BIOLOGY
OF
REPRODUCTION
19,
Endometrial STANLEY
790-796
Lipid
F. GOULD’,
(1978)
Inclusions
in the Estrogen
MAURICE
Departments
H. BERNSTEIN
of Anatomy
and
Wayne
State
Stimulated
and
Uterus
ROBERTA
G. POURCHO
Gynecology/Obstetrics’, University,
of Medicine, Michigan 48201
School
Detroit,
ABSTRACT Cytochemical studies have been made and without estrogen stimulation. The fixative has made possible the retention epithelium of estrogen stimulated mouse These granules range in excess of 1 m
of the uterine epithelium incorporation of malachite
and
visualization
endometrium in diameter
lumen. It is suggested that this granule pattern may phospholipid metabolism after estrogen stimulation. appearance of lipid granule patterns in the endometrium
of a granule
that has and appear reflect In
of ovariectomized mice, with green into a glutaraldehyde
population
in the
not been previously to be released into
apical
described. the uterine
the biochemically described increase in the absence of estrogen stimulation, the of spayed mice was consistent with earlier
reports. phospholipid
INTRODUCTION
Light
microscopic
histochemical
studies
in the malachite
on
the lipid droplets of rodent uterine epithelium have shown a cyclic variation to lipid localizations which appear to be hormone related (Boshier Elftman,
and Holloway, 1963; Davis
spayed lipid
of
animals, in the
cell
the
Stainable scanty
neutral amounts
appear
to be present.
(Davis
and
lipids is greatest Fine structure 1959)
of
Under
the
picture.
both
apical
However, under the
parallel influence
estrogens
a
within
only
of
the
apical lipid
In
1972 for
Teichman the
et
visualization
al.
studied
in
use
are
et
this tech-
of
of malachite
lipids
reproductive
in
tissues
other
tract. that
histochemically
extracted
from
uterine
malachite green staining patterns of lipid distribution
spayed
both
animals,
and
with
Studies
light and
were
electron
MATERIALS
cell.
techwere without
performed
microscopes.
or exogenous loss
in
the
inclusions. visible lipid cell
described
of aqueously
METHODS
operatively. Following the postoperative “rest” period the ovariectomized animals were divided into two
This was
groups. Group 1 received 2 received no hormonal
cytoplasm
inclusions
AND
Uterine tissue was obtained from sexually mature, virgin, albino mice. All animals underwent bilateral oophorectomy and were held for 3-4 weeks post-
1 g
estradiol-17j3 i.p. Group stimulation and served as
ovariectomized controls. Twenty h after estrogen injection, ether anesthetized and sacrificed
were
perfusion perfused
a techsoluble
of fixative. with 2.5%
cacodylate was
buffer, perfused
fixative in which June March
of
stimulation.
group
Accepted Received
the
visualize
Pourcho
of
The
in
the
seen. nique
and
applicability
lipids
niques.
with
epithelium
significant
occasional
retain recently
of the possibility
application
estrogen
occur
regions
lipid in
and the dye, fixative,
tissue during routine tissue processing for fine structure studies, we have investigated lipid inclusions in the endometrium through the
spayed
inclusions
of endogenous
the
staining
male
to
More
expanded
shown the
spermatozoa
microscopic
of uterine
demonstrable decrease
apparent
where
basal
studies
showed
amount of estrogen-induced most
lipid
and
the
have
In view
of es-
from
light
than
able
lipids.
demonstrable
cell cytoplasm. (Nilsson, b958a,b,
the
Numerous
the
influence
epithelium
confirmed
in
1959).
and only phospholipid
in phospholipids oca decrease in neutral increase in phospho-
in the apical studies
uterine
have
Alden,
green
in
By incorporating into a glutaraldehyde
were
soluble
al. (1978) nique and
amounts of and basal
lipid predominates of demonstrable
trogen, a marked increase curs (Elftman, 1963) with and unsaturated lipids. The
animals
large apical
authors
water
1973; Smith, 1970; and Alden, 1959). In
and diestrous are visualized
regions
the
elements epididymis. green,
12, 1978 21, 1978
immediately
proximal 790
before
one-third
One-half glutaraldehyde
pH
all animals
by
of
were
intracardiac
each group was in 0.06 7 M sodium
6.8. The other half of each with the same glutaraldehyde
0.1%
malachite use.
of each
green
Tissue
samples
uterine
horn
was
dissolved
from the were removed
LIPIDS
ENDOMETRIAL
and fixed respective postfixed veronal
fixative.
in acetate
One
2
m
were
ESTROGEN
h at 4#{176}C in their were
sections
were
by
contrast
phase
screened
by
basal
method
2b).
mized
and
between and
viewed
When
animals,
fixed
green, were
apical
In this case
contains
only
apical
from
These
one
occupies
band a
position.
free
to
of
can
tissues
with
fixed (Fig.
stimulated 2c,
b)
2a,
metrium
fixed
large,
irregular
granules.
and
with
these
granules A
smaller,
population
is seen
Comparable tissue fixed the
number
basal
granule
of
apical
adsorbed
microvilli. of
epithelium
exhibits
with (Fig.
