BIOLOGY
OF
REPRODUCTION
Subcellular
20,
1133-1145
Location
of Progesterone
A Biochemical, SUSAN
J.
(1979)
Morphological
QUIRK1,
Nuffield
DAVID GEOFFREY
Department
in the
Bovine
Corpus
and Cytochemical L. WILLCOX, DILYS D. THORBURN’ of Obstetrics
and
Luteum:
Investigation M.
PARRY2
and
Gynaecology
and
Nuffield
Institute
John
for
Radcliffe
Medical
Hospital,
Headington,
Research,
Headley
Oxford,
Way
U.K.
ABSTRACT
The subcellular biochemical, homogenates this particulate
location
of progesterone
in the bovine
cytochemical up to one-third of banded discretely
morphological showed that progesterone
and
corpus
luteum has been investigated by Differential centrifugation of tissue the total progesterone could be sedimented. Most of at low density on sucrose gradients. Marker enzyme techniques.
studies showed that it was not associated with either the microperoxisomes, lysosomes or mitochondria nor appreciably with the vesiculated subcellular components such as plasma membrane and microsomes. Morphological examination of tissue sections showed that the luteinized cells of the bovine corpus luteum contain electron dense granules. Three types of granules were present. Some of these granules were identified as microperoxisomes and lysosomes by cytochemical techniques, but a third type of granules was also present. Morphological and cytochemical studies of different regions of the sucrose density gradient confirmed the distribution of subcellular organelles as judged by marker enzyme techniques. Electron dense granules were observed in material fixed from the progesterone peak, while microperoxisomes and lysosomes were found in denser regions of the gradient and separate from the progesterone
band.
These results support the hypothesis that some of the progesterone is sequestered within electron dense granules.
INTRODUCTION
are electron
The secretion of steroids from steroidogenic tissue is usually assumed to occur by diffusion from the site of synthesis through the plasma
number
membrane
related
to the
into port
the blood, the steroids 1973). In the
extracellular
space
phase, creases.
is when High
Accepted Received ‘Present
Department Queensland 2Present rics and
Headington,
maximal
and
thence
the
these
to
the
regression
(PGF2y),
luteolytic
progesterone biosynthesis derates of progesterone biosynthesis
September 25, 1978. June 6, 1978. address: University of Physiology, St.
into of
bovine
presence some of
extracellular
by
of
of
occurs
and
granule
(Gemmell
et al.,
1976;
Stacy
tissue corpus When F2a
progesterone
ceases
secretion et al.,
granule
colchicine
is
the
prostaglandin
decrease
and
The
exocytosed
activity
induced
by
space.
being
secretion
decreased
of many which are
as regression of the (Gemmell et al., 1974).
marked
progesterone
luteum
corpus
granules
secretory
is a
the
with the granules,
and decreases luteum proceeds
between the progesterone the
associated dense
expelled
where binding proteins transto their site of action (Enders, corpus luteum, marked differ-
ences in morphology are observed midluteal phase of the cycle when biosynthesis
and
in
1976).
Both
production
are
to ewes
administration
(Gemmell and Stacy, 1977). These results suggest that the secretion of progesterone from the luteal cell might occur by exocytosis of hormone containing granules. Polypeptide hormones hormones from the
Queensland, Lucia, Brisbane. of
4067, Australia. address: Nuffield Department Gynaecology, John Radcliffe Oxford, U.K.
of ObstetHospital,
1133
such as insulin, glucagon and the of the anterior pituitary are secreted cell by this mechanism. Intracellular
transport
of
granules
mediated
by
microtubules;
chicine,
which
interfere
to
the
cell
drugs, with
periphery such
is
as col-
microtubular
1134
QUIRK
also
function
interfere
these 1971;
hormones Leclerq-Meyer
Edds, In
1976). this
and
et
study
fractionation,
we
that
the
of
the
presence
al.,
al.,
1974;
of
Farquar, Stephens and
used
centrifugation studies
sedimentable
luteum
is
to
proges-
associated
with
of granules.
MATERIALS
chemicals
All
AND
were p-nitrophenol HEPES
13-D-glucuronide, N’-2-ethane-sulphonic
4,
METHODS
DCPIP
Analar
grade.
