close

Вход

Забыли?

вход по аккаунту

?

Uterine simple and complex nuclear bodies are separate structural entities.

код для вставкиСкачать
THE ANATOMICAL RECORD 205119-130 (1983)
Uterine Simple and Complex Nuclear Bodies Are Separate
Structural Entities
HELEN A. PADYKULA AND SHTRWIN M. POCKWINSE
Department of Anatomy, University of Massachusetts Medical School,
Worcester, MA 01605
ABSTRACT
In rat uterine luminal epithelial cells, nuclear bodies occur in
the euchromatin in varying numbers in relation to the nuclear concentration
of the estrogen receptor (Clark et al., 1978; Padykula et al., 1981, 1982). This
functional responsiveness indicates that nuclear bodies may be useful indicators of the degree of cellular estrogenization. Because these filamentous bodies
vary in size (200-1200 nm), shape, and composition, quantitative analysis of
frequency of their occurrence has been difficult. A fundamental division into 2
categories can be made by the following criteria: 1)simple nuclear bodies (200500 nm) consisting of a protein mesh of microfilaments, and 2) complex nuclear
bodies (200-1200 nm) composed of a n outer filamentous protein capsule enclosing a lucent core that may contain granules. Previous quantitative analyses
at the electron microscopic level has excluded “simple bodies” because they
might actually be ultrathin sections through the filamentous capsule of complex bodies (Le Goascogne and Baulieu, 1977; Clark et al., 1978). To resolve
this sampling problem, we have performed serial ultrathin section analysis of
nuclear bodies in hyperestrogenized luminal epithelial cells. Ultrastructural
evidence presented here demonstrates that simple and complex nuclear bodies
are anatomically separate entities. Ultrathin sections through the capsule of
complex nuclear bodies will be misidentified as profiles of simple bodies during
quantitative analysis. This anatomic distinctness of simple and complex nuclear bodies correlates with their differing responses to estrogenic stimulation
and withdrawal (Fitzgerald and Padykula, pp. 131-141, this volume). Thus the
existence of these two major categories should be taken into consideration
during quantitative analyses.
The frequency of observation of nuclear bodies have been recognized (Bouteille et al.,
bodies in rat uterine luminal epithelial cell 1974). Two principal populations of nuclear
has been correlated with the degree of estro- body profiles are easily recognizable by size
genic stimulation (LeGoascogne and Bau- and structural differences, i.e., nuclear bodlieu, 1977; Clark et al., 1978; Padykula et al., ies and complex nuclear bodies (Fig. 1). Sim1981).Because nuclear bodies are not readily ple bodies are small (diameter 200-500 nm)
visible by light microscopy, quantitative and structurally quite homogeneous; they are
analysis is performed by direct visualization composed of a protein (Krishnan et al., 1967;
in ultrathin sections by electron microscopy Dupuy-Coin et al., 1972) mesh of microfila(Padykula and Clark, 1981). The diameter of ments and possibly microtubules. By conthese rat uterine nuclear bodies ranges from trast, the complex nuclear bodies are strucapproximately 200-1200 nm, and thus in turally heterogeneous and have a greater
routine ultrathin sections (approximately 50 range in size (200-1200 nm), shape, and comnm) only slices through them are observed in position. They consist of a n electron dense
electron micrographs. Such ultrathin sec- filamentous capsule that encloses a n electron
tions are called “nuclear body profiles.”
lucent core which may contain granules of a
This sampling problem is further compli- varying size, density, and shape. Cytochemicated by the structural heterogeneity of nuclear bodies. Various subcategories of nuclear
Received August 2, 1982; accepted October 25, 1982.
0003-276X/83/2052-0119$03.50
0 1983 ALAN R. LISS, INC.
QUANTITATIVE ANALYSIS OF NUCLEAR BODIES
Diagram
illustrating sampling problem
Nuclear Profile-Luminol Epithelial Cell
Immature rat uterus
50 nm
f
50nm
250nm
800nm
L
II
5$nm
7,000nm
I
Fig. 1. The difficulty of specific identification of simple nuclear bodies in routine
ultrathin plastic sections is shown here.