large
cytoplasm,
a diameter
3b). with
Many
of
mitochon-
DISCUSSION
light
had
been
1970;
1973). retention
1
spherical,
the
from mala-
Elftman,
Use of uterine
population
malachite
1963;
of
the
use
light
(Teichman green
et et indicate
in
histo-
1959; and
facilitates
lipid
elements can
electron
at.,
Smith, Holloway,
green which
the
previously
microscopic
postfixation
Pourcho
a
Alden,
labile
of
a
epithelium. The of lipid inclusions
Boshier
normally
into visualiza-
population
malachite
osmium
1972;
reports of
by and
epithelium
by
following al.,
of
granule
(Davis
visualized
portion
permitted
structure
suggested
chemistry
and
of
has
stimulated uterine of such a population
2a).
granules
fine
green
malachite
apical
estrogen presence
(Fig.
basal
the
of
apical
sections without
of fixative
undescnibed
endo-
in excess
in the
be
apical
associated
(Fig.
malachite
population
pm
3c)
inset).
glutaraldehyde
exhibits
more
epithelium. stimulated show
the
measure
of the estrogen
small
green
epithelium
granule
green
the
stimulated
in
of the
3b,
be
Incorporation
estrogen
support
granules
diameter.
a
(Fig.
to
3d) to
granules
0.1
seen
of
(Fig.
microvilli
of the
in the
uterine
or
nonstimulated
malachite
the
lumen
No
density
of
Estrogen
regions
and
dna
of
coat
no
3b).
cell mem-
granule
endometrium data.
chite
the
are
tips appears
dense
small
the
granules
(Fig.
granules
approximately
tion
d)
microscopic
in
of
these
cytoplasm
apical
micrographs
(Figs.
of
to
the
the
stimulated
numerous
be seen in nonestrogen-treated (Fig. ic) or without malachite
electron
perinuclear
estrogen
bands;
position,
the
in
ic).
on
with (Fig.
within
discrete
to
Electron
of malachite
in shape
as
surface
that
is released
membrane
material
stained
estrogen
lumen.
variegated
magnification
addition
densely
luminal
suggests
masses
is found
luminal
the
In
fixed
contrast
the
along
estrogen
the
large
limiting
both
3b). At higher green stained
id).
Survey
(Figs.
as a capping
(Fig.
osmiophilic
differences
distribution
pm
In
material
and
osmiophilic
a supranuclear
Similar
be
a
granules
in two
endometrium,
appears
Most
appear
to
of
cytoplasm
the
highly
material
3a)
material
into the
in
granules
stainable
show
evidence
the
estrogen-treated
(Fig.
epithelium
uterus,
green
of
green
the
animals
from
malachite
number
subjacent
significant
basal
Endometrium
granules
basal
immediately
a
osmiophilic
of
estrogen-treated
green
perinuclear
different
granules.
brane
the
in
malachite
green
Such
granules,
found
nonestrogen-treated
presence a larger
other
are
endometrium.
in the contains
ib).
are rare.
markedly
stimulated
epithelium
(Fig.
of
endometrium
the
to
the
and
granules
2a).
stimulated
micrographs
of
apical apical
proximity
distribution
from
contrasts
of osmiophilic
occasional
granules
Tissue
appears
green
which
position.
perinuc-
This
stimulated
a paucity
of
cell.
which
pattern
ovariectomized
the
in
(Fig.
without
distinct
of
seen
(Fig.
The
cytoplasm
in basal,
of the
estrogen
majority
and
granules
close
green
The
the
shows
was
estrogen
fixed
2d).
None
the
differences
malachite
in
consistently
in as
receive
tissue (Fig.
groupings.
epithelium
malachite
the
seen
malachite
displays
the
were
regions
without
tissue
stained
throughout
with
fixed
of
not
apparent
distribution
appears surface
the stimulated
presence
densely
la). Granules
sharply
microscopy, estrogen
the
in
displayed
and
light from
distributed
(Fig.
lear
by
epithelium
did no with
The by
of malachite green from ovariecto-
reveal fixed
2b).
(Fig.
unaffected
epithelium
green
malachite
basal
be
comparable
control RESULTS
size
to
which
tissue
2c)
791
or absence
Uterine
mice
granule
uterine
2a)
stimulation
only those blocks in which the uterine lumen was clearly identifiable were used. Thin sections were prepared, stained with uranyl acetate and lead citrate and viewed in the electron microscope.
same
appear
(Fig.
(Fig.
optics.
UTERUS
the
granules
presence
examined
this
STIMULATED
approximately
subsequently tetroxide buffered with for electron microscopy.
staining
blocks
18
tissues
plastic
additional
tissue
for
All
2% osmium and processed
to
without All
immersion
by
IN THE
then
in be
microscope (Teichman
1978).
al., 1974) that
on the
et
Previous the
use
material
of
792
with
GOULD
Plate
1. Uterine
FIG. 0.1%
is. Ovaniectomized malachite green.
this slightly FIG. philic
FIG.
30%
oblique lb. Same
granules
Dense
are ic.
shorter FIG.
phase
section. as la but largely
are than
id. similar
seen
without
confined
to
the Same
both
estrogen as
to
mouse. in
that
ic
X 800.
contrast.
mouse, 20 h after injection of estradiol-173. Tissue fixed in 2.5% glutaraldehyde Darkly stained granules are distributed profusely through the cytoplasm as seen in
Ovariectomized
granules
essentially
epithelium,
ET AL.
malachite green. The granule population the basal region of the epithelial cells. Control.
and
stimulated but
observed
Tissue
supranuclear without in
fixed basal
endometrium malachite similar
tissue
in
shown green. fixed
2.5%
regions in
Number in
the
glutaraldehyde
of
the
Figs. and
presence
with
epithelial la
and
less dense. The
is much
cells.
