies to the luminal epithelial cells of the uterus from rats
sacrificed immediately after delivery (0
hour post-partum).
At eight hours after parturition (fig. lo),
most of the luminal cells showed a marked
hypertrophy of the Golgi complex. The
lamellae were more numerous and were
arranged in concentric layers, and many
of them contained abundant lead deposits
indicative of acid phosphatase activity.
Primary lysosomes, such as Golgi vesicles,
and secondary lysosomes of the dense
body-type, showed an intense acid phosphatase reaction which was also detected
in larger vacuoles present in the Golgi
region.
Twenty-four hours after parturition
(fig. l l ) , the hypertrophy of the Golgi
complex still persisted, but the acid phosphatase reaction in the lamellae was decreased in comparison with the eight hour
specimens. The number and complexity
of the dense bodies in the Golgi region, on
the other hand, was moderately increased.
Progressive concentration of lysosomes
in the surface epithelial cells three days
after parturition is illustrated in a low
power view (fig. 12). On the fourth day
post-partum, many of the epithelial cells
LYSOSOMES O F UTERINE EPITHELIUM IN INVOLUTION
were occupied by closely packed clusters
of lysosomes (fig. 13). The heavy lead deposits present in these bodies (fig. 14)
serve to emphasize the magnitude of their
hydrolytic capacity.
Increased lysosome activity in the luminal epithelial cells during post-partum
involution, resulted not only in the enhancement of activity in the Golgi elements and in the development of numerous
dense bodies, but also in the formation of
autophagic vacuoles.
The autophagic vacuoles showed variations in the magnitude of the cytoplasmic
area encapsulated within the limiting
membrane, as well as in the degree of
breakdown of segregated cell organelles.
The autophagic vacuole illustrated in
figure 15, contains a large number of cell
organelles, chiefly mitochondria, clusters
of ribosomes, lysosomes and cisternae of
the rough endoplasmic reticulum. Although
some of these organelles are stiU wellpreserved, others, particularly some of the
mitochondria, show marked structural alterations, especially of their cristae.
Stages in the progressive dissolution of
the entire contents of the mitochondria
with the apparent formation of electronlucent vacuoles can also be seen in figure
15.
In more advanced stages of their lifecycle (fig. 16), the contents of the autophagic vacuoles appeared completely autolyzed with the exception of some enlarged
lysosomes, which seemed to outlive a l l the
other cell organelles originally segregated
within the vacuoles.
Conventional electron microscopical
preparations, that is sections not incubated
for acid phosphatase, were also examined.
In specimens from uteri not undergoing
involution (seventeenth day of gestation;
0 hour and 17th day post-partum), structures that might represent lysosomes were
seldom detected.
In the epithelial cells from uteri in involution, numerous electron-dense bodies,
surrounded by a single membrane, were
present. None of these bodies contained
material that might be confused with the
lead deposits seen in the histochemical
preparations.
233
DISCUSSION
The present results are in agreement
with the reported light microscopy histochemical findings of Lobel and Deane
('62) on the activation of lysosomal enzymes in the uterine epithelium during
post-partum involution.
Our electron-cytochemical observations
have served to characterize, at the subcellular level, the different functional
types of lysosomes which developed during
involution. The presence of acid phosphatase activity in Golgi elements; the development of primary lysosomes of the Golgi
vesicle-type as well as secondary lysosomes such as dense bodies (Gordon, Miller and Bensch, '65) and autophagic vacuoles, is in accordance with the postulated
role of the Golgi apparatus in the distribution of hydrolytic enzymes to the various
kinds of lysosomes (Novikoff, '63; de Duve,
'63; Gordon et al., '65; de Duve and Wattiaux, '66).
Unusually large autophagic vacuoles,
such as that illustrated in this paper, have
been described as 'massive cytosegresomes'
in injured flounder tubules (Trump and
Bulger, '67).
The observed changes in the distribution of lysosomes in the uterine epithelium
during involution correlate with the concept (de Duve, '63, '64; Van Lancker, '64)
that concentration of hydrolases in areas
of cytoplasmic degradation, such as dense
bodies and autophagic vacuoles, is characteristic of involution and of tissue regression in general.