The two outermost sections of a spherical
cal analysis indicates that the granules in
complex nuclear bodies are composed of protein and that some may also contain RNA
(Kierszenbaum, 1969; Dupuy-Coin et al.,
1972). Since the protein capsule of the complex nuclear bodies resembles closely the
substance of the simple bodies, the possibility has existed that so-called simple bodies
may represent only a surface section through
the capsule of a nuclear body (Fig. 1).Thus,
the existence of simple bodies has not been
established.
Because of this limitation in interpretation, quantitative analyses of the frequency
of occurrence of nuclear body profiles during
different endocrine states have been limited
to counts of complex nuclear bodies (Le-
A
I
complex nuclear body (800 nm) represent
solely the capsular material which would
appear as filamentous meshwork. (Taken
from Padykula et al., 1981.)
Goascogne and Baulieu, 1977; Clark et al.,
1978) or to counts of total nuclear body profiles which combined complex and so-called
simple nuclear bodies (Padykula et al., 1981).
When we subsequently analyzed the data
from this latter investigation, in which the
observations a t the electron microscope had
been recorded as the frequency of simple or
complex nuclear bodies, it was evident that
distinctly different functional responses by
complex and simple nuclear bodies occur in
response to a single injection of estradiol or a
single injection of the nonsteroidal estrogen
antagonist, nafoxidine (Fitzgerald and Padykula, pp. 131-141, this volume). Since the
view has been advanced that simple nuclear
bodies may be regularly present in nuclei
UTERINE SIMPLE AND COMPLEX NUCLEAR BODIES
(Bouteille et al., 19741, it became necessary
to solve this structural problem.
To confront this sampling problem, we have
prepared serial ultrathin sections (80 nm) of
hyperestrogenized uterine luminal epithelial
cells to determine whether or not simple bodies are distinctly separate entities. Our analysis demonstrates that simple nuclear bodies
are structurally different from complex nuclear bodies. In the accompanying report
(Fitzgerald and Padykula, pp. 131-141, this
volume), quantitative evidence is presented
which indicates that simple and complex
bodies respond differently to a single injection of the estrogen antagonist-agonist, nafoxidine, and to a single injection of estradiol.
MATERIALS AND METHODS
Hyperestrogenized immature rat uterine
luminal cells were selected for serial section
analysis because nuclear bodies occur in high
frequency (Padykula et al., 1982). Estradiolcontaining silastic tubes were implanted subcutaneously in nape of 21 day old female rats
and removed 72 hours later. During this period of steady estrogenic stimulation, the
uterine luminal epithelial cells hypertrophy
to twice (30 pm) the control height (15 pm),
and a t least one profile of nuclear bodies is
observed in 48% of the nuclear profiles observed in ultrathin sections.
Procedure for obtaining serial ultrathin
sections through nuclear bodies
Blocks of uterine cross sections were chosen to include a n expanse of the luminal
epithelium sectioned along the longitudinal
axes of the cells. Ultrathin sections (beige,
approximately 80 nm thick) were prepared
on a Sorvall MT-1 ultramicrotome and placed
on 0.5% formvar coated copper one-slot grids.
A ribbon of serial sections was cut, and one
or two sequential sections were placed on
each coated grid. The grids were touched to
a piece of filter paper to remove excess water
and then sequentially placed into a n LKB
grid storage box. The grids were then stained
in 4% aqueous uranyl acetate for 3 minutes
and, one by one, carefully rinsed by dipping
into three beakers of distilled water. A grid
was slowly immersed perpendicularly to the
meniscus, raised and lowered ten times under the the meniscus, and slowly brought
perpendicularly out of the distilled water.
They were then stained in Reynolds lead citrate for 5 minutes and rinsed as before. After
removal of excess water by touching the grid
121
to a piece of filtering paper, the grid was
placed in a n LKB grid storage box.