0.1% The
osmio-
malachite epithelium
green. is
about
lb.
distribution of malachite
of
darkly green
stained (Fig.
ic).
granules
is
ENDOMETRIAL
Plate in
2.5% FIG.
dense FIG. FIG.
Densely
stained
apical
cytoplasm
the
portion
malachite treated
2a. in
FIG, few
epithelium with 0.1%
glutaraldehyde
present basal
2. Uterine
of 2b.
the
Same
granules 2c.
cells. as can
Uterine
but
be
seen
material
the
basket
without of
more
is visible apical
by
h after
the
through one
Spherical
cytoplasm.
ovariectomized
affected
in
STIMULATED
20
surface.
green.
are seen in both without malachite is not
dispersed luminal
malachite
the
epithelium
mouse
near
ESTROGEN
mouse,
is broadly
and
in
IN THE
of ovariectomized malachite green.
A nucleolar 2a
green. Dense granules 2d. Same as 2c but ovariectomized
LIPIDS
mouse,
Smaller, of
the
presenlce
injection
of
epithelium. more
nuclei.
793
estradiol-17j3. Tissue
Large,
rounded,
irregular
granules
fixed
granules are
are
seen
in
the
X 2,700.
granules
are
seen
at
the
base
of
the
cells.
Only
X 3,300. control,
basal and supranuclear green. Number and the
UTERUS
or absence
fixed regions distribution of
in
2.5%
glutaraldehyde
of
the cells. of granules
X 3,300. in the
malachite
green.
X 4,000.
with nonestrogen-
0.i%
a
794
GOULD
:-‘‘‘.
ET
AL.
ENDOMETRIAL
stabilized
by
malachite
phospholipid
of
has
Various
of
fatty
minimal
amounts
facilitates
in
Elftman,
Gorski
and to
least
1969).
al.,
apical which
surface. are not
an
trium which
(Goswami
of
acid
hematein
et
Histochemical
indicate
that
neutral
estrogen the
Alden,
at., has
phospholipids
procedure
of
an
(Bourgeois
and
The
classic
mouse
studies
uterine
Nilsson
et
Nilsson,
al.
They
tetroxide
in
fixation
reported estrous
spayed
mice were
vacuolated
estrogen
Fuxe
and
an cell
height
(Nilsson, observed,
hum,
or
with
the
estradiol-treated
spayed
some
de
by in
FIG.
This
and
work
05384.
The
mouse,
20
limited.
3b.
this
(arrow).
FIG. seen FIG. apical
of
At
is evident
micrograph The
Profiles
distention.
those
Electron
3c. in
3d.
microvilli
rough X 12,000.
At
apical
higher (arrow).
variety
of
shows
magnification, X 85.000.
green
the are
of
granules
malachite
cytoplasm.
of
reticulum a population
Inset
of
character
endoplasmic
magnification,
Accumulations the
showing
amorphous
with
malachite material
of
are
fate of green.
It
is derived from other
conversion the
uterine
epithe-
from
more
remote
small within
affinity
green granule granules
mitochondria. material
by
are of Mr.
of
discrete
stained
grateful Wesley
NIH
estradiol-17#{231}3.
to
the
are
in
suggests
that
exhibit
frequently
associated
the
RR-
excellent
Tissue
seen
luminal
material
frequently
Grant
for the A. Heiple.
granules
adjacent
population
and
abundant apical
supported
h after injection
malachite large
was
authors
assistance
technical
epithelium.
data
ACKNOWLEDGMENTS
spaces.
uterine
an
or ultimate
mobilization
may
phospholipid
experimental
by
release
study
indicating in
origin
populations via
The
visualized
synthesis,
FIG. 3a. Plastic section, 1-2 m thick, slightly oblique. Numerous the epithelium. The apical population is often observed to be closely granules are seen within the lumen of the uterus (arrow). X 960. of
no
that
lysosomal
present
data
the
is
sites.
which
aggregations
the
upsurge
the
to
elements.
visualized
novo
but
suggested
related
by
yet,
that
lipid
in areas
Plate 3. Uterine epithelium of ovariectomized glutaraldehyde with 0.1% malachite green.
2.5%
material
granule
of
membrane
the
lipid
granules
typically
As
(1958a)
lipid
b958b),
and
seen
evidence
1963)
are
biochemical
regarding
is possible
in
study
the
Similar osmium
also mouse,
uterine lumen has not Indeed, the earlier
Nilsson,
lipids
stimulated
available
were
estrous
of membrane
to
granules in location.
is morphological
suggested
synthesis.
estrogen
Nilsson
as compared
contained
by
membranes. here. Using
decrease
mouse
performed
i958a,b;
procedures,
In a subsequent
granules
were
epithelial
a marked
relate
effects
described
of intracellular are noted
observations
on
estrogen
(Nilsson,
increase
development
mouse.
of
fine structure
1963).
related
and
of material
the
elements green. endome-
pm)
granules
into the reported.
observed
degradation
1965).
larger, to
stimulated
the
a
stained with malachite green the uterine lumen. Visualization
(Fuxe
the
has
extend are labile malachite
estrogen
there
exhibits
mouse
granules without
as
the
in a subapical
small (0.1-0.2 intramitochondrial
release been
reports
have
modifications
mouse
which
(1958a) in spayed mouse.