Very little is presently known about the
mechanisms that may be responsible for
triggering the activation of the lysosomal
system during involution, or its si@cance in the economy of the epithelial
uterine cells.
Changes in the hormonal environment
seem to play a role in the enhancement
of lysosomal activity, as judged from the
similarities of response after hormone withdrawal in ovariectomized rats treated with
estrogens and progesterone (Lobel and
Deane, '62). In this study, as more than
nine young were suckling, it is possible
that a progesterone effect might have influenced the results.
A similar enhancement of lvsosomal activity related to hormone withdrawal has
234
E. ANTON, D. BRANDES AND
been observed in prostatic cells after orchiectomy, where the process of histologic
involution and depression of functional
activities of the organ were accompanied
by the development of dense bodies and
autophagic vacuoles (Brandes, '66). Acid
phosphatase activity in the Golgi, and development of autophagic vacuoles in relation to the secretory process, has also been
reported in mammotrophic cells of the
anterior pituitary gland of the rat, presumably in relation to the control of production of secretory products (Smith and
Farquhar, '66).
Cellular autophagia is also a prominent
feature in tissue remodeling processes during embryogenesis, and formation of autophagic vacuoles and dense bodies has been
observed after various types of injurious
attacks to the cell (de Duve and Wattiaux,
'66).
Focal degradation within autophagic
vacuoles and dense bodies in the epithelium of the uterus during involution may
represent a mechanism for controlled intracytoplasmic breakdown of excess organelles and certain materials accumulated
during gestation in order to meet the increased metabolic demands imposed upon
the pregnant uterus. Similar autophagic
mechanisms during involution are involved
in the breakdown of the hyperplastic cell
organelles accumulated in the smooth muscle fibres during pregnancy (Brandes and
Anton, '68).
LITERATURE CITED
Brandes, D. 1966 The fine structure and histochemistry of prostatic glands in relation to sex
hormones. Internat. Rev. Cytol., 20: 207-272.
Brandes, D., and E. Anton 1969 Lysosomes in
uterine involution: Intracytoplasmic degradation of myofilaments and collagen. J. Geront.,
24: 55-69.
de Duve, C. 1963 The lysosome concept. In:
Ciba Foundation Symposium on Lysosomes.
(A. V. S. de Reuck and M. P. Cameron, eds.),
Little, Brown and Company, Boston, pp. 1-31.
1964 Lysosomes and cell injury. In:
Injury, Inflammation and Immunity. (L.
Thomas, J. W. Uhr and L. Grant, eds.). The
Williams and Wilkins Company, Baltimore, pp.
283-311.
S. BARNARD
de Duve, C., and R. Wattiaux 1966 Functions
of lysosomes. Ann. Review Physiol., 28: 435492.
Gomori, G. 1952 Microscopic Histochemistry :
Principles and Practices, Chicago Univ. Press,
Chicago, p. 193.
Gordon, G. B., L. R. Miller and K. G. Bensch
1965 Studies on the intracellular digestive
process in mammalian tissue culture cells. J.
Cell Biol., 25: 41-55.
Hayashi, M. 1965 Histochemical demonstration
of N-acetyl-p-glucosaminidaseemploying naphthol AS-BI N-acetyl-p-glucosaminidase as substrate. J. Histochem. Cytochem., 13: 355-360.
Hayashi, M., T. Shirahama and A. S. Cohen
1968 Combined cytochemical and electron
microscopic demonstration of p-glucuronidase
activity in rat liver with the use of a simultaneous coupling azo dye technique. J. Cell
Biol., 36: 289-297.
Lobel, B. L., and H. W. Deane 1962 Enzymic
activity associated with post-partum involution
of the uterus and its regression after hormone
withdrawal in the rat. Endocrinology, 70: 567578.
Novikoff, A. B. 1963 Lysosomes in the physiology and pathology of cells: Contributions of
staining methods. In: Ciba Foundation Symposium on Lysosomes. (A. V. S. de Reuck and
M. P. Cameron, eds.) Little, Brown and Co.,
Boston, pp. 36-73.
Pearse, A. G. E. 1961 Histochemistxy-Theoretical and Applied. Little, Brown and Company,
Boston, p. 913.
Reynolds, E. S. 1963 The use of lead citrate
at high pH as an electron opaque stain for
electron microscopy. J. Cell Biol., 17: 208-212.
Sabatini, D. D., K. Bensch and R. J. Barrnett
1963 Cytochemistry and electron microscopy.
The preservation of cellular ultrastructure and
enzymatic activity by aldehyde fixation. J. Cell
Biol., 17: 19-58.
Slater, T. F., A. L. Greenbaum and D. Y. Wang
1963 Lysosomal changes during liver injury
and mammary involution. In: Ciba Foundation
Symposium on Lysosomes. (A. V. S. de Reuck
and M. P. Cameron, eds.) Little, Brown and
Company, Boston, pp. 311-334.
Smith,R. E., and M. G. Farquhar 1966 Lysosome function in the regulation of the secretory
process in cells of the anterior pituitary gland.
J. Cell Biol., 31: 319-347.
Trump, B. F., and R. E. Bulger 1967 Studies
of cellular injury in isolated flounder tubules.
I. Correlation between morphology and function of control tubules and observations of
autophagocytosis and mechanical cell damage.
Lab. Invest., 16: 3, 453-482.
Van Lancker, J. L. 1964 Concluding remarks.
Federation Proc., 23: 1050-1052.
Woessner, J. F.,Jr. 1965 Acid hydrolases of rat
uterus in relation to Dregnancv. Dost-Dartum
involution, and collagen Geakdbwn. Biochem.
J., 97: 855-866.
PLATES
PLATE 1
EXPLANATION OF FIGURES
Endometrium, seventeenth day of gestation. No lysosomes are visible in the
luminal epithelium (arrow). LU, lumen. Acid phosphatase stain. X 350.
Endometrium eight hours after parturition. Lysosomes in the luminal epithelial
cells (arrow) are very rare and small (black lead deposits). LU, lumen. Acid
phosphatase stain. X 350.
Endometrium, 24 hours post-partum. Marked concentration of lysosomes in the
epithelium (arrow). Intense reaction is present in stromal macrophages. LU,
lumen. Acid phosphatase stain. X 350.
Endometrium, 48 hours post-partum. Greater concentration of lysosomes in the
epithelial cells (arrow) and in stromal macrophages. LU, lumen. Acid phosphatase stain. X 350.
Figs. 5-7 Endometrium, 72 hours post-partum. Other hydrolases of the lysosomal
enzymatic complex, such as p-glucuronidase, N-aoetyl-p-glucosaminidase and E6O0 resistant esterase (cathepsin) are increased during this period.
5
Positive-stained lysosomes in luminal epithelial cells (arrow). LU, lumen. p-glucuronidase reaction. X 350.
6
N-acetyl-p-glucosaminidasereaction: postively stained lysosomes in luminal epithelium (arrow). LU, lumen. X 350.
7 Intense EEoOresistant esterase reaction (cathepsin) in luminal epithelium (arrow).
LU, lumen. X 350.
8
236
Endometrium, 17 days post-partum. The reaction for acid phosphatase has returned to pre-involution stages (compare with fig. 1). LU, lumen; arrow, luminal
epithelium. X 350.
LYSOSOMES OF UTERINE EPITHELIUM I N INVOLUTION
E. Anton, D. Brandes and S. Barnard
PLATE 1
237
PLATE 2
EXPLANATION OF FIGURE
9 Uterine epithelium, seventeenth day of gestation. Very few lysosomes
are present at this stage. The Golgi complexes ( G ) , shown in detail
in inset, are poorly developed and show no reaction. LU, lumen. Acid
phosphatase stain, Gomori’s lead salt method, 20‘ at 37°C. x 17,900;
inset, X 30,000.