Mode o f study at the transmission electron
microscope (JEOL lO@S)
At 8000 times magnification, a grid from
the middle of the series of sections was
scanned for serial longitudinal sections of nuclei that contain nuclear bodies. Starting
with the number 3 or number 4 grid of a
partially serially sectioned nucleus, a small
homogeneous profile of a nuclear body was
located in a nucleus of a representative longitudinal section of uterine epithelial cell and
photographed a t 8000 times magnification
and then 5000 times magnification to record
the entire nuclear profile. After scanning the
same nucleus for other nuclear bodies, which
usually were present, each location was noted
and photographed. To aid in locating the
same cell and nucleus in sequential.sections,
the shapes of the surrounding cells around
and below the chosen cell were noted. Proceeding to serial sections on either side of the
starting point, the adjacent profiles of the
chosen cell, its nucleus, and the nuclear bodies were photographed a t 8000 and 5000
times magnification. Several nuclear bodies
were traced through five, six, seven, or eight
serial sections in this manner.
Fourteen different nuclei were thus analyzed photographically; some of these lacked
one or two sections from the serial sequence.
Another 15-20 were viewed a t the electron
microscope, but were not suitable for photographic record because of various technical
limitations.
RESULTS
Structural and functional characteristics
of hyperestrogenized uterine luminal
epithelial cells
A physiologic state in which nuclear bodies
are numerous was selected for the serial section analysis. In a recent investigation, we
observed that, during 72 hours of sustained
estrogenic stimulation (subcutaneous silastic-estradiol implant) of the immature rat
uterus, the luminal epithelial cells double in
height (from 15 to 30 pm), and that twice as
many nuclear profiles contain a t least one
complex nuclear body profile (Padykula et
al., 1982).In control cells, approximately 25%
of the nuclear profiles contain a t least one
complex nuclear body profile, whereas after
72 hours of steady estrogenic stimulation approximately twice as many nuclear profiles
122
H.A. PADYKULA AND S.M. POCKWINSE
Figure 2 represents section 4 in a series of
six serial sections that are illustrated in Fig.
3a, b. Note the four profiles of nuclear bodies
which are numbered. Of these NB1, 2, and 4
are complex bodies composed of capsules and
central cores. However, NB3 is smaller, without a distinct core, and composed of filamentous and tubular components. To determine
whether or not this profile represents a simple nuclear body, study of the six serial sections (Fig. 3a, b) indicates that NB3 is
different from NB1, 2, and 4, and that NB3
is a simple body rather than a component of
a complex nuclear body. Its entire extent is
most likely demonstrated in S1 through S5
(diameter 400 nm).
Figure 4 represents section 3 in a series of
five nuclear profiles. It contains a nuclear
profile with four nuclear bodies which might
be classified as follows: NB5 and 6, simple;
NB8, small complex; NB7, ?. Serial section
analysis in Figure 5 confirms that NB5 and
6 are simple nuclear bodies. NB7 appears to
be a simple body also, although for determination of its complete structure a 6th section
would be necessary. NB8 is a small complex
body with a distinct core which is evident in
section 3; it appears to be more angular in
Serial section analysis of nuclear bodies
its shape than NB5,6, and 7.
in hyperestrogenized uterine luminal
The problem of distinguishing between
epithelial cells
simple and complex nuclear bodies in ultraThe goal of this analysis was to determine thin sections is illustrated in Figure 3a, b.