of lipid previously
(Adams,
phospholipids
with
the
contains are often
Furthermore,
Oil Red 0) fatty acids
of
stimulated granules
that material released into
of
lipids
spayed
intramitochondrial
been
specificities
the
distribution
granules
These retained
populations
of
and
the
estrogen
by Nilsson not in the
that
and estrogen stimuexhibit differences
spherical
addition,
The
1963;
definitive
stains (Sudan Black, for neutral fats and for
and
and
effect
because
In
estrogen
fats
(Davis
containing
demonstrated
also
at
A
Choline
been
the
present
1963).
useful
Hubbard,
irregular
Nicolette,
classic lipid most useful
of the
1959).
this
Whereas
population more
and
size
granules.
band,
et
obtain
the
amounts
Goswami
of
difficult
and
indicates
characteristics
in
Holloway,
1958;
demonstration
is
epithelium
795
in spayed epithelia
solubility
as
well
and
phospholipids
1959;
in the
major
diestrus
UTERUS
study
maximal
Alden,
depletion
present
that
studies
a
increase
and
The
mouse
the
STIMULATED
of lipid granule lated uterine
challenge.
triglycerides)
estrus
at
and
Davis
uterine
Boshier
and are
the
that
(i.e.,
1975;
lipids
in
agreed
fat
biochemical
the are
ESTROGEN
probably
a difficult
in the
1970)
stainable
1963;
is
lipids
be
have
(Hall,
Smith,
of
to
neutral
acids
1973;
and
role
lipids
as
present
of
the
proven
authors
fraction
green
IN THE
in nature.
Analysis uterus
LIPIDS
fixed
throughout
surface. apical
it is not a moderate with
in
Other
cytoplasm membrane cisternal
mitochondria
X 46,000. within
the
lumen
are
identical
in appearance
to
X 10,800. malachite
green
stainable
material
is seen
adsorbed
onto
the
surface
of
796
GOULD
ET AL.
early pregnancy.
REFERENCES Adams,
C.W.M.
(1969).
Lipid
hiscochemistry.
Adv.
in
Lipid Res. 7, 1-62. Boshier, D. P. and Holloway, H. (1973). Effects of ovarian steroid hormones on histochemically demonstrable lipids in the rat uterine epithelium. J. Endocrinol. 56, 59-67. Bourgeois, C. and Hubbard, B. (1965). A method for the simultaneous demonstration of choline-con-
taining
phospholipids J. Histochem.
sections.
J.
Davis,
S.
and
influence
Rec. Elftman, lipids Elftman, Golgi
Fuxe,
Gorski,
R.
lipids
in
(1963).
and lipid the
normal
cycle.
Anat.
Nilsson,
0.
subcellular
fractions
of
rat
Arch.
uteri.
Biochem. Biophys. 104, 418-423. Goswami, A., Kar, A. B. and Chowdhury, (1963). Uterine lipid metabolism in mice oestrous
replacement 295. K. (1975).
cycle:
Effect
therapy.
during
of ovariectomy
J. Reprod.
Fert.
and
6, 287-
in
the
mouse
uterus
during
effect
mouse
uterine estrogenic
of estrogen
administered
J. Ultrastruct.
Res.
2,
185-
0. (1959). Ultrastructure of mouse uterine surface epithelium under different estrogenic influences. 4. Uterine secretion. J. Ultrastruct,
histology 1.
of
effects
uterine Cell
of estrogen
epithelium
microscopy Exptl.
Pourcho,
of Res.
26,
Stain
Technol.
(1970).
M.S.R.
decomposing
53, 29-3
mouse uterus Reprod. Fert. 22, 461-467. R. J., Fujimoto,
(1972).
7,
lipid
malachite
A
previously
M.
and
green
on J.
Yanagimachi,
it.
unrecognized
spermatozoa and
5.
observations oestrus cycle.
material as
pyronine.
in
revealed Biol.
Reprod.
73-81.
Teichman, R. J., Cummins, J. M. and Takei, (1974). The characterization of a malachite stainable,
577,
the
mouse.
334-343.
Histochemical during the
the
Teichman,
on
of the
G., Bernstein, M. H. and Gould, S. F. Malachite green: Applications in electron
it. (1978).
Smith,
The
the
Electron
granules.
in
Lipids
different
animals.
mammalian
R.
of
under
3. Late
spayed
by
S.
Ultrastructure
epithelium
microscopy.
(1963). The lipid granules of the uterine epithelium in the spayed mouse. J. Ultrastruct. Rca. 8, 379-390. J. and Nicolette, J. A. (1963). Early estrogen effects on newly synthesized RNA and phospho-
the
(i958b).
Res. 2, 331-341. Nilsson, 0. (1962),
146,
Rec.
65, 23 3-243.
199.
Estrogen-induced
apparatus rat in
in
to
of the phospho62, 410-41 5.
changes in the of the uterine epithelium
0.
surface influences,
Nilsson,
725-737.
139-144. K. and
lipids
Hall,
tissue
Nilsson,
571-578. Hormonal
13,
(1959). of rat uterus. Anat.
H.
(1958). Estrogen control of the uterus. Endocrinology
H.
of the
Alden,
on lipid metabolism
134,
H.
and neutral Cytochem.
J. Endocrinol.
Nilsson, 0. (1958a), Ultrastructure of mouse uterine surface epithelium under different estrogenic influences. I. Spayed animals and oestrous animals, J. Ultrastruct. Res. 1, 375-396.
rabbit
glutaraldehyde spermatozoa.
extractable Biol.
G. green
phospholipid Reprod.
10,
565-
H.
