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Structural changes in nuclear chromatin in rat pituitary after chronic stress of low intensity.

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THE ANATOMICAL RECORD 220:125-131(1988)
Structural Changes in Nuclear Chromatin in Rat
Pituitary After Chronic Stress of Low Intensity
DYMITR KOMITOWSKI, SHUHEI MUTO, fiRGEN WEISS, BERTOLD S C H M I n , AND
GEORGE T. TAYLOR
Institute for Experimental Pathology, German Cancer Research Center, 0-6900Heidelberg,
FRG (D.K., S. M., B.S.); Center for Molecular Biology, University of Heidelberg, 0-6900
Heidelberg, FRG (J.W.);
Laboratory for Psychobiology, University of Missouri-St. Louis,
‘ :‘1:)
St. Louis, MO 63121 (G. 1
ABSTRACT
Acute, intense sources of “psychogenic” stress clearly modify the
structure and function of the hypophysis, and there are concomitant changes in
many peripheral physiological systems. Less dramatic sources of stress yield more
equivocal results. An experiment is reported in which nuclear morphology of adenohypophyseal cells from 49 male rats exposed to a chronic, low-intensity stressor was
examined both by conventional histological and computer-assisted-image-processing
methods. The hypothesis tested was that an unequivocal pattern of morphological
changes in the nucleus and nuclear chromatin would be revealed by image processing. Rats were killed after living for a year in a relatively low-stress environment,
“crowded” in groups of five animals per cage. The control condition was a minimal
stress environment of two rats per cage. Results suggested few signs of pathology
from peripheral measures of hypophyseal activity, and direct light microscopic
examination of the gland revealed no differences between the two groups. Analysis
of computer-enhanced images of the pars distalis nuclei from the adenohypophysis,
on the other hand, generated findings that were statistically and biologically significant. Nuclear size increased in the stress condition, the number of chromatin and
area occupied by the particles increased, and the position of chromatin shifted
toward the periphery of the nucleus. Perhaps more important, optical density analysis indicated that chromatin was less tightly packed in the experimental animals.
Implications are that chronic, low-intensity stress modulates nuclear structural
changes from a dormant to an active state that portend changes in the peripheral
systems influenced by the hypophysis.
Recent progress in the study of the pituitary has confirmed functions that were suggested five decades ago.
Moreover, there is now a clear picture of such features
as cellular localization and functional consequences of
structural changes in the pituitary. A key question that
remains unanswered is the interrelation among different cell types (Kovacs, 1984; Tixier-Vidal et al., 1984).
The anterior lobe of the gland contains distinctive cell
types that influence a notable array of physiological
systems. Somatotropic and thyrotropic cells influence
metabolism, gonadotropic and lactotropic cells affect fertility, and adrenocorticotropic cells influence various homeostatic processes (Martin, 1985). Stress can influence
all these peripheral systems (McGrady and Chakraborty, 1983; Munck et al., 1984; Pollard, 19841, and there
is general agreement that the stress effects are mediated
by the hypophysis (Hennessy and Levine, 1979).
The possibility that everyday sources of “psychological” stress may influence the genesis and onset of pathological states has been entertained for years, but there
is little empirical support (Locke et al., 1984). Experi0 1988 ALAN R. LISS, INC.
mental findings with animal models, most commonly
rodents, indicate that acute, intense stressors can induce
pathological changes in liver (Hidalgo et al., 19861, testes
(Collu et al., 1979), digestive tract (Glavin, 19851, and
circulatory system (Fokkema and Koolhaas, 1985). Direct histological analysis of acutely stressed animals is
unusual, but Moriarty et al. (1975) reported cytoloplasmic changes in parenchymal cells of the intermediate lobe of the rat hypophysis. The most reliable of the
acute stressors, in comparison with a nonstressed control animal, are immobilization by restraint (Quirce and
Maickel, 1981) and electric footshock (Friedman and
Iwai, 1976). Subtle manipulations, on the other hand,
have yielded more equivocal results. For example, both
predictable and unpredictable footshock (Maier et al.,
Received April 13, 1987; accepted August 11, 1987.
Address reprint requests to Prof. Dr. Dymitr Komitowski, Institute
for Experimental Pathology, German Cancer Research Center, Im
Neuenheimer Feld 280, D-6900 Heidelberg, FRG.
126
D. KOMITOWSKI ET AL.
Fig. 1. A general microscopic view (a) of hypophysis from a male rat framed indicates the region of measurements made by image analysis.
showing the three major anatomical subdivisions: pars distalis (PD), H.E. x 125. Higher-magnification photomicrographs of pars distalis
pars intermedia (PI), and pars nervosa (PN). The section of PD that is from a representative (b) control and stressed (c) rat. H.E. ~ 5 0 0 .