whether simple nuclear bodies are: 1) sepa- The following profiles of complex nuclear
rate structural entities, and/or 2) tangential body profiles observed in isolation might be
sections through the capsule of complex nu- classified erroneously as simple nuclear
clear bodies.Using ultrathin sections that in- bodies: NB1 in S1, S2, S5; NB2 in S5; and
clude luminal epithelial cells cut in the NB4, S3. From Figure 1, it is evident that
longitudinal plane, nuclear profiles that con- the two sections tangential to the surface of
tained a t least one small homogeneous nu- a complex nuclear body would usually inclear body were selected for serial section clude primarily filamentous capsular matestudy. The nuclear diameter (transverse axis)
is approximately 4000 nm (Fig. 1). Hence,
the six serial sections through the first nucleus (Figs. 2 and 3a,b) represent approximately 12% of its volume (six sections x 80
Figs. 2-5. Serial ultrathin sections (approximately
nm = 480 nm). Within this region, four nu- 80 nm)
through uterine luminal epithelial cells from
clear bodies occur as conspicuous components immature rats which had received steady estrogenic
of the euchromatin (Fig. 2). Heterochromatin stimulation for 72 hours from a subcutaneous implanted
is sparse and localized primarily along the silastic tube filled with estradiol.
inner surface of the nuclear envelope. PeriFig. 2. (~17,500)
The nuclear profile illustrated here
chromatin and interchromatin granules is one of six nuclear serial profiles. Regions containing
(Monneron and Bernhard, 1969) contribute profiles of these four nuclear bodies are enlarged in
to the overall granularity of the largely eu- Figure Za, b. Here in section 4 (S4),four nuclear bodies
are evident. NBl, 2, and 4 are complex nuclear
chromatic nucleus. Note the halo, a rela- (NB)
bodies as identified by a n electron lucent core surtively clear area, that immediately surrounds rounded by a more opaque capsule. Note that NB 1 and
2 are bipartite. NB3 is small and lacks a distinct core.
nuclear bodies.
(48%) contain a t least one complex nuclear
body profile. Profiles of simple nuclear bodies
occur a t a relatively low frequency which
varies around 25%.
This cellular growth and differentiation occurs during a period when the cytoplasmic
estrogen receptor complexed with estradiol
has been largely translocated to the nucleus
(Padykula et al., 1982). During the 72 hour
steady exposure to estradiol, the concentration of nuclear estrogen receptor reaches
maximal level within 24 hours and stays
high for the remaining 48 hours.
This hyperestrogenization produces tall
columnar cells with highly euchromatic nuclei and large nucleoli. The cytoplasm acquires the ultrastructural features associated
with protein synthesis for secretion in that
the rough endoplasmic reticulum is voluminous (Figs. 2 and 4) and the supranuclear
Golgi complex is large. Free polysomes are
abundant. The overall appearance correlates
well with the condition of sustained high concentration of nuclear estrogen receptor,
which promotes a functional state of cellular
growth and differentiation through heightened transcription and translation.
UTERINE SIMPLE AND COMPLEX NUCLEAR BODIES
123
s1
s2
s3
NB1
NB2
NB3
Fig. 3a, b. ( ~ 2 7 , 5 0 0 Serial
)
section analysis of the
four nuclear bodies shown in Figure 2. Note the designation of the section number along the top and the nuclear body number along the left margins of Figure 3, a
and b. Start structural analysis with Section 4, and refer
to Figure 2 for low power orientation. NB1 and NB2
occur in five consecutive sections and, hence, have a
diameter of approximately 400 nm; also, they appear to
be similar in shape. NB1 is shown in its entirety, whereas
only part of NB2 is present. NB1 and NB2 are small
complex bodies with electron lucent cores. NB3 occurs in
five sections and differs distinctly form NB1 and 2 in
shape, size, and absence of a distinct core. Thus, NB3 is
a simple body comprised almost entirely of filamentous
material. NB4 is a large complex nuclear body with
electron opaque components in its core. It is only partially included in the six sections illustrated here. In
section 3, the plane-of-section passes through the capsule
of this complex body; this image might be misconstrued
as a simple body.
126
H.A. PADYKULA AND S.M. POCKWINSE
UTERINE SIMPLE AND COMPLEX NUCLEAR BODIES
127
128
H.A. PADYKULA AND S.M. POCKWINSE
UTERINE SIMPLE AND COMPLEX NUCLEAR BODIES
rial, as shown for NB4 in Section 3 and
somewhat in Section 4. The profile of NB4 in
Section 3 might be misidentified as a simple
nuclear body except for its large size.