OF
REPRODUCTION
19,
Endometrial STANLEY
790-796
Lipid
F. GOULD’,
(1978)
Inclusions
in the Estrogen
MAURICE
Departments
H. BERNSTEIN
of Anatomy
and
Wayne
State
Stimulated
and
Uterus
ROBERTA
G. POURCHO
Gynecology/Obstetrics’, University,
of Medicine, Michigan 48201
School
Detroit,
ABSTRACT Cytochemical studies have been made and without estrogen stimulation. The fixative has made possible the retention epithelium of estrogen stimulated mouse These granules range in excess of 1 m
of the uterine epithelium incorporation of malachite
and
visualization
endometrium in diameter
lumen. It is suggested that this granule pattern may phospholipid metabolism after estrogen stimulation. appearance of lipid granule patterns in the endometrium
of a granule
that has and appear reflect In
of ovariectomized mice, with green into a glutaraldehyde
population
in the
not been previously to be released into
apical
described. the uterine
the biochemically described increase in the absence of estrogen stimulation, the of spayed mice was consistent with earlier
reports. phospholipid
INTRODUCTION
Light
microscopic
histochemical
studies
in the malachite
on
the lipid droplets of rodent uterine epithelium have shown a cyclic variation to lipid localizations which appear to be hormone related (Boshier Elftman,
and Holloway, 1963; Davis
spayed lipid
of
animals, in the
cell
the
Stainable scanty
neutral amounts
appear
to be present.
(Davis
and
lipids is greatest Fine structure 1959)
of
Under
the
picture.
both
apical
However, under the
parallel influence
estrogens
a
within
only
of
the
apical lipid
In
1972 for
Teichman the
et
visualization
al.
studied
in
use
are
et
this tech-
of
of malachite
lipids
reproductive
in
tissues
other
tract. that
histochemically
extracted
from
uterine
malachite green staining patterns of lipid distribution
spayed
both
animals,
and
with
Studies
light and
were
electron
MATERIALS
cell.
techwere without
performed
microscopes.
or exogenous loss
in
the
inclusions. visible lipid cell
described
of aqueously
METHODS
operatively. Following the postoperative “rest” period the ovariectomized animals were divided into two
This was
groups. Group 1 received 2 received no hormonal
cytoplasm
inclusions
AND
Uterine tissue was obtained from sexually mature, virgin, albino mice. All animals underwent bilateral oophorectomy and were held for 3-4 weeks post-
1 g
estradiol-17j3 i.p. Group stimulation and served as
ovariectomized controls. Twenty h after estrogen injection, ether anesthetized and sacrificed
were
perfusion perfused
a techsoluble
of fixative. with 2.5%
cacodylate was
buffer, perfused
fixative in which June March
of
stimulation.
group
Accepted Received
the
visualize
Pourcho
of
The
in
the
seen. nique
and
applicability
lipids
niques.
with
epithelium
significant
occasional
retain recently
of the possibility
application
estrogen
occur
regions
lipid in
and the dye, fixative,
tissue during routine tissue processing for fine structure studies, we have investigated lipid inclusions in the endometrium through the
spayed
inclusions
of endogenous
the
staining
male
to
More
expanded
shown the
spermatozoa
microscopic
of uterine
demonstrable decrease
apparent
where
basal
studies
showed
amount of estrogen-induced most
lipid
and
the
have
In view
of es-
from
light
than
able
lipids.
demonstrable
cell cytoplasm. (Nilsson, b958a,b,
the
Numerous
the
influence
epithelium
confirmed
in
1959).
and only phospholipid
in phospholipids oca decrease in neutral increase in phospho-
in the apical studies
uterine
have
Alden,
green
in
By incorporating into a glutaraldehyde
were
soluble
al. (1978) nique and
amounts of and basal
lipid predominates of demonstrable
trogen, a marked increase curs (Elftman, 1963) with and unsaturated lipids. The
animals
large apical
authors
water
1973; Smith, 1970; and Alden, 1959). In
and diestrous are visualized
regions
the
elements epididymis. green,
12, 1978 21, 1978
immediately
proximal 790
before
one-third
One-half glutaraldehyde
pH
all animals
by
of
were
intracardiac
each group was in 0.06 7 M sodium
6.8. The other half of each with the same glutaraldehyde
0.1%
malachite use.
of each
green
Tissue
samples
uterine
horn
was
dissolved
from the were removed
LIPIDS
ENDOMETRIAL
and fixed respective postfixed veronal
fixative.
in acetate
One
2
m
were
ESTROGEN
h at 4#{176}C in their were
sections
were
by
contrast
phase
screened
by
basal
method
2b).
mized
and
between and
viewed
When
animals,
fixed
green, were
apical
In this case
contains
only
apical
from
These
one
occupies
band a
position.
free
to
of
can
tissues
with
fixed (Fig.
stimulated 2c,
b)
2a,
metrium
fixed
large,
irregular
granules.
and
with
these
granules A
smaller,
population
is seen
Comparable tissue fixed the
number
basal
granule
of
apical
adsorbed
microvilli. of
epithelium
exhibits
with (Fig.