Trizma
phosphate,
Ficoll
was
Hopkins
and
Centre,
from
Williams
obtained Pharmacia,
and
Ci/mmol)
Amersham.
3’, and
were purchased
(80-110
Radiochemical (4-phenylspiro was obtained reagent from
(3,
tetrahydrochloride)
(2,6-dichlorophenol-indophenol)
l’FII-progesterone
7.0,
p-nitrophenol-
(N-2-hydroxyethylpiperazineacid), diaminobenzidine
4’-tetraaminobiphenyl
from Sigma. sucrose from
S,. was retained. In some experiments aliquots of the cytosol were loaded on to sucrose density gradients as described below.
1, 2,
6,
from
7. the
Fluorescamine
lfuran-2 (3), l’-phthalanl-3, from Roche and Folin-Ciocalteau Fison Scientific.
3’-dione) phenol
Density Centrifugation
After evaluation of several different density gradients, linear 20-40% w/w buffered sucrose gradients were prepared using an ISCO Model 570 gradient former. The gradients were of 36 ml capacity with a 1.5 ml underlay of 50% w/w sucrose. After
loading,
Chemicals p-nitrophenol,
AL.
Sucrose Gradient
subcellular
cytochemical
the
corpus
secretion
1968;
gradient and
demonstrate
the
et
have
density
morphological
terone
with
(Lacy
ET
the
gradients
were
centrifuged
in a Beckman
SW-27 rotor for 2.25 h at 25,000 rpm (82,500 X gay), and eluted in 2 ml fractions by upward displacement with 60% w/w sucrose using an ISCO Model 184 tube-holding and piercing mechanism coupled to a nonperistaltic pump. The sucrose concentration or density (20#{176}14#{176}) of individual fractions was determined by refractometry (Bellingham and Stanley refractometer, Tunbridge Wells, U.K.). Fractions were stored at -20#{176}Cuntil further analysis was carried out. In some experiments, fractions of identical density from more than one gradient were pooled. Some pooled gradient fractions were fixed directly by glutaraldehyde (see below) for morphological and cytochemical examination. The fixed material was collected by centrifugation and treated as described below for tissue sections.
Assays Tissue
Manipulations
Midluteal phase bovine corpora lutea were obtained from 2 local abattoirs. The material was placed on ice within 10 mm of death of the cows. All subsequent manipulations were performed at 0-4#{176}C. In the laboratory, some 60-90 mm later, the corpora Iutea were decapsulated, minced using a Climpex tissue mincer (fitted with a stainless steel grid perforated by 1.7 mm diameter holes) and weighed. All sucrose solutions were buffered with either 0.01 M Trizma 7.0 (pH 7.5; 5#{176}C) or 0.01 M HEPES, pH 7.4. A 10% w/v homogenate in buffered 0.25 M sucrose was obtained (H,) with 3-5 strokes of a Potter-Elvehjem homogenizer (Thomas, Philadelphia, PA; No. C44491, 0.177 mm clearance) driven by an MSE homogenizer at quarter speed. The homogenate (H,) was centrifuged at 600 X umax for 5 mm (MSE, Mistral 6L) to remove nuclei, large pieces of cell debris and incompletely homogenized and unbroken cells. After centrifugation, the supernatant (5,) was decanted and recentrifuged at 10,000 X av for 30 mm (Beckman Spinco L2-65B). Floating lipid was removed with a clean dry glass rod. The supernatant (S2) was decanted and the pellet resuspended in buffered 0.25 M sucrose (H2, 10,000 X g pellet) by 3 handstrokes of a Potter-Elvehjem homogenizer (No. A8427, 0.127 mm clearance) to a total volume in ml equivalent to the number of g of original minced corpus luteum tissue (after allowing for sampling). Between 1.0 and 1.5 ml of this 10,000 X av pellet (H2) was loaded on to sucrose density gradients as described below. A microsomal fraction (H,, 100,000 X g pellet) was obtained by centrifuging supernatant 2 at 96,300 for 30 mm and resuspending the pellet in buffered sucrose. The supernatant or cytosol fraction,
Progesterone was measured by radioimmunoassay et al., 1973). Pregnenolone, 17-hydroxyprogesterone, 20n-hydroxy-pregn-4-en-3-one, cortisol and estradiol-17j3 showed less than 1.5% cross reaction with the progesterone antiserum. The solvent blanks were routinely equivalent to 1.116). Progesterone levels were
(2) (1)
The
assayed.