In Figure 6, nearly the full extent of a
complex body is illustrated in 11 serial sections which span a nuclear area that is 880
nm thick. Visual reconstruction of this complex body suggests that it is somewhat irregularly spherical in shape. It largest diameter
(S7 and 58) is 600 nm. However, as estimated
from section thickness, it would be somewhat
larger than 880 nm. Note that S11 and the
section preceding S1 seen in isolation would
be identified as simple bodies. Thus, the
magnitude of error in counts of the frequency
of occurrence of simple nuclear bodies would
vary with both the shape, number, and size
of the complex bodies. In the least, two erroneous profiles per complex nuclear body
would be introduced in estimations of the
frequency of occurrence of simple nuclear
bodies.
DISCUSSION
This new evidence demonstrates, for the
first time, that simple nuclear bodies exist as
entities which are structurally distinct from
complex nuclear bodies in rat uterine luminal epithelial cells. Since Bouteille et al.
(1974) stated that simple bodies have been
reported “in most tissues in which they have
been carefully sought,” it seems possible that
they may be regularly present in nuclei. In
our experience they are more easily identifiable in highly euchromatic nuclei, rather
than in heterochromatic nuclei.
The structural similarity between the filamentous substance of simple nuclear bodies
and the capsule of the complex nuclear bodies suggests interrelationship, especially
since the center of a simple body is sometimes more lucent. In our recent investigation of the effect of 72 hours of steady
estrogenic stimulation in the immature rat
luminal epithelial cells, we observed a linear
increase in frequency of observation of complex nuclear bodies, while the simple bodies
Fig. 6. ( ~ 2 5 , 0 0 0The
)
extent of complex body is partially illustrated in 12 consecutive 80 nm serial sections.
Although its diameter in this plane is approximately
600 nm, it would be, in another plane, at least 800 nm
since it occurs in 10 sections. Note the presence of small
electron opaque granules in the cores of profiles S5,6,7,
and 8.
129
remained a t approximately the base-line
level (Padykula et al., 1982). Th‘is serves as
a n example of differing functional response
by the two types of nuclear bodies to the
same hormonal stimulus. Whether or not a
functional relationship exists between simple and complex nuclear bodies cannot be
analyzed until their rate of turnover has been
determined.
The possibility that simple bodies may be
precursors of complex bodies has been
brought forth from review of the literature
(Bouteille et al., 1974). The formation of increasing numbers of complex nuclear bodies
in uterine target cells can be achieved by
steady maintenance of high nuclear concentrations of estrogen receptor by steady estrogenic stimulation (Padykula et al., 1981;
Padykula and Clark, 1981). Once the estrogenic stimulation is removed, the nuclear estrogen receptor concentration decreases
rapidly and is accompanied by a steady decrease in the frequency of observation of complex nuclear bodies (Padykula et al., 1982).
The formation and disappearance of complex
bodies is without evident change in the frequency of simple bodies.
Since so little is known about the origin
and function of complex nuclear bodies, speculation is appropriate. They are essentially
protein structures, as demonstrated by trypsin digestion (Krishnan et al., 1967). Our experimental analyses indicate that complex
nuclear bodies increase during progressive
cellular hypertrophy brought about by
heightened transcriptional activity, and conversely decrease in number during cellular
atrophy. Whether or not these complex bodies arise from simple bodies remains to be
determined experimentally. The filamentous
capsule contains protein, as demonstrated by
protease digestion (Krishnan et al., 1967; Dupuy-Coin et al., 1972). The filamentous components of both simple and complex nuclear
bodies may reflect linkage to the “fibrogranular” acidic polypeptide network known as
the nuclear matrix (Berezney, 19791, which
possesses specific binding sites for estrogens
and androgens (Barrack and Coffey, 1980).