large
cytoplasm,
a diameter
3b). with
Many
of
mitochon-
DISCUSSION
light
had
been
1970;
1973). retention
1
spherical,
the
from mala-
Elftman,
Use of uterine
population
malachite
1963;
of
the
use
light
(Teichman green
et et indicate
in
histo-
1959; and
facilitates
lipid
elements can
electron
at.,
Smith, Holloway,
green which
the
previously
microscopic
postfixation
Pourcho
a
Alden,
labile
of
a
epithelium. The of lipid inclusions
Boshier
normally
into visualiza-
population
malachite
osmium
1972;
reports of
by and
epithelium
by
following al.,
of
granule
(Davis
visualized
portion
permitted
structure
suggested
chemistry
and
of
has
stimulated uterine of such a population
2a).
granules
fine
green
malachite
apical
estrogen presence
(Fig.
basal
the
of
apical
sections without
of fixative
undescnibed
endo-
in excess
in the
be
apical
associated
(Fig.
malachite
population
pm
3c)
inset).
glutaraldehyde
exhibits
more
epithelium. stimulated show
the
measure
of the estrogen
small
green
epithelium
granule
green
the
stimulated
in
of the
3b,
be
Incorporation
estrogen
support
granules
diameter.
a
(Fig.
to
3d) to
granules
0.1
seen
of
(Fig.
microvilli
of the
in the
uterine
or
nonstimulated
malachite
the
lumen
No
density
of
Estrogen
regions
and
dna
of
coat
no
3b).
cell mem-
granule
endometrium data.
chite
the
are
tips appears
dense
small
the
granules
(Fig.
granules
approximately
tion
d)
microscopic
in
of
these
cytoplasm
apical
micrographs
(Figs.
of
to
the
the
stimulated
numerous
be seen in nonestrogen-treated (Fig. ic) or without malachite
electron
perinuclear
estrogen
bands;
position,
the
in
ic).
on
with (Fig.
within
discrete
to
Electron
of malachite
in shape
as
surface
that
is released
membrane
material
stained
estrogen
lumen.
variegated
magnification
addition
densely
luminal
suggests
masses
is found
luminal
the
In
fixed
contrast
the
along
estrogen
the
large
limiting
both
3b). At higher green stained
id).
Survey
(Figs.
as a capping
(Fig.
osmiophilic
differences
distribution
pm
In
material
and
osmiophilic
a supranuclear
Similar
be
a
granules
in two
endometrium,
appears
Most
appear
to
of
cytoplasm
the
highly
material
3a)
material
into the
in
granules
stainable
show
evidence
the
estrogen-treated
(Fig.
epithelium
uterus,
green
of
green
the
animals
from
malachite
number
subjacent
significant
basal
Endometrium
granules
basal
immediately
a
osmiophilic
of
estrogen-treated
green
perinuclear
different
granules.
brane
the
in
malachite
green
Such
granules,
found
nonestrogen-treated
presence a larger
other
are
endometrium.
in the contains
ib).
are rare.
markedly
stimulated
epithelium
(Fig.
of
endometrium
the
to
the
and
granules
2a).
stimulated
micrographs
of
apical apical
proximity
distribution
from
contrasts
of osmiophilic
occasional
granules
Tissue
appears
green
which
position.
perinuc-
This
stimulated
a paucity
of
cell.
which
pattern
ovariectomized
the
in
(Fig.
without
distinct
of
seen
(Fig.
The
cytoplasm
in basal,
of the
estrogen
majority
and
granules
close
green
The
the
shows
was
estrogen
fixed
2d).
None
the
differences
malachite
in
consistently
in as
receive
tissue (Fig.
groupings.
epithelium
malachite
the
seen
malachite
displays
the
were
regions
without
tissue
stained
throughout
with
fixed
of
not
apparent
distribution
appears surface
the stimulated
presence
densely
la). Granules
sharply
microscopy, estrogen
the
in
displayed
and
light from
distributed
(Fig.
lear
by
epithelium
did no with
The by
of malachite green from ovariecto-
reveal fixed
2b).
(Fig.
unaffected
epithelium
green
malachite
basal
be
comparable
control RESULTS
size
to
which
tissue
2c)
791
or absence
Uterine
mice
granule
uterine
2a)
stimulation
only those blocks in which the uterine lumen was clearly identifiable were used. Thin sections were prepared, stained with uranyl acetate and lead citrate and viewed in the electron microscope.
same
appear
(Fig.
(Fig.
optics.
UTERUS
the
granules
presence
examined
this
STIMULATED
approximately
subsequently tetroxide buffered with for electron microscopy.
staining
blocks
18
tissues
plastic
additional
tissue
for
All
2% osmium and processed
to
without All
immersion
by
IN THE
then
in be
microscope (Teichman
1978).
al., 1974) that
on the
et
Previous the
use
material
of
792
with
GOULD
Plate
1. Uterine
FIG. 0.1%
is. Ovaniectomized malachite green.
this slightly FIG. philic
FIG.
30%
oblique lb. Same
granules
Dense
are ic.
shorter FIG.
phase
section. as la but largely
are than
id. similar
seen
without
confined
to
the Same
both
estrogen as
to
mouse. in
that
ic
X 800.
contrast.
mouse, 20 h after injection of estradiol-173. Tissue fixed in 2.5% glutaraldehyde Darkly stained granules are distributed profusely through the cytoplasm as seen in
Ovariectomized
granules
essentially
epithelium,
ET AL.
malachite green. The granule population the basal region of the epithelial cells. Control.
and
stimulated but
observed
Tissue
supranuclear without in
fixed basal
endometrium malachite similar
tissue
in
shown green. fixed
2.5%
regions in
Number in
the
glutaraldehyde
of
the
Figs. and
presence
with
epithelial la
and
less dense. The
is much
cells.
0.1% The
osmio-
malachite epithelium
green. is
about
lb.
distribution of malachite
of
darkly green
stained (Fig.
ic).
granules
is
ENDOMETRIAL
Plate in
2.5% FIG.
dense FIG. FIG.
Densely
stained
apical
cytoplasm
the
portion
malachite treated
2a. in
FIG, few
epithelium with 0.1%
glutaraldehyde
present basal
2. Uterine
of 2b.
the
Same
granules 2c.
cells. as can
Uterine
but
be
seen
material
the
basket
without of
more
is visible apical
by
h after
the
through one
Spherical
cytoplasm.
ovariectomized
affected
in
STIMULATED
20
surface.
green.
are seen in both without malachite is not
dispersed luminal
malachite
the
epithelium
mouse
near
ESTROGEN
mouse,
is broadly
and
in
IN THE
of ovariectomized malachite green.
A nucleolar 2a
green. Dense granules 2d. Same as 2c but ovariectomized
LIPIDS
mouse,
Smaller, of
the
presenlce
injection
of
epithelium. more
nuclei.
793
estradiol-17j3. Tissue
Large,
rounded,
irregular
granules
fixed
granules are
are
seen
in
the
X 2,700.
granules
are
seen
at
the
base
of
the
cells.
Only
X 3,300. control,
basal and supranuclear green. Number and the
UTERUS
or absence
fixed regions distribution of
in
2.5%
glutaraldehyde
of
the cells. of granules
X 3,300. in the
malachite
green.
X 4,000.
with nonestrogen-
0.i%
a
794
GOULD
:-‘‘‘.
ET
AL.
ENDOMETRIAL
stabilized
by
malachite
phospholipid
of
has
Various
of
fatty
minimal
amounts
facilitates
in
Elftman,
Gorski
and to
least
1969).
al.,
apical which
surface. are not
an
trium which
(Goswami
of
acid
hematein
et
Histochemical
indicate
that
neutral
estrogen the
Alden,
at., has
phospholipids
procedure
of
an
(Bourgeois
and
The
classic
mouse
studies
uterine
Nilsson
et
Nilsson,
al.
They
tetroxide
in
fixation
reported estrous
spayed
mice were
vacuolated
estrogen
Fuxe
and
an cell
height
(Nilsson, observed,
hum,
or
with
the
estradiol-treated
spayed
some
de
by in
FIG.
This
and
work
05384.
The
mouse,
20
limited.
3b.
this
(arrow).
FIG. seen FIG. apical
of
At
is evident
micrograph The
Profiles
distention.
those
Electron
3c. in
3d.
microvilli
rough X 12,000.
At
apical
higher (arrow).
variety
of
shows
magnification, X 85.000.
green
the are
of
granules
malachite
cytoplasm.
of
reticulum a population
Inset
of
character
endoplasmic
magnification,
Accumulations the
showing
amorphous
with
malachite material
of
are
fate of green.
It
is derived from other
conversion the
uterine
epithe-
from
more
remote
small within
affinity
green granule granules
mitochondria. material
by
are of Mr.
of
discrete
stained
grateful Wesley
NIH
estradiol-17#{231}3.
to
the
are
in
suggests
that
exhibit
frequently
associated
the
RR-
excellent
Tissue
seen
luminal
material
frequently
Grant
for the A. Heiple.
granules
adjacent
population
and
abundant apical
supported
h after injection
malachite large
was
authors
assistance
technical
epithelium.
data
ACKNOWLEDGMENTS
spaces.
uterine
an
or ultimate
mobilization
may
phospholipid
experimental
by
release
study
indicating in
origin
populations via
The
visualized
synthesis,
FIG. 3a. Plastic section, 1-2 m thick, slightly oblique. Numerous the epithelium. The apical population is often observed to be closely granules are seen within the lumen of the uterus (arrow). X 960. of
no
that
lysosomal
present
data
the
is
sites.
which
aggregations
the
upsurge
the
to
elements.
visualized
novo
but
suggested
related
by
yet,
that
lipid
in areas
Plate 3. Uterine epithelium of ovariectomized glutaraldehyde with 0.1% malachite green.
2.5%
material
granule
of
membrane
the
lipid
granules
typically
As
(1958a)
lipid
b958b),
and
seen
evidence
1963)
are
biochemical
regarding
is possible
in
study
the
Similar osmium
also mouse,
uterine lumen has not Indeed, the earlier
Nilsson,
lipids
stimulated
available
were
estrous
of membrane
to
granules in location.
is morphological
suggested
synthesis.
estrogen
Nilsson
as compared
contained
by
membranes. here. Using
decrease
mouse
performed
i958a,b;
procedures,
In a subsequent
granules
were
epithelial
a marked
relate
effects
described
of intracellular are noted
observations
on
estrogen
(Nilsson,
increase
development
mouse.
of
fine structure
1963).
related
and
of material
the
elements green. endome-
pm)
granules
into the reported.
observed
degradation
1965).
larger, to
stimulated
the
a
stained with malachite green the uterine lumen. Visualization
(Fuxe
the
has
extend are labile malachite
estrogen
there
exhibits
mouse
granules without
as
the
in a subapical
small (0.1-0.2 intramitochondrial
release been
reports
have
modifications
mouse
which
(1958a) in spayed mouse.
of lipid previously
(Adams,
phospholipids
with
the
contains are often
Furthermore,
Oil Red 0) fatty acids
of
stimulated granules
that material released into
of
lipids
spayed
intramitochondrial
been
specificities
the
distribution
granules
These retained
populations
of
and
the
estrogen
by Nilsson not in the
that
and estrogen stimuexhibit differences
spherical
addition,
The
1963;
definitive
stains (Sudan Black, for neutral fats and for
and
and
effect
because
In
estrogen
fats
(Davis
containing
demonstrated
also
at
A
Choline
been
the
present
1963).
useful
Hubbard,
irregular
Nicolette,
classic lipid most useful
of the
1959).
this
Whereas
population more
and
size
granules.
band,
et
obtain
the
amounts
Goswami
of
difficult
and
indicates
characteristics
in
Holloway,
1958;
demonstration
is
epithelium
795
in spayed epithelia
solubility
as
well
and
phospholipids
1959;
in the
major
diestrus
UTERUS
study
maximal
Alden,
depletion
present
that
studies
a
increase
and
The
mouse
the
STIMULATED
of lipid granule lated uterine
challenge.
triglycerides)
estrus
at
and
Davis
uterine
Boshier
and are
the
that
(i.e.,
1975;
lipids
in
agreed
fat
biochemical
the are
ESTROGEN
probably
a difficult
in the
1970)
stainable
1963;
is
lipids
be
have
(Hall,
Smith,
of
to
neutral
acids
1973;
and
role
lipids
as
present
of
the
proven
authors
fraction
green
IN THE
in nature.
Analysis uterus
LIPIDS
fixed
throughout
surface. apical
it is not a moderate with
in
Other
cytoplasm membrane cisternal
mitochondria
X 46,000. within
the
lumen
are
identical
in appearance
to
X 10,800. malachite
green
stainable
material
is seen
adsorbed
onto
the
surface
of
796
GOULD
ET AL.
early pregnancy.
REFERENCES Adams,
C.W.M.
(1969).
Lipid
hiscochemistry.
Adv.
in
Lipid Res. 7, 1-62. Boshier, D. P. and Holloway, H. (1973). Effects of ovarian steroid hormones on histochemically demonstrable lipids in the rat uterine epithelium. J. Endocrinol. 56, 59-67. Bourgeois, C. and Hubbard, B. (1965). A method for the simultaneous demonstration of choline-con-
taining
phospholipids J. Histochem.
sections.
J.
Davis,
S.
and
influence
Rec. Elftman, lipids Elftman, Golgi
Fuxe,
Gorski,
R.
lipids
in
(1963).
and lipid the
normal
cycle.
Anat.
Nilsson,
0.
subcellular
fractions
of
rat
Arch.
uteri.
Biochem. Biophys. 104, 418-423. Goswami, A., Kar, A. B. and Chowdhury, (1963). Uterine lipid metabolism in mice oestrous
replacement 295. K. (1975).
cycle:
Effect
therapy.
during
of ovariectomy
J. Reprod.
Fert.
and
6, 287-
in
the
mouse
uterus
during
effect
mouse
uterine estrogenic
of estrogen
administered
J. Ultrastruct.
Res.
2,
185-
0. (1959). Ultrastructure of mouse uterine surface epithelium under different estrogenic influences. 4. Uterine secretion. J. Ultrastruct,
histology 1.
of
effects
uterine Cell
of estrogen
epithelium
microscopy Exptl.
Pourcho,
of Res.
26,
Stain
Technol.
(1970).
M.S.R.
decomposing
53, 29-3
mouse uterus Reprod. Fert. 22, 461-467. R. J., Fujimoto,
(1972).
7,
lipid
malachite
A
previously
M.
and
green
on J.
Yanagimachi,
it.
unrecognized
spermatozoa and
5.
observations oestrus cycle.
material as
pyronine.
in
revealed Biol.
Reprod.
73-81.
Teichman, R. J., Cummins, J. M. and Takei, (1974). The characterization of a malachite stainable,
577,
the
mouse.
334-343.
Histochemical during the
the
Teichman,
on
of the
G., Bernstein, M. H. and Gould, S. F. Malachite green: Applications in electron
it. (1978).
Smith,
The
the
Electron
granules.
in
Lipids
different
animals.
mammalian
R.
of
under
3. Late
spayed
by
S.
Ultrastructure
epithelium
microscopy.
(1963). The lipid granules of the uterine epithelium in the spayed mouse. J. Ultrastruct. Rca. 8, 379-390. J. and Nicolette, J. A. (1963). Early estrogen effects on newly synthesized RNA and phospho-
the
(i958b).
Res. 2, 331-341. Nilsson, 0. (1962),
146,
Rec.
65, 23 3-243.
199.
Estrogen-induced
apparatus rat in
in
to
of the phospho62, 410-41 5.
changes in the of the uterine epithelium
0.
surface influences,
Nilsson,
725-737.
139-144. K. and
lipids
Hall,
tissue
Nilsson,
571-578. Hormonal
13,
(1959). of rat uterus. Anat.
H.
(1958). Estrogen control of the uterus. Endocrinology
H.
of the
Alden,
on lipid metabolism
134,
H.
and neutral Cytochem.
J. Endocrinol.
Nilsson, 0. (1958a), Ultrastructure of mouse uterine surface epithelium under different estrogenic influences. I. Spayed animals and oestrous animals, J. Ultrastruct. Res. 1, 375-396.
rabbit
glutaraldehyde spermatozoa.
extractable Biol.
G. green
phospholipid Reprod.
10,
565-
H.