recovered
progesterone,
negligible
dehydrogenase
activity
5%
13-glucuronidase,
alkaline phosphatase, reductase and inosine The latter 3 enzyme
acid
of
were
catalase
of
the and
than phosphatase,
and
pletely terone
tube
less
NADH-cytochrome c diphosphatase activities. activities were spread
at
8
low
100%.
onwards
from
tube
8
highest
from
tube
resolved from the peak, while a small
onwards
14
the
lysosomes, were com-
particulate proportion
progesof the
microsomes and plasma membrane overlapped in this region. Compared with the homogenate, the enrichment (relative specific activity) of progesterone was
greatest
ment of experiment
in
tubes
7-8-fold shown
experiments ranged from
Maximum
the enrichment 7-17-fold. The
2, which entered
enrich4-6 in the In 4 other this other
were obtained and inosine
enzyme is also of mitochondria.
located Tubes
progesterone showed
gradient,
at
densities
progesterone mum
peak,
enrichment
position endoplasmic
greater
of
and usually
maximal reticulum
than the
position
corresponded
on 1 had
that a much
enrichment owing to the presence of protein which did not enter the gradient. All marker enzymes showed higher ments
region signi-
microsomal marker enwas, however, less than
contained the
in only
in this region c reductase
the enrichment
diphosphatase, zymes. This
and
3-8.
occurred in tubes in Figs. 4-6.
ficant enrichments for NADH-cytochrome
not
density
50%
percent
set
onwards, whereas acid phosphatase, j3-glucuronidase, catalase and succinate dehydrogenase
2-fold. The former the outer membrane
organelles.
sucrose, about
succinate
of
about
13-glucuronidase
presumably
activity,
enzyme
=
tubes
acid
some
100,000
(sucrose density >1.146). Thus mitochondria and microperoxisomes
Gradients
Figs.
centrif-
or activity
mass
Pellet
(cytosol)
activities Distribution
5.7 0.9 0.7 7.0 4.7
6.1 5.1
X g pellet
significant
containing
chosen
density
100,000
(not
10,000
differential
or enzyme activity in each fraction is expressed relative to the homogenate, mean ± SD; the number of experiments is indicated in parentheses.
a
enrichment
terone
by
obtained
c reductase
insensitive)
Inosine
activities
enzyme
homogenate.a
lutea
lower soluble enrich-
that
of of
the
maxito
the
enzyme activity. The and plasma membrane
PROGESTERONE
markers and
thus
This ation
were
spread
throughout
not
heavily
enriched
distribution in vesicle
during elements
homogenization of the cell.
separate
lysosomes,
the
denser
the
gradient
medium,
fraction.
terone
because density of
No
of the variwhich arises
these
membranous was made
attempt
and
the enrichment of the gradient
was less than if the gradient to effect a separation. Prolonged centrifugation
had
of the to
micro-
been
h)
of
Linear
as
gradients
Ficoll range 0.25
above
were
also
2.25,
18
or
h,
at the
top
gradients
nor
of
the
use
of
the
midcycle
extensive
endoplasmic
corpus
network
reticulum
of
characteristic
secretory tissues and an abundance of dense granules ranging in size from
30
75
20
50
10
25
Ficoll
1
4
7
10 TUBE
13
16
19
NO
Thus,
gradient.
centrifugation the
of an
very similar gradients.
considerable alkaline and acid phosactivities remained with the progesprolonged
cells
the
and
However, phatase
peak
Cytochemistry
in
progesterone obtained
terone
proges-
20-
resulting to those
neither
of
evaluated
20.25
profiles were with sucrose
separation
Luteum
contained
of steroid electron
0-20% w/w and 5-25% w/w M sucrose. They were centrifuged
for
and
luteal
agranular
extended
(17.5
The
the
enzymes.