238
LYSOSOMES OF UTERINE EPITHELIUM IN INVOLUTION
PLATE 2
E. Anton, D. Brandes and S. Barnard
239
PLATE 3
LXPLANATION OF FIGURE
10
240
Uterine epithelium, eight hours after parturition. The Golgi complexes ( G ) of two adjacent cells, show marked hypertrophy. Intense
acid phosphatase reaction in lamellae (arrow heads); primary lysosomes (Golgi vesicles), vacuoles (VA) and lysosomes of the dense
body-type (LY). Acid phosphatase stain, Gomori’s lead salt method,
20‘ at 37°C. X 33,400.
LYSOSOMES OF UTERINE EPITHELIUM I N INVOLUTION
E. Anton, D. Brandes and S. Barnard
PLATE 3
24 1
PLATE 4
EXPLANATION O F FIGURE
11
242
Uterine epithelium, 24 hours after parturition. Golgi complexes (G),
with less reaction products (arrow head) than in the eight hour
specimen. Lysosomes of the dense body-type (LY) are more numerous
and show a complex configuration. Acid phosphatase stain, Gomori’s
lead salt method, 20’ at 37°C. X 42,300.
LYSOSOMES OF UTERINE EPITHELIUM IN INVOLUTION
E. Anton, D. Brandes and S. Barnard
PLATE 4
243
PLATE 5
EXPLANATION OF FIGURE
12
244
Endometrium, 72 hours post-partum. Survey picture of luminal epithelium showing the abundance of lysosomes (LY), usually arranged
in clusters. Acid phosphatase activity is also seen in Golgi elements
(arrow heads). N, nuclei. Acid phosphatase stain, Gomori’s lead
salt method, 20’ at 37°C. X 8,300.
LYSOSOMES OF UTERINE EPITHELIUM IN INVOLUTION
E. Anton, D. Brandes and S. Bamard
PLATE 5
245
PLATE 6
EXPLANATION OF FIGURES
13 Endometrium, 96 hours post-partum. Basal region of surface epithelium showing abundant conglomerates of lysosomes ( L Y ) , which are
also present in some mesenchymal cells (arrows). Acid phosphatase
stain, Gomori's lead salt method. X 7,300.
14 Detail of some of the types of lysosomes shown i n figure 13. The
lysosomes of the dense body-type (1.Y) contain abundant lead deposits
which points to high levels of enzyme activity. Positive Golgi elements including lysosomes of the Golgi-vesicle type, are signaled by
arrow heads. Acid phosphatasc stain, Gomori's lead salt method, 20' a t
37OC. X 26,900.
246
LYSOSOMES OF UTERINE EPITHELIUM IN INVOLUTION
E. Anton, D. Brandes and S. Barnard
PLATE 6
247
PLATE 7
EXPLANATION OF FIGURE
15 Endometrium, 72 hours post-partum. Recently formed, complex, autophagic vacuole (AV), delimited by a membrane (Me). Sequestered
organelles consist of: mitochondria ( M ) ; rough endoplasmic reticulum (RER); and lysosomes of the dense body ( L Y ) or Golgi types
(arrow heads). Mitochondria ( M ) show varying degrees of structural
alteration. The electron-lucent cavities ( * ) may represent the final
stage in the solubilization of the mitochondria1 content. Acid phosphatase stain, Gomori's lead salt method, 20' at 37°C. X 23,000.
LYSOSOMES OF UTERINE EPITHELIUM I N INVOLUTION
E. Anton, D. Brandes and S. Barnard
PLATE 7
2.49
PLATE a
EXPLANATION O F FIGURE
16 Endometrium, 96 hours post-partum, autophagic vacuole (AV) with
advanced autolysis of contents. It contains no recognizable structures, except several lysosomes (LY). Confluent cavities with electronlucent solubilized material, presumed to derive from digested structures, occupy the rest of the vacuole. G, Golgi elements. Acid
phosphatase stain, Gomori’s lead salt method, 20‘ at 37°C. x 26,700.
250
LYSOSOMES OF UTERINE EPITHELIUM I N INVOLUTION
E. Anton, D. Brandes and S. Barnard
PLATE 8
25 1
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