The remarkable rapid formation and disappearance of complex nuclear bodies in direct
relation to increasing and decreasing concentration of nuclear estrogen receptor suggests
that they may be transient differentiations
of a pre-existing protein framework. They
disappear from the nuclear scene without
signs of progressive degradation associated
130
H.A. PADYKULA AND S.M. POCKWINSE
with the accumulation of visible debris, as it
occurs during cytoplasmic autophagy.
ACKNOWLEDGMENTS
The assistance of Christopher D. Hebert in
the photographic work is gratefully acknowledged. This research was supported by NIH
research grant HD13941.
LITERATURE CITED
Barrack, E.R., and D.S. Coffey 119801The specific binding of estrogens and androgens to the nuclear matrix
of sex hormone responsive tissues. J. Biol. Chem.
255:7265-7275.
Berezney, R. (1979) Dynamic properties of the nuclear
matrix. In: The Cell Nucleus, Vol. VII. H. Busch, ed.
Academic Press, New York, pp. 413-456.
Bouteille, M., S.R. Kalifat, and J. Delarue (1967) Ultrastructural variations of nuclear bodies in human diseases. J. Ultrastruct. Res.,19:474-486.
Bouteille, M., M. Laval, and A.M. Dupuy-Coin (1974)
Localization of nuclear functions as revealed by ultrastructural autoradiography and cytochemistry. In: The
Cell Nucleus, Vol. I. H. Busch, ed. Academic Press,
New York, pp. 1-74.
Clark. J.H.. J.W. Hardin, H.A. Padykula, and C.A. Cardasis (1978)Role of estrogen receptor binding and transcriDtiona1 activitv in the stimulation of hvperestrogenism and nuclear bodies. Proc. Nat. Acid: Sci.,
752781-2784.
Dupuy-Coin, A.M., S.R. Kalifat, and M. Bouteille (1972)
Nuclear bodies as proteinaceous structures containing
ribonucleoproteins. J. Ultrastruct. Res., 38:174-187.
Fitzgerald, M., and H.A. Padykula (1983) Differing
Functional responses of simple and complex nuclear
bodies in uterine luminal epithelial cells following estrogenic stimuli. Anat. Rec. 205:131-141.
Kierszenbaum, A.L. (1969)Relationship between nucleolus and nuclear bodies in human mixed salivary tumors. J. Ultrastruct. Res., 29:459-469.
Krishnan, A., B.G. Uzman, and E.T. Hedley-Whyte (1967)
Nuclear bodies: A component of cell nuclei in hamster
tissues and human tumors. J. Ultrastruct. Res., 19563572.
LeGoascogne, C., and E.E. Baulieu (1977). Hormonally
controlled “nuclear bodies” during the development of
prepubertal rat uterus. Biol. Cell., 30:195-206.
Monneron, A,, and W. Bernhard (1966) Fine structural
organization of the interphase nucleus in some mammalian cells. J. Ultrastruct. Res., 27266-288.
Padykula, H.A., L.G. Coles, C. Weston, W.C. Okulicz, W.
Robidoux, and W.W. Leavitt (1982) Correlated responses of nuclear bodies and nuclear estrogen receptor to estradiol exposure and withdrawal in uterine
epithelial cells. Abstract #1076, Program of the 64th
Annual Meeting of the Endocrine Society, p. 348.
Padykula, H.A., and J.H. Clark (1981)Nuclear bodies as
functional indicators in the target cells of sex steroid
hormones. In: Cell Nucleus, Vol. IX. H. Busch, ed.
Academic Press, New York, pp. 309-339.
Padykula, H.A., M. Fitzgerald, J.H. Clark, and J.W. Hardin (1981) Nuclear bodies as structural indicators of
estrogenic stimulation in uterine luminal epithelial
cells. Anat. Rec., 201:679-696.
Документ
Категория
Без категории
Просмотров
2
Размер файла
1 411 Кб
Теги
simple, structure, complex, nuclear, entities, bodies, separate, uterine
1/--страниц
Пожаловаться на содержимое документа