marker
Corpus
1137
LUTEUM
improved from
luteum
obtained (2-4-fold)
CORPUS
Morphology
40% w/w sucrose gradients gave maximal enrichments of progesterone and marker enzymes at the same densities as found for the shorter centrifugation time. Thus, by 2.25 h in a 20-40% w/w sucrose gradient, equilibrium had been attained. over the buffered
BOVINE
in any
mitochondria
so that regions
peroxisomes, in
occurs size and
IN THE
LOCATION
of
sucrose
as
density
>
FIGS. 1-3. The distribution of progesterone. enzyme activities and protein on 20-40% w/w sucrose density gradients. Aliquots of the 10,000 X av pellet, prepared from an homogenate of bovine corpora lutea, were loaded on to 20-40% w/w sucrose gradients. Centrifugation was carried out for 2.25 h at 82,500 X
0
20
gay.
The from
abscissa the
denotes
gradient. each tube
the
On the is plotted
tube ordinate,
number the
as eluted mass of
in as a percentage mass or activity recovered from the gradient. The percentage of the original homogenate mass or activity in the 10,000 X g pellet and the mass or enzyme activities in each aliquot applied to the gradient were, respectively: progesterone, 26.3%, 6.06 Lg; protein, 21.4%, 20.05 mg; succinate dehydrogenase, 5 8.8%, 25.0; acid phosphatase, 29.8%, 0.819; i3-glucuronidase, 39.3%, 0.206; catalase, 19.2%, 0.767; alkaline phosphatase, 23.3%, 0.318; NADH-cytochrome c reductase, 40.8%, 7.64; inosine diphosphatase, 28.5%, 0.534. The percentage recovery of the applied mass or activities from the gradient ranged from 80-120%. 1) #{149}, progesterone; 0, protein; 0, % sucrose, w/w. 2) #{149}, acid phosphatase; 0, catalase; #{149}.P-glucuronidase; 0, succinate dehydrogenase. 3) #{149}, inosine diphosphatase; A, NADH-cytochrome reductase; A, alkaline phosphatase. activity
10 TUBE
or the
total
13
16
19
NO
30
20
>>
10 C
0
20 I 1
4
I
I 7
I
I
10
TUBE
I
II
13
NU
I
16
19
QUIRK
1138
ET AL.
0.1-0.4
pm,
in
Many
shape.
cell
some
periphery
of
being
(Fig.
of
of
the
and
appeared
expelled
the
and
in
>
ing
C
4
these
C
phatase
(size
Some
I
I
I
I
I
I
I
10
7
TUBE
I 13
I
I
I 16
>5
granules
were
C
The in
In
may 1
7
4
10
TUBE
16
13
19
NO
FIGS. 4-6. The relative specific activity of progesterone and marker enzymes on 20-40% w/w sucrose density gradients. Details of the experimental procedures are given in the legend to Figs. 1-3. The results shown are from the same representative experiment. The abscissa denotes the tube number as eluted from the gradient; the ordinate value is the relative specific activity or enrichment. The relative specific activity is the percentage of mass or activity recovered in each tube divided by the percentage of protein covered in that tube (H1 = 100%). 4) ., progesterone; 0, % sucrose, w/w. 5) S acid phosphatase; 0, catalase; 0, succinate dehydrogenase; #{149}, #{216}-glucuronidase. 6) #{149},inosine diphosphatase; A, NADH-cytochrome c reductase; A, alkaline phosphatase.
FIG.
7.
A luteal
cell
from
a cow
at
Day
13
of
the
estrous
present, some of which are irregular in shape (arrows) and cell (double arrows). The cytoplasm is packed with smooth secreting Golgi regions can be seen (G). X 7400.
lu-
the
range
Golgi
and
been
enrichment
caused
shape
of
irregular.
with in
by
various
the
usually
increase
also
of
sections,
consistent
The
where
fractions
vesicles
was
dense
5-6
These
tissue
sucrose
electron
tubes
gradient
was
from
that
10).
to the
sections.
have
in
fractions
membrane
contrast
size
were corpus
observed
in
(Fig.
from
tissue
phos-
acid
(lysosomes)
fixed
present
many
sizes.
of
exocytosis
Few
concentration
contained
generally
bovine
showed
maximal
granules
and
8).
was
of
gradients
were
exo-
were
9).
density
>2
pm) (Fig. granules
progesterone
C4
any
luteum. (size
undergoing
midluteal
Examination
6
other In 24 0.1-0.2
from
granules
staining
(Fig.
cisterni
19
NO
a
were stained with alkaline and thus were identified as None of the positive stain-
0.2-0.4
the
(Reddy, Gulyas
1973;
them
observed
in
been
have
al., 1976) and out to identify
observed
positive
teum. 4
process
exocytosis
lysosomes
corpus granules
nonstaining
was
observed Y
these
were
The
larger >
1
at the
the
by
Novikoff,
and
granules
cytosis.
Il
in
cell
1975; Gemmell et study was carried
pm) a fifth of diaminobenzidine microperoxisomes.
6
irregular seen
tissues
these and to distinguish granules in the bovine fields containing 400
NO
be
steroidogenic
Novikoff
1973;
and Yuan, cytochemical
10
to
from
Microperoxisomes
TUBE
were were
7).
identified >
which
granules
that
found
pleomorphism
the
experimental
manipulations. Electron dense granules were observed in material from tubes 11-12 and tubes 17-18 of the gradient. These granules were not associated with the presence of progesterone and were subsequently shown by cytochemical peroxisomes
techniques or lysosomes.
contained with
endoplasmic
consisted
mainly
lysosomes
and
cycle.
11-12
also
that
Large
Tubes
mitochondria
17-18
some
plus
microperoxisomes.
of
studies very
numbers
few
of
of
the the
densly
are in the process of being surfaced endoplasmic reticulum
many
micro-
mainly
reticulum. of
Cytochemical showed
be
Tubes
many membrane vesicles consistent presence of plasma membrane and
the
agranular
to
fixed granules
staining
fractions in
granules
frac-
are
expelled from the and many actively
PROGESTERONE
IN THE
BOVINE
CORPUS
LUTEUM
1139
of a luteal cell from a cow at Day 13 of the estrous cycle incubated with alkaline diaminoto demonstrate microperoxisomes. The section is not counterstained and the dense osmiophilic reaction product can be seen over a microperoxisome (MP). Nonreacting granules can also be seen (G) some in the process of exocytosis (arrows). X 11,247. FIG.
benzidine
8. Portion
LOCATION
QUIRK
1140
tions
5
and
6
stained
DAB (not contained
shown). substantial
positively
staining
sional 11).
In
observed
the
vast
tubes
17-18,
of the gradient. observed only
the
densest
maximal
the
in
sample zone at the top of any progesterone entering
in this
dense were
granules
were
(tubes
distri-
tubes
where
located
were
also
observed.
nor
The
microperoxi-
the
Our results Gemmell We
(1975).
et
have
of the total is particulate, centrifugation.
Since
have
homogenization.
the
some
and some
proges-
total
present enrichment
in
sucrose,
d
=
up to of liver
We
observed which
(1971)
has
progesterone When
subjected
most
was
shown
density fraction
lysosomes It could
similarly
containing to density
of the
distorted
a
released
granules. gradient
sedimentable
in
well
centrif-
progesterone
entered the gradient. The portion found in the sample zone at the top of the gradient, along with some of the lysosomal and micro-
was low
was that
associated with were visible in of
the
the
progesterone-
dense
these
granules irregular
enrichment were in
of the progesterone-enriched was low compared with
Granules
gradients.