1986)similarly increased the release of glucocorticosteroids and ACTH, and reliable dose-response changes in
physiology with different stress intensities have proved
difficult to demonstrate (Murison et al., 1986; Natelson
et al., 1987). One problem may be that the acute stressors that have been employed bear questionable relation
to the evolutionary history of the experimental animal
(Brain and Benton, 1979; Domanski et al., 1986). More
“ecologically valid” sources of stress may yield more
reliable data.
In a n initial attempt to establish a more valid stressor,
we reported recently on the effects of manipulating aggressiveness of male rats as a source of chronic social
stress (Taylor et al., 1987). The experimental paradigm
employed was notable mostly for its complexity. A simpler chronic social stressor would be to house different
numbers of animals in similar-size cages (Brayton and
Brain, 1984; Gamallo et al., 1986). The apparent low
intensity of “overcrowding,” however, provokes few reliable changes in pituitary or peripheral structures
127
STRESS AND CHROMATIN STRUCTURE
Fig. l(c).
(Benton et al., 1979; Shalicky et al., 1984). Rather than
being a disadvantage for the present experiment, however, the unreliability of manipulating housing conditions was a mechanism by which to test the effectiveness
of computer-assisted image processing. Using subvisual
quantitative parameters, the system can describe very
subtle morphological changes (Curcio and Sloan, 1986;
Panno and Nair, 1986). The basic function of the adenohypophysis is its hormonal activity, and function is related to structure of both nucleus and nuclear chromatin
(Alberts et al., 1983; Thrall et al., 1978).The hypothesis
tested was that there would be systematic changes in
the morphology of pars distalis cell nuclei, as revealed
by image processing, in the hypophysis of male rats
housed chronically in a low-stress environment.
cessing. Hypophyses prepared for routine light microscopy were fixed immediately in buffered formaldehyde
(pH 7.4). The tissue was imbedded in paraffin blocks, cut
at 2 pm (Autocut 1140, Reichert-Jung), and stained with
haematoxylin and eosin. In order to differentiate cell
types, from every four Feulgen-stained sections, one section was stained by the Glenner and Lillie (1957) procedure and a second section was stained by PAS (Pearse,
1968). For image analysis, serial paraffin sections from
hypophyses were prepared as described above for light
microscopy and were stained by Feulgen's method
(Pearse, 1968). In the Feulgen reaction the fluorescent
staining displays DNA in nuclear chromatin (Ross and
Reith, 1985).
MATERIALS AND METHODS
Animals and Maintenance
The system for image analysis includes a light microscope (Axiomat, Zeiss), plumbicon scanner, Quantimet
720 analyzer (Cambridge Instruments Inc., Dortmund),
and color display. Computers were VAX 11/750,PDP 11/
34, and LSI 11/23 models (Digital Equipment Corp.,
Maynard). There are several discrete steps used by the
computer to analyze tissue. Details are presented in
more technical publications by Komitowski and associates (Komitowski and Zinser, 1985; Komitowski et al.,
19831, but a brief description of the process employed for
the present research follows; the major steps are presented in the second figure.
Initially, an image of the Feulgen-stained section of
the nuclei from a section of adenohypophyseal tissue
was generated by microscopic illumination. The images
were digitized in a matrix of 768 x 512 picture elements, or pixels. The optical density of each pixel was
evaluated by the comDuters relative to "may" levels
remesentin; 64 different levels of light intensity (Fig.
2a.b).
Each &el corresDonds to 36 n& of the obi&t. agd
,
the images 'were segmLntd (Fig. 2c,d) by a polir ckrdinate method (Zinser and Komitowski, 1983).Parameters
The animals were male Wistar rats man: Wistaroutbred stock) that were killed and examined at 14
months of age, 1 year after being housed in groups of
two (n=24) or five (n=25) rats per cage. Cages were
clear plastic with inside dimensions of 37.7 cm x 21.5
cm x 15 cm (total available space = 12,158 cm3).Room
conditions were standardized with 22 f2"C, 55 f 5 % humidity, and 12-hour light: 12-hour darkness that were
controlled automatically. Water and food (Ssniff, Soest)
were freely available. At the end of a year in the environment, all animals were killed by carbon dioxide
asphyxiation.
Tissue Preparation
At necromv the animals were weighed. adrenal glands
and hypophises were excised, andU wet weight; were
obtained. Testes were removed, weighed, and prepared
for sperm counts by using a method described earlier
(Taylor et al., 1983). Hypophyses from each group were
prepared for conventional light microscopy or image pro-
Image Processing
,
~
128
D. KOMITOWSKI ET AL.
Fig. 2. Representative nuclei from control (a,c, and e) or stressed (b,
d, and f) rats demonstrating the steps involved in computer-assisted
image analysis (see text). ~9,000.
of nuclear contents were described by using a n algorithm that enables the localization of condensed chromatin regions within the matrix based on locally
adaptive thresholding. These were defined as regions
with high optical density or chromatin particles. Each
chromatin particle could be identified and the number
of particles per fixed area determined; evaluation of
parameters such a s area occupied by chromatin and
position of the particle within the nucleus was also possible (Fig. 2e,f). The final step is optical density analysis, or the amount of light passing through the chromatin that provides a n accurate measure of the relative degree of chromatin condensation or packing (Yaniv and
Cereghini, 1986).
A total of 889 and 1,266 nuclei were analyzed in this
manner from the control and experimental groups, re-
STRESS AND CHROMATIN STRUCTURE
129
Fig. 2(e,f).
Quantitative Image Analysis
spectively. As described previously (Komitowski et al.,
1986), a n area of visual field was selected a t random
from tissue sections, and all nuclei within that visual
field were analyzed. We did not distinguish among cell
types for the analysis because the cells are functional
interdependent (Thrall et al., 19781, and our interests
are in the structural properties that are common to all
pars distalis cells (Allen et al., 1977).Also, we examined
only the nucleus and its chromatin because structural
changes at that level determine transcriptional processes that modulate hormone synthesis and morphological changes (Alberts et al., 1983) in the gland and,
subsequently, in peripheral physiological systems.
RESULTS
Light Microscopy
Excised glands and adenohypophyseal tissue were prepared for conventional histological analyses. Two pathologists in the histodiagnostic division of the German
Cancer Research Center independently examined tissue
sections without knowledge of which group the animals
belonged to. Results were described in the same manner
in which other tissues are described during routine histological service to researchers in the center. There were
infrequent pathological changes in peripheral tissues
including pneumonia, endocardia1 fibrosis, and nephrosis, but these findings were not related to differences in
housing conditions.
Morphology of individual hypophyseal cells, including
integrity of the nuclear envelope, membranes, cytoplasm, and nucleus, appeared quite normal in both sets
of animals (Fig. 1). Percentages of acidophilic, basophilic, and chromophobic cells relative to the total cell
population did not differ among the rats nor from those
values reported in the literature (Nakane, 1973).
Analyses of nuclei and nuclear chromatin were the
primary interest. The parameters examined in each nucleus were size of nucleus, number of particles and area
occupied by chromatin, position of particles relative to
the periphery of the nucleus, and, finally, the degree of
packing of chromatin fibers. Area occupied by chromatin was calculated in pm2 of nucleus to allow comparisons of nuclei of different sizes. The capacity of chromatin packing to organize with higher-order foldings of
DNA (Nicolini, 1983) allows number, area, and density
of chromatin to be calculated independently of each
other. The data were combined for the animals in each
group and appear in Table 1.
A series of t-tests at 5% significance level were used to
analyze those data, and the results revealed statistically
reliable values for each of the five parameters. Animals
from the experimental group had adenohypophyseal nuclei that were larger than those from the control group.
Experimental males also had higher numbers of chromatin, and the area occupied by chromatin particles was
greater. The position of chromatin within the nucleus
was more peripherally located, and chromatin particles
were more loosely packed in experimental males. Multivariate analysis of variance indicates that nuclei from
the two groups were clearly from different statistical
populations, F(5, 2149) = 289.91, P< .01.
Finally, representative nuclei were selected from adenohypophyseal tissue obtained from a rat from each
group, and the image-analysis process is shown in Figure 2. Figure 2a,b shows the initial image of a typical
nucleus projected by computer. Figure 2c,d demonstrates the intial image of the nucleus with its detected
contour. Figure 2e,f is the computer-generated digitized
image in which one can note clearly different areas
occupied by densely compacted chromatin.
130
D. KOMITOWSKI ET AL
TABLE 1. Computer-assisted quantitative image analysis findings from nuclei and
nuclear chromatin from adenohypophysisof rats maintained under chronic low-intensity
stress
Mean
Control
Measure
Nuclear area (pm')
Number of particles
Area occupied by
particles (pm')
Optical density of
particle (per pm')
Percentage of particles
in periphery
SEM
t-value
Change (%)
33.54
13.52
13.19
+31.4
0.2'
+ 0.03'
+ 16.7
+ 16.7
26.2 & 0.4
17.8 & 0.2l
20.57
-32.1'
60.6 rt 0.3
61.8 f 0.3'
3.04
group
22.3 f 0.2
25.1 & 0.2
4.02 k 0.04
Stress group
29.3
29.3
4.69
0.1'
+2.0
'Significant difference from the control group ( P < .01).
'Decrease in this parameter value indicates more light is passing through the nucleus.
Peripheral Measures
Of secondary interest was a general measure of each
of the broad functions of adenohypophyseal cells. Specifically, body weight was used as a gross measure of
disruptions in metabolism, and a t-test on those data
indicated that there were no differences between the
groups, mean = 541 f SEM = 12 gm, for controls and
522 f 11 for experimental animals. Similarly, there
were no differences in a crude measure of adrenal-pituitary interactions; wet weights in mgI100 gm body
weight of adrenal and pituitary glands were 10.5f0.6
and 2.2k0.2, respectively, for the controls and 10.7f0.4
and 2.350.2, respectively, for the experimental animals.
The measure of fertility was number of sperm; again the
groups did not differ significantly. The values of control
males and experimental males in x106 per gm testicular tissue were 82.8k8.6 and 70.6 f3.3, respectively.
DISCUSSION
Data from the present experiment suggest that adenopophyses of male rats undergo structural modification
during exposure to stress. There were two contributions
from the present study to the literature on stress-pathology relations. First, a computer-assisted examination of
sectioned pars distalis tissue revealed systematic quantitative changes that were undetected with conventional
analyses. Second, a chronic social stressor of relatively
low intensity, living in crowded conditions for a year,
induced structural modifications that were reliable and
common to the varied cell types in the gland.
Previous findings from animals maintained under
mildly stressful conditions have proved difficult to replicate (Brain and Benton, 1979). The apparent conclusion is that mild stress provokes no systematic
pathological changes or that the results reflect individual differences to stress Nogel, 1985). If only typical
markers of the effects of stress on peripheral systems
were used, the present data would have presented a
similar picture. Body weights, wet weights of glands,
and sperm counts did not differ under the two housing
conditions. Conventional histological analyses with light
microscopy were no more helpful. Characterization of
adenohypophyseal tissue suggested that there were no
reliable differences between the two ~ O U D Sof rats.
Quantitative image processing on the z h e r h a n d , sug-
gested clearly that the stress provoked systematic
changes in adenohypophyseal nuclei.
There were five parameters of the nuclei that were
analyzed with computerenhanced image processing, and
differences between the groups were statistically and
biologically significant. Size of the nucleus increased by
approximately 30% under stress. Number of chromatin
particles increased, as well a s the area within the nucleus occupied by chromatin. Location of chromatin
changed with a small, but statistically significant, shift
toward the periphery of the nucleus. Finally, there was
a loosening in the packing of chromatin. These latter
data may prove to be the most important finding and
may demonstrate the usefulness of image processing as
a powerful supplemental tool for special problems in
histology (Panno and Nair, 1986).
Structural change in the hypophysis has profound implications on the integrity of varied peripheral physiological systems. There were no differences, at least none
detected by traditional measures, between the peripheral systems of the two groups. The chromatin data,
however, suggest that the pituitary was undergoing
structural changes that later would be clearly revealed
in both central and peripheral systems.
There is evidence from postmortem analyses of the
hypophysis that many people harbor small "silent" adenomas without exhibiting detectable clinical symptoms
(Hausson et al., 1982;Krieger, 1984).Although we found
no obvious tumors, there were changes in the nucleus
that suggest that cells from rats under stress were in a
state of activation. There are data in the literature indicating that transcriptionally active nuclei are characterized by increased size of the nuclear envelope (Franke
et al., 1981), and nuclei of stressed animals were hypertrophic. Stressed animals also exhibited increased chromatin, which suggests higher DNA content (Wolley et
al., 1982) and activation of the nucleus (Haag et al.,
1977; Nicolini et al., 1982). Although movement of chromatin toward the periphery of the nucleus in crowded
rats is of uncertain biological significance, the capacity
of optical density analysis to demonstrate a loosening of
tightly bound chromatin is of special interest.
Only a small fraction of the DNA in nuclei of a cell is
transcriDtionallv active (Darnell. 1982). Although some
of thesg genes &e likely permanently' inactivGed dur-
STRESS AND CHROMATIN STRUCTURE
ing the developmental process, the remaining inactive
genes must await signals that convert inactive chromatin into its active counterpart (Yaniv and Cereghini,
1986).The suggestion that psychosocial stress can act as
one of these signals remains a n attractive hypothesis
without empirical support (Grossarth-Maticek et al.,
1985).The present data offer some support for that idea.
A prominent feature that distinguishes active from inactive chromatin is the packing of the particles. Active
chromatin is more loosely packed (Nicolini, 1983). The
present data suggest that low-intensity chronic social
stress can activate formerly inactive chromatin.
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