Published August 1, 1966
SOME
OBSERVATIONS
STRUCTURE
OF THE
OF X-IRRADIATED
BOJAN
FLAKS
ON THE
LUTEIN
RAT
FINE
CELLS
OVARY
and P A U L
BRESLOFF
From the Department of Experimental Pathology and Cancer Research and the Steroid Laboratory
Department of Chemical Pathology, the School of Medicine, Leeds, England
ABSTRACT
INTRODUCTION
Mandl and Zuckerman (9) have shown that adult
rats enter a phase of constant vaginal cornification
after irradiation of their ovaries by sterilizing doses
of X-rays which produce complete destruction of
follicles and oocytes and that, in these circumstances, the resulting constant vaginal cornification can be terminated by total ovariectomy. It
has been suggested that even normal cyclic estrus
can occur in the absence of the follicular system
of the ovary (12-16).
It would appear, then, that the cells remaining
in the ovary, after treatment with large doses of
X-rays, must be responsible for production of the
factor which causes constant vaginal cornification.
This view is supported by the work of Westman
(18) which involved parabiosis between normal
and X-ray-sterilized rats.
The present study was undertaken in order to
observe what morphological changes were present,
in the ovarian tissues remaining after X-irradiation, concurrent with constant vaginal cornification.
MATERIALS
AND METHODS
The animals used were albino rats of a Sheffield
Wistar strain, bred at the Mcdical School in Leeds
England.
At 10 to 13 wk of age, rats were subjected to bilateral exteriorization of thcir ovaries, using a dorsal
approach, under sodium amytal anesthesia. The
technique of Mandl and Zuckerman (9) was followcd,
227
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T h e ovaries of 10- to 13-week-old rats werc extcriorized and irradiated with sterilizing doses
of X-rays. Following treatment, the animals cntercd a phase of constant vaginal cornification. Animals were killed 8 to 12 wk after the onset of this phase, and their ovaries were
prepared for electron microscopy. Tissue was fixed in glutaraldehyde, postfixcd in osmium
tetroxide (Millonig's, phosphate-buffered), and embedded in Epon. Lutein cells from these
ovaries were compared with those from sham-irradiated controls. The cytoplasm of lutein
cells from experimental animals was characterized by an increase in the amount of agranular
endoplasmic rcticulum and by an increase in the numbcr of mitochondria. These mitochondria are more variable in external form and often possess increased numbers of villiform
cristae. Other features noted were a decrease in the amount of cytoplasmic lipid granules
and an increase in cell size and surface irregularity. The significance of the morphological
findings is discussed in relation to ovarian hormone production in animals sterilized by
X-irradiation.
Published August 1, 1966
OBSERVATIONS
Light Microscopy
Light microscope e x a m i n a t i o n of serial sections
of paraffin preparations reveals the total absence
228
of oocytes a n d follicles in the X - i r r a d i a t e d r a t
ovaries. T h e picture presented by these ovaries is
d o m i n a t e d by large luteinized cells usually organized in bodies resembling corpora lutea. No
corpora albicantes were observed in sections.
T h e luteinized cells observed in X - i r r a d i a t e d
rat ovaries resemble the granulosa lutein cells of
n o r m a l corpora lutea, with respect to their large
pale staining nuclei with p r o m i n e n t nucleoli, a n d
a b u n d a n t cytoplasm (Figs. 1 a n d 2),
Electron Microscopy
Comparison between lutcin cells from irradiated
a n d control ovaries shows a n u m b e r of differences.
T h e lutein cells of the former are considerably
larger, the increase in size being almost entirely
due to a n increase in the a m o u n t of cytoplasm.
T h e cell m e m b r a n e of control lutein cells (Fig.
3) shows some folds a n d projections. T h e surface
irregularity is most m a r k e d o n cell margins which
face o n perivascular spaces surrounding the
endothelial cells of capillaries. T h e cell m e m branes between adjacent lutein cells are generally
smooth. T h e extent of surface irregularity is increased in irradiated cells (Fig. 4) a n d often extends between adjacent cells, appearing as folds
a n d microvillous projections,
Lutein Cells from Control Ovaries
T h e cytoplasm of lutein cells from control animals (Fig. 5) shows elements of the a g r a n u l a r
endoplasmic rcticulum in the form of b o t h vesicles
a n d irregularly b r a n c h i n g tubules. I n all animals
examined, the a m o u n t of endoplasmic reticulum
varied considerably from one cell to another.