For
of
mucosa (Trotman
band
intestinal 1.17-1.18 we
which higher
at
example,
of usually
shape. The granular that of other
contain
which with these
polypeptide
density the
in
granules
a high
rather
granules. hormones
the the
gradient.
electron
and
that
in
observed in The endowere in fact
cell organelles. The low density at progesterone banded would be compatible the sequestration of progesterone within
ties
some
reticulum. enzymes were and although
fraction, the large endoplasmic reticulum in was responsible for the
contained
progesterone;
during
homogenizer
15% of the homogenate.
progesterone
that
of
fractions
Duve
from
apart
contamination
micrographs peak
activities
experi-
density,
to data were
amounts and showed higher at higher densities (3 1-36% w/w 1.132-1.156). Thus, it is unlikely
organelle our
lower
up
greater
minor
probably
enrichment a narrow density d = 1.094-1.117)
considerable vesicle contamination the progesterone-enriched peak. plasmic reticulum marker enzymes
enzyme
during
sequestered
was considerable, and morphological other cell organelles
microsomal
cytosol,
most
over sucrose,
the progesterone-enriched volume of agranular the corpus luteum
enriched
lysosomal
been
progesterone
that progesterone membrane vesicles
tissue
have
the gradient. the sucrose
with the endoplasmic the microsomal marker throughout the gradient,
marker
occurred De
damage preparation
ugation,
a third
large
use of a Potter-Elvehjem
can
from
contain
progesterone
must
the
and
of
this
remained
also
or membrane.
enrichment Enzymatic that no
electron
both
procedures,
that
cells
would
which
least
x g centrifugation
600
unbroken
in
mental
be
the
at
the corpus luteum be sedimented by is probably an
in
can figure
since
recovered
damage
that
This
microperoxisomal
were
extend the findings and Kramers et al.
shown
progesterone since it
underestimate step removed fragments terone.
confirm and al. (1974)
must
peak
at
overlap However, spread
found,
DISCUSSION
and
an organelle
occurred reproducibly range (23-28% w/w
somes.
of
gradient
within
enzyme
those
lysosomes
density
and the 17-fold. showed
levels
neither
the
was presumably of the 10,000 We estab-
centrifugation.
cytosolic
The
activity from
cytochemical
marker in
progesterone
latter
(Fig.
Thus
gradient
the
and
Furthermore,
electron
progesterone
of
present
morphological
confirmed
butions.
that
because
Acid phosphatase material obtained of
lished
microperoxisomes
probably
in
region
occa-
12).
the
findings
observed
of mitochondria
part was
Thus
less
enzymes, resuspension
(which many an
AL.
peroxisomal marker leached out during x g pellet or during
alkaline
only
were
field,
per
Fig.
and
granule,
number
17-18,
with
11-12 activity),
granules
nonstaining
were
positively
In tubes catalase
ET
gastrin
in
sucrose
granules
band in sucrose at d et al., 1976). The densi-
found
for
the
other
cell
=
organelles
FIG. 9. Portion of a luteal cell from a cow at Day 15 of the estrous cycle incubated to demonstrate the presence of acid phosphatase activity. The section is not counterstained and reaction product can be seen in the Golgi cisternae and in lysosomes (L). Other granules (G) can be seen which are not positively stained. X 17,368. FIG.
(G,
arrows)
10.
Fixed pellet from are present, together
tubes
with
5-6
vesicles
of a 20-40%
of various
w/w
sucrose
sizes and other
density
gradient.
membranous
Dense
components.
staining granules X 27,388.
PROGESTERONE
LOCATION
IN THE
BOVINE
CORPUS
LUTEUM
1141
QUIRK
1142
of
the
bovine
those
corpus
found
gradients
strated
in
of
sections
of
Both
and these
cellular
in
whereas
space, granules
are
(Parry,
vitro
are
which
secrete
released
into
adrenal,
ACTH
the
tion
and
(Rubin
Rubin,
1974;
authors
also
secreted
fixed
In
suggested
that
a protein
in
parts
of
corresponded tive
to
marker
that
contain
for
enzymes
electron
dense
microperoxisomes
or
chemical
data
electron
dense
addition,
FIG.
support
11.
Fixed
is
tissue
likely
pellet
the
per-
and These were
gradient
other Their the
these
organelles. which did
lysosomes. the
that
from
did
granule
the is
production
also contain cyto-
that
these
progesterone. a protein
11-12
the
biochemical
and
also
might cytochemical
contain results
support the concept that progesterone is localized within electron dense granules which are neither microperoxisomes nor lysosomes. Further confirmation of the hypothesis that steroid
hormone
secretion
occurs
via exocytosis
ACKNOWLEDGMENTS The
These
tubes
levels
cycle of that relaxin
than
respec-
not
contention contain
of
location
for
granules,
relaxin
of steroid containing granules must await the purification of granules in quantities sufficient to investigate their composition more fully.
sucrose density and lysosomes
fractions,
granules it
the
form.
observed
concomitant
However, that high
gradient
1977).
peak.
progesterone-enriched
In
the
the estrous therefore
by relaxin
that
regions relaxin. Our
glucocorticoids
progesterone-enriched
and
during unlikely
porcine
observed
cat
increase
an
into
fractions from microperoxisomes
the
of
perfused
Laychock
in granule
location
the electron dense granules in the progesterone-enriched
the
produc-
al.,
in
de-
(1970),
possibility that which we observed
within
et
granules
pregnancy were who suggested
progesterone
the
that
to
Gueriguian
was
ewe, and
progesterone
protein
similar
a
corpus
a protein the
1974;
Gemmell
located
the
of al.,
bovine
within these granules. al. (1975) reported
not coincide sheep. It is
for
fell
that
glucocorticoid
et
al.
yet-investigated the on density gradients.
dense
to
et
Abel
in
and
corpus luteum of Belt et al. (1971)
related
the
by
and progesterone secretion observed by Gemmell et al. (1974). Since an assay for bovine relaxin is not available, we cannot exclude the
caused
granules
release
with
gradients,
In
increased
the
fusate
were
incubate.
of
an
space.
rose
stimulation
number with
along
also
the of
not
circulating
luteum,
reported
concomitantly
the
shown
pericellular
we have component
them
is
evidence
data)
Leymarie
render
candidate
preliminary protein
was contained Chamley et
electron
progesterone,
decrease
or
pen-
unpulished
exocytosed
that
and
the
corpus
from
granules
have
Electron
These
of
have
but this
Gulyas
Willcox,
(1977)
We
by
also
the
al.
increase
an
secretion
in
et
(1977).
to
possible described
(unpublished
We have
secreting
protein
scribed
1976).
group
secreted
granules
A
luteum
(Reddy,
ovine
preparations of
number with
of
into
Abel
similar
and
slices
granules
Recently, in
Quirk
that actively
observations)
a third
the
dense.
luteum.
1973;
secreted.
within
present. reported
into
present electron
progesterone-binding
been
secreted
AL.
granules
tissues
al.,
et
never
demon-
are
Novikoff,
Gemmell
are
dense
corpus
have
and
1975;
organelles
bovine lysosomes
steroidogenic
Novikoff
Yuan,
dense
organelles
in
1973;
electron of
Microperoxisomes
previously
with sucrose
we have
studies,
3 types
present
broadly
1973).
cytochemical that
and
agree
discontinuous
(Gospodarowicz,
In our are
luteum
using
ET
must
be
of
a 20-40%
We are indebted for expert technical assistance to Misses Georgina Pooley and Elaine Johnston and to Mr. Ian Cookson. We are very grateful to the staff of R. A. Rixon Ltd. and F.M.C. (Thame) Ltd. for material. The supply of dated bovine corpora lutea from Dr. S. Dixon, A.R.C., Compton is gratefully acknowledged. We thank Dr. G. Jenkin for helpful discussions and Mrs. Jill Iredale for her help in preparation of the typescript. G.D.T. was a member of the External
w/w
sucrose
density
gradient,
after
incubation
with
alkaline diaminobenzidine to demonstrate microperoxisomes. The section is not counterstained and the dense osmiophilic reaction product can be seen over microperoxisomes (arrows). An occasi9nal nonstaining granule can be seen (G, double arrows). Membrane vesicles constitute the remainder of the field. X 34,650. FIG. 12. Fixed pellet from tubes 17-18 of a 20-40% w/w sucrose density gradient incubated to demonstrate the presence of acid phosphatase activity. The section is not counterstained. The dense reaction product identifies a lysosome (L); some nonbackground stain is observed. The rest of the field is comprised mainly of mitