/ . Embryol. exp. Morph. Vol. 32, 1, pp. 593-602, 1974
Printed in Great Britain
593
Mitotic cell death and delay
of mitotic activity in guinea-pig embryos following
brief maternal hyperthermia
By M. J. EDWARDS, 1 R. MULLEY, 1 SHIELA RING 1
AND R. A. WANNER 1
From the Department of Veterinary Medicine,
The University of Sydney
SUMMARY
Pregnant guinea-pigs were exposed to an environmental temperature of 42-0-42-5 °C for
1 h on day 21 of gestation. Their embryos were removed at periods from 45 min of heating to
48 h following exposure. Histological preparations of embryos showed clumping of nuclear
chromatin and subsequent death of cells which were at about the stage of mitosis. Affected
cells were particularly numerous in the central nervous system. Further mitotic activity was
inhibited for 6-8 h. Squash preparations of the telencephalon at 1 h after heating showed an
increase from 3 to 86 % in the number of mitotic cells showing damage in the form of nuclear
clumping; this number fell progressively to 30% by 24 h after heating. The proportion of cells
in various stages of mitosis changed considerably at 1-8 h after heating, but had returned to
pre-heating values by 24 h. The proportion of cells in prophase fell markedly, while the
proportion of metaphase cells was doubled at 4 h after heating, indicating blocks to the
cell generation cycle before prophase and in metaphase.
INTRODUCTION
Newborn guinea-pigs which have been exposed to maternal heating during
days 20-23 of gestation usually have a number of serious developmental defects,
including severely retarded development of their brains and less severely
retarded general bodily development (Edwards 1969a, b). The smaller brains of
heated young contain fewer cells and probably fewer neurones (Edwards,
Penny & Zevnik, 1971) than control animals. The deficit in brain development
is not compensated for in post-natal growth, and at maturity these micrencephalic guinea-pigs perform poorly in learning experiments, compared with
control animals (Lyle, Jonson, Edwards & Penny, 1973). Both at birth and at
maturity, the shape of the smaller brain is generally normal, and the histological
appearance does not show any marked deviation from that of controls.
The present experiments were designed to observe the effect of heat on the
1
Authors'1 address: Department of Veterinary Medicine, University of Sydney, Private
Bag, Camden 2570, N.S.W. Australia.
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M. J. EDWARDS AND OTHERS
Hyperthermia and mitotic activity
595
developing guinea-pig embryo, and particularly on the dividing cells of its
central nervous system soon after the heat stress was applied. Preliminary
observations showed that after a day or more post-heating few significant
changes were to be found, so examinations were concentrated within the first
few hours after heating.
MATERIALS AND METHODS
Details of the methods used in determining the stage of gestation and the
induction of hyperthermia have been described elsewhere (Edwards, 1967,
1969tf). On day 21 of gestation guinea-pigs were exposed for 1 h in a forceddraught egg-incubator set at 42-0-42-5 °C dry bulb and 23-0-27-5 °C wet bulb.
In the histological study some embryos were also recovered after 45 min of
maternal heat exposure. Deep rectal temperatures were recorded immediately
before and after exposure. The mean elevations in body temperature of the heated
groups were 3-1-4-0 °C. Embryos were obtained from control mothers and from
mothers after 45 min of exposure, or at short intervals between 0 and 48 h
following exposure. The mothers were anaesthetized by pentobarbital sodium
solution given by intraperitoneal injection, and the embryos were placed immediately in Bouin's fixative. After 24 h they were washed in tap water and
stored in 70 % alcohol. Sections were cut at 6 jum and stained with haematoxylin
and eosin or cresyl violet.
Brain squash preparations were made by obtaining embryos at 21 days of
gestation as before, immersing them in isotonic citrate and quickly excising the
walls of the telencephalic vesicles. The excised portions were cut into pieces of
less than 1 mm diameter, fixed in methanol: acetic acid (3:1) for 10 min and
stained on a glass slide with lacto-acetic orcein for 15 min. They were then
squashed by firm pressure under a glass coverslip, which was sealed to the slide
with molten paraffin.
Embryos were sampled from control mothers and from groups of heated
mothers at 1, 4, 8, 12,16 and 24 h after the end of heat exposure. There were five
mothers in each group and one embryo was examined from each mother. A
number of squash preparations was made, and a total of 500 mitotic nuclei
FIGURES
1-6
Figs. 1, 2. Sections of ependyma of developing brains of control 21-day guinea-pig
embryos. Note mitotic figures confined to cell layer adjacent to ventrical. x 1300.
Fig. 3. Ependyma after 45 min of heating. Clumping of chromatin in cells adjacent
to ventricles, x 1300.
Fig. 4. Vacuolation around clumped chromatin 2 h after heating, x 1300.
Fig. 5. Fragmentation of clumped chromatin 2 h after heating, x 1300.
Fig. 6. Mitotic activity recommenced 6 h after heating. Some cells showing clumping
of chromatin deep in the ependyma. x 800.
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M. J. EDWARDS AND OTHERS
counted from each embryo. Nuclei were classified as prophase, metaphase,
anaphase, telophase or damaged.
RESULTS
Histological study
Sections of control 21-day guinea-pig embryos showed many cells in mitosis
in the ependyma adjacent to the lateral, third and fourth ventricles of the brain,
and adjacent to the central canal of the spinal cord (Figs. 1, 2). The mitotic cells
were not evenly distributed throughout the ependyma. They appeared most
numerous around the lateral ventricles and in the dorsal part of the ependyma of
the spinal cord. Fewer were found in the third and fourth ventricles, the retina
adjacent to the choroid, in the cranial and spinal ganglia, and fewer still in sites
other than the nervous system.
Cellular abnormalities in the nervous system of heated embryos were most
numerous in the regions corresponding to the areas which had shown the most
marked proliferative activity in control embryos.
Some cells adjacent to the ventricles in the developing ependyma of the telencephalon, mid-brain and spinal cord showed a clumping of nuclear chromatin
after 45 min of exposure to heat (Fig. 3). The nuclei of these cells later became
irregular and pyknotic, and their cytoplasm became eosinophilic and then
vacuolated (Fig. 4). By 6-8 h after the end of heat exposure the nucleus showed
karyorrhexis (Fig. 5). At this stage a small number of cells situated some layers
deep in the mantle zone, and well away from the ventricular lining, also showed
pyknotic changes in the nucleus (Fig. 6). Some mesenchymal cells were seen with
similar pyknotic changes, or with two or more nuclei or nuclear particles.
Normal mitotic figures were infrequently found in the ependyma until about
6 h after the end of heat-stress. After this period, numerous mitotic cells were
found both in the ependyma lining the ventricles, and (Fig. 6) some were also
found in the mantle layer.
A similar series of changes occurred in some nuclei of the proliferative zone of
the retina, in the cranial and spinal ganglia and in the developing ear.
By 24 h after exposure, much of the nuclear debris had disappeared, but some
small pieces of chromatin-like material could usually be found about 3-4 cells
deep in the mantle zone. At this stage mitotic activity appeared to be proceeding
briskly.
Squash preparations
As the histological study revealed many cells with clumping of chromatin in
the ependymal layer lining the ventricles, nuclei with this appearance in squash
preparations were assumed to be at about the stage of mitosis, and were
classified as damaged mitotic nuclei. In the control embryonic brains about 3 %
of nuclei were damaged. The proportion was increased to 86 % 1 h after heat
exposure, and progressively decreased to 30 % at 24 h (Table 1). The apparently
597
Hyperthermia and mitotic activity
Table 1. The proportions of apparently normal and damaged mitotic cells in squash
preparations of the telencephalon, at various periods of time after exposure of
21-day guinea-pig embryos to heat
Cells in mitosis
A
heating (h)
Damaged (%)
1
4
8
12
16
24
Controls
(no heat treatment)
Normal (%)
86-4
64-9
43-8
46-2
36-4
30-4
3-3
13-6
351
56-2
53-8
63-6
69-6
96-7
Prophasc
Metaphase
Anaphase
Tclophase
Control
8
12
16
Time after heat exposure (h)
Fig. 7. Changes occurring in the proportion of brain cells in various stages of
mitosis, after exposure of 21-day guinea-pig embryos to heat.
undamaged nuclei, classified according to stage of mitosis, showed a marked
decrease in the proportion of prophase figures, particularly at 4 h, and a decrease of anaphase and telophase nuclei 1 h after exposure (Fig. 7). However,
the proportion of metaphase figures doubled at 4 h. By 24 h the proportions of
all phases of mitosis had returned to control levels.
Figure 8 shows the appearance of normal metaphase chromosomes in a squash
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M. J. EDWARDS AND OTHERS
12
Hyperthermia and mitotic activity
599
preparation from a control embryo. The appearance of the damaged cells in
squash preparations resembled that in the histological sections, and is illustrated
in Figs. 9-12. There were few apparently normal mitotic figures until 6 h after
the end of heat-stress. By 8 h there were numerous chromatin-like fragments
amongst many apparently normal mitotic nuclei. A number of chromatin-like
fragments were still present 24 h after the end of heating. In some squash
preparations, clumped chromatin appeared to be extruded through the nuclear
membrane leaving 'ghost' nuclei containing no chromatin-like material.
DISCUSSION
The results from the present study indicate that both cell death and delay in
mitosis could contribute to the deficit of cells in the brains of newborn guineapigs following heating during early embryonic development.
Most cells which were killed were situated in the ependyma adjacent to the
ventricles, and appeared to be at about the stage of mitosis. In the developing
brain, mitosis normally occurs only at this site (Fujita, 1960; Berry, Rogers &
Eayres, 1964). Cells with clumped chromatin were found in embryos exposed to
45 min of maternal heating. The prevalence of these cells appeared to increase
up to 1 h after exposure and then to diminish up to 6-8 h after the end of exposure.
At this stage, a small number of cells with clumped chromatin or groups of
chromatin fragments were found about 7-8 cells deep in the mantle layer of the
brain. Whether these lesions represent cells which have died in an intermitotic
phase subsequent to heating, or represent cell remnants following heat damage
at about mitosis, is uncertain.
Sections from embryos sampled at the end of the heat-stress period were
usually devoid of cells in mitosis and further mitotic activity was inhibited for
6-8 h. This period of inhibition appeared to be followed by a period of intense
mitotic activity, which might represent a degree of synchronization of mitosis which can be induced by heat-shocks in cultures of unicellular organisms
(Scherbaum & Zeuthen, 1954) or, less efficiently, with cold-shocks in mammalian
cells (Newton, 1964).
The relative contribution of death of cells at about mitosis, and of inhibition
FIGURES
8-12
Fig. 8. Normal metaphase chromosomes of squash preparation from control
embryo. x2000.
Fig. 9. Squash preparation of telencephalon 30 min after heating. Chromosomes
shortened and thickened, x 2000.
Fig. 10. Aggregations of chromatin in squash preparation 30 min after heating.
x 2000.
Fig. 11. Large clumps of chromatin within nuclei 1 h after heating, x 2000.
Fig. 12. Chromatin clumps fragmenting 2 h after heating, x 2000.
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M. J. EDWARDS AND OTHERS
of mitosis, to the cell deficit which exists in the brain at birth is difficult to assess
from the data available, as it is possible that the apparent burst of mitotic
activity 6-8 h after the end of heating might compensate for the period of
inhibition.
If it is assumed that most cells which were injured will show clumping of
chromatin by 1 h after heating, and that few additional cells will show these
changes after 1 h, the data of Fig. 7 indicate that the cell generation cycle is
blocked before prophase and in metaphase. As the proportion of cells in
prophase falls sharply by 1 h and is halved by 4 h, the block before prophase
appears to be caused by some interference with a process occurring in the G 2
phase and perhaps in the S phase.
The block occurring in metaphase might result from a denaturation of the
spindle. A reversible change in the nature of the spindle of chick embryo cells
has been produced by exposure to incubation temperatures which were higher
than normal (Lewis, 1933). Although it would be convenient to propose simply
that the clumping of chromatin found in cells at about mitosis might result from
such a denaturation of the metaphase spindle and collapse of the unsupported
chromosomes, this mechanism does not appear to be likely in all cases, as
cells in metaphase do not appear to be the only ones involved. For a period after
the heat treatment, a nuclear membrane still enclosed the clumped chromatin of
some affected cells. The nuclear membrane normally disappears toward the end
of prophase and is re-formed during telophase (Mazia, 1961).
Fertilized sea-urchin eggs (Psammechinus miliaris) heated to temperatures
which block cell division (25-27 °C) show a nuclear cycle in which the nuclear
envelopes dissolve, but chromosomes do not split into chromatids and a single
nucleus is reconstituted. Similar irreversible damage to mitotic cells occurs also
in Limnaea and Schizosaccharomyces. It was considered that the damage to cells
in mitosis and the set-back of interphase cells of these cultures were both due to
damage to microtubules (Zeuthen, 1972). Cultures of Tetrahymena pyriformis
can be prevented from entering mitosis by heat-shocks induced by changing the
temperature of incubation from the optimum of 28-29 °C to a sublethal temperature, 32-34 °C (Scherbaum & Zeuthen, 1954). Rao & Engelberg (1966) found
that Hela cells stopped dividing immediately after a change in incubation
temperature from 37 to 41 °C. There was also a lag of 4 h, during which no
divisions occurred, after a shift from 34 to 37 °C, which suggests that the
relative change in temperature might be important in the delay imposed on
mitotic activity.
Many other possible causes of the delay in mitosis following heat treatment of
cultures have been advanced, including disintegration of the many preparatory
processes required for division (Prescott, 1961); the inactivation of a specific
division-linked protein (Scherbaum, 1963; Zeuthen, 1963); transitions in the
state of intracellular water or changes in the properties of lipid membranes
(Rao & Engelberg, 1966); prevention of synthesis and destruction of division-
Hyperthermia and mitotic activity
601
associated RNA (Moner, 1967) and ultrastructural lesions affecting the nucleolus (Amalric, Simard & Zalta, 1969; Simard, Amalric & Zalta, 1969; Love,
Soriano & Walsh, 1970). However, heat-induced cell death is popularly believed
to be due to protein denaturation. Rosenberg, Kemeny, Switzer & Hamilton
(1971) showed a good numerical correlation between thermodynamic parameters
of protein denaturation and cell death rates. Westra & Dewey (1971) believed
that heat inactivation of cells results primarily from denaturation of proteins.
They showed cultures of Chinese hamster ovary to be most sensitive to the lethal
effects of heat during the S phase and mitosis. It was recently calculated that
0-2 % of cells cultured from lungs of Chinese hamsters and incubated at 37 °C
were irreversibly lost from the proliferative population as a result of heat injury.
The theoretical upper temperature limit for the growth of these cells was found
to be 40-6 °C, and rapidly proliferating cells were more sensitive than slowly
proliferating cells (Johnson & Pavelec, 1972).
The set-back to interphase cells by heat-shock might represent a mechanism
of some importance in the protection of a proliferative cell population against
the lethal effects of heat. The damage produced by moderate elevations of
temperature during interphase appears to be largely reversible and it prevents cells from entering mitosis when this increased temperature might cause
irreversible damage.
This study was supported by grants from the Wellcome Foundation, the Australian
Research Grants Committee and the World Health Organization.
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LYLE,
(Received 4 December 1973)