A b u n d a n t free ribosomes are present, usually
aggregated into groups of four to six units. R i b o somes are also found, less frequently, a t t a c h e d to
m e m b r a n e s of the endoplasmic reticulum, which
then takes the form of parallel arrays of eisternal
elements. T h e n u m b e r of m e m b r a n e pairs, which
are a r r a n g e d in this m a n n e r , is usually restricted
to two or three in these cells, as seen in sections.
I n control lutein cells, the Golgi zones a p p e a r
to be widely distributed in the cytoplasm a r o u n d
the nucleus a n d consist of small stacks of cisternae
with very few associated vesicles.
N u m e r o u s m i t o c h o n d r i a are present in the cytoplasm of the lutein cells from the controls. T h e i r
outline is most c o m m o n l y r o u n d or oval, although
occasional cup-shaped or elongated forms are
seen, a n d they possess villiform or t u b u l a r cristae,
T ~ s JOURNAL OF CELL BIOLOGY • VOLUME 30, 1966
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in which the remainder of the animal's body is
shielded by lead. Irradiation of the exposed ovaries
was then carried out, with unfiltered soft X-rays at
200 kv and 15 ma, having a half-layer value of 0.5 m m
of copper. The focus-to-source distance was 20 cm.
The animals received total doses of 4700 -4- 300 R,
during a continuous period of 9 min, at a dose rate of
approximately 525 R per minute. Dose rate was
measured with a Baldwin-Farmer substandard X-ray
dosimeter (Baldwin Instrument Company, Ltd.,
Dartford, England), and the total dose with Con-Rad
LiF, type N and Cou-Rad T L D thermoluminescence
dosimeters (Controls for Radiation, Inc., Cambridge, Massachusetts) (2). Control animals were
sham-irradiated by subjecting them to the full surgical procedure, but the X-ray source was not activated.
Following operation, vaginal smears were taken
daily in order to monitor vaginal cornification. Experimental animals were sacrificed 8 to 12 wk postoperatively, several weeks after they had entered the
phase of constant vaginal cornification.
The controls were sacrificed when at full estrus, as
shown by vaginal smear. At necropsy the ovaries were
removed for processing for both light and electron
microscopy.
For electron microscopy, the tissue was cut into 1
m m 3 blocks and fixed for 4 hi" at 0 to 4 C in 5 % glutaraldehyde (6), buffered with 0.067 M cacodylate at
p H 7.2. After being washed for a minimum period
of 16 hr with cold 0.25 M sucrose in 0.1 M cacodylate
buffer, the tissue was postfixed in cold, phosphatebuffered, 1% osmium tetroxide (10) for 2 hr.
Rapid dehydration was carried out according to
the method of Luft (8) and the tissue was embedded
in Epon 812. Thin sections were cut on a PorterBlum ultramicrotome and stained with lead tartrate
(11), either alone or preceded by uranyl acetate
staining (19). The sections were examined in an RCA
EMU-3G, with accelerating voltages of either 50 or
100 kv.
Thick parallel Epon sections were cut on the Porter-Blum ultramierotome, stained with 1% toluidine
blue in ethanol, and examined by light microscopy so
that the sections examined in the electron microscope
could be correlated with the histological appearance
of the tissue under investigation.
For light microscopy, tissue was fixed in formaldehyde-saline and embedded in paraffin wax. Sections
were stained with haematoxylin and eosin and examined for evidence of luteinization and destruction
of follicular structures.
Published August 1, 1966
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FIOURE 1 Sham-irradiated control ovary showing portion of corpus luteum. The upper part of the field
shows closely packed granulosa lutein cells (GL). Photomicrograph. X 400.
FIGURE 2 X-irradiated ovary showing luteinized tissue, the cells of which resemble the lutein cells of Fig.
1. Photomicrograph. X 400.
Published August 1, 1966
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:FIGURE 3 Lutein cell from ovary of sham-irradiated rat. The cell surface is seen to be relatively smooth.
Large numbers of lipid granules are present. X 12,000.
FIGIYnE 4 Ovary of X-irradiated rat showing portions of two lutein cells. Cell surfaces are irregular and
have numerous invaginations (v). The cytoplasm has many tubules and vesicles belonging to the smooth
endoplasmic reticulum. The mitochondria (m) show villiform cristae, characteristic of these ceils. Their
numbers appear to be increased and their shape and size more variable. X 1~,000.
Published August 1, 1966
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FIGVRE 5 Lutein cell from ovary of sham-irradiated rat showing smooth endoplasmic retieulum, Golgi
zone (g), and numerous free ribosomes, the majority of which are associated as polysomes. The nuclear
membrane is interrupted by nuclear pores (arrows). X 16,500.
FIGVaE 6 Cytoplasm of X-irradiated lutein cell is filled with smooth endoplasmic reticulum (ser). The
interiors of the mitochondria show increased numbers of villiform cristae. Numerous lysosomes (ly) are
present in the peripheral cytoplasm. The cell surface (arrows) is seen to be highly irregular. X 16,500.
Published August 1, 1966
considered to be characteristic of the mitochondria
of steroid-producing cells (1, 7). They possess a
dark matrix and usually contain a number of
intramitochondrial granules. Less frequently,
more elongate mitochondria having lamelliform
cristae are found. These form only a small proportion of the mitochondrial population of these
cells.
T h e cytoplasm of many control lutein cells
contains large numbers of lipid droplets (Fig. 3),
which appear to be accumulated in one area.
Zutein Cells from X-Irradiated Ovaries
232
DISCUSSION
The preceding observations compare the fine
structure of lutein cells from ovaries of control
animals with that of lutein cells from ovaries of
animals that received sterilizing doses of X-rays.
In both cases, ovaries were removed from animals
in estrus, the criterion for this being maximal vaginal cornification.
We have used the term "lutein cells" to describe
the cells observed, in this study, in both control
and experimental ovaries. Clearly this term could
be open to the criticism that the origin of the cells
found in X-irradiated ovaries may not be from
the granulosa lutcin cells of normal ovaries. However, in view of the resemblance in histological
preparations of the former to normal granulosa
lutein cells and the similarity of their uhrastructure to that of the stimulated lutein cells described
by Enders and Lyons (5), we consider our ter-
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The lutein cells from irradiated animals are
larger than those from controls and show some
striking differences in cytoplasmic appearance.
The number of mitochondria per cell appears
to be greatly increased and the proportion of the
cytoplasm which they occupy is also increased.
The mitochondria are commonly enlarged and
are more variable in form (Figs. 7 to 9) than those
from control lutein cells. While many mitochondria appear to be spherical in shape, others are
elongated or cup-shaped. These forms are more
common than in control lutein cells. The matrix
of these mitochondria is darker than the ground
cytoplasm and they possess abundant villiform
cristae (Fig. 8) which are often tortuous and
closely packed. Those mitochondria which have
this type of structure are usually of the spherical
type and possess numerous intramitochondrial
granules. Lamelliform cristae are also found, but
are more commonly present in the elongated type
of mitochondrion. Different forms of mitochondria
are found in the cytoplasm of any one cell.
Lutein cells from X-irradiated ovaries contain
abundant agranular endoplasmic reticulum. This
frequently occupies most of the cytoplasm and is
the dominating structure of these cells (Figs. 6
and 10). The amount of endoplasmic reticulum
present is consistently much greater than in control cells. The reticulum takes the form of tortuous
branching tubules or vesicles (Fig. 4), often closely
associated with mitochondria and containing variable quantities of finely granular material. Tubular and vesicular forms of reticulum are often
observed in the same cell, and on this basis no
significant difference between different lutein cells
from ovaries of X-irradiated animals could be
found. The vesicular form is a common feature of
lutein cells from all animals examined, although
it is more frequently found in experimental ani-
mals than in controls. It is to be found in cells
which possess well preserved lipid granules, in
preparations showing well preserved erythrocytes.
For this reason we do not consider the presence
of vesicular agranular reticulum to be necessarily
indicative of inadequate fixation.
Since there appears to be little constancy in the
size of the elements of the endoplasmic reticulum,
the recognition of Golgi zones is difficult. When
observed, the Golgi zones do not appear to differ
in structure or number from those of controls.
Free ribosomes are numerous, but their numbers and state of aggregation are similar to those
of control lutein cells. Cisternal element3 of endoplasmic reticulum with attached ribosomes are to
be found in the cytoplasm, near the nucleus. No
difference is observed between lutein cells from
experimental and control ovaries, with respect to
this organelle.
The number of lipid globules present in the
cytoplasm appears to be reduced in lutein cells
from irradiated ovaries. Those globules that remain are more variable with respect to their
density and size. They are usually scattered diffusely throughout the cytoplasm, and many are
found in close apposition to mitochondria (Fig. 8).
Lutein cells from both X-irradiated and control
ovaries possess large nuclei which are somewhat
irregular in outline. The perinuclear cisternae are
perforated by numerous nuclear pores. In all cells
examined, prominent skeinlike nucleoli were present.
Published August 1, 1966
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FIGun~ 7 X-irradiation. Cytoplasmic detail from lutein cells showing interdigitating cytoplasmic processes of adjacent cells. X 15,000.
Fmuu~ 8 Mitochondria from lutein cell of X-irradiated rat ovary are closely associated with lipid globules. The mitochondria possess abundant villifo~an cristae in a finely granular matrix. X 20,000.
Published August 1, 1966
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Frumpy. 9 X-irradiated ovary. Detail of cytoplasm of lutein cell showing mitochondria and agranular
endoplasmie reticulum. X 48,000.
l~otmE 10 X-irradiated ovary, showing a portion of cytoplasm filled with agranular endoplasmic l~tieulure. X 48,000.
Published August 1, 1966
This feature is an apparent increase in the number of mitochondria per unit of cell area and per
cell, particularly of the enlarged and spherical
types of mitochondria, and in the number of their
villiform cristae. This feature suggests that these
cells may be highly active, especially since lipid
globules are commonly associated closely with
these mitochondria. Although the agranular endoplasmic reticulum appears to be the organelle
which is directly responsible for steroid synthesis in
the cells under consideration, the energy and reduced pyridine nucleotides required for this process
must be produced by mitochondrial activity. We
therefore regard the increase in the number of
mitochondria, which parallels the increase in the
amount of agranular endoplasmic reticulum, as
having some significance as an index of cellular
activity, though not necessarily specific for steroid
production. The decrease in the number of lipid
globules also suggests that lipid is being utilized by
the active lutein cells.
None of the changes described can be regarded
as being caused specifically by X-irradiation. The
general appearance of the cytoplasm of lutein ceils
from experimental animals resembles, in most respects, that of LTH-stimulatea cells and is compatible with that of active steroid-secreting cells.
It is realized that the normal lutein cells described
above as controls may have been taken from inactive corpora lutea.
As the lutein cells described in this study are
apparently the only cells present in X-ray sterilized
ovaries which appear, morphologically, to have the
capacity for steroid production, we consider it probable that these ceils are responsible for the constant vaginal cornification observed. At present,
the sole criterion of estrogenic activity in the rat is
vaginal cornification. In order to overcome this
limitation, work is now in progress that is designed
to estimate the output of estrogens by normal and
X-irradiated rat ovaries, using in vitro biochemical
techniques.
The authors wish to thank Mr. G. Zanelli, Department of Medical Physics, for assistance with the Xirradiation of animals, and Miss Jennifer A. Moody
for photographic assistance and for typing the manuscript.
Received for publication 28 December 1955.
B. FLAKSAND P. BRESLOFF Fine Structure of Luteln Cells
235
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minology to Icejustified, in the absence of evidence
to the contrary.
The appearance of the control lutein cells is
similar to that described by previous workers (4,
7), the characteristic mitochondria with villiform
cristae and dense matrix and the abundant agranular endoplasmic reticulum being features which
make these cells relatively easy to recognise in
electron micrographs. Enders and Lyons (5) suggested that the abundance of agranular endoplasmic reticulum may have functional significance in the lutein cells, and the relevance of this
organelle to steroid production has been considered by Christensen and Fawcett (3). Enders and
Lyons (5) reported an increase in the amount of
agranular endoplasmic reticulum in lutein cells of
rats treated with luteotrophic (or mammotrophic)
hormone (LTH), comparable to the increase
found in X-irradiated rat ovaries in the present
study. This increase in amount of endoplasmic
reticulum is, in our opinion, of particular relevance to steroid secretion in these ovaries.
Other similarities between lutein cells of Xirradiated and LTH-stimulated rat ovaries are
also evident. The most notable are an increase in
cell size, the more frequent occurrence of cupshaped and otherwise "abnormal" mitochondria,
and the decrease in the amount of lipid granules
present. Enders and Lyons (5) considered these
features to be those of active lutein cells.
It is possible that X-ray sterilization of rat
ovaries, by destroying the normal structure of the
ovary, may interfere with a feedback mechanism
operating on the anterior pituitary and thus permit uncontrolled secretion of pituitary gonadotrophins (18). The constant vaginal cornification,
which results from X-ray sterilization, could be
explained if it could be shown, by biochemical
studies, that the stimulated lutein cells are secreting estrus-producing hormones. Tentative
support for this suggestion is supplied by the clinical observations of Smith and Emerson (17), who
found an increase in urinary estrogen in postmenopausal women with mammary carcinoma
whose ovaries had been X-irradiated.
Irradiated rat ovary lutein cells, in the present
study, exhibit a feature which is not described by
Enders and Lyons (5) in LTH-stimulated cells.
Published August 1, 1966
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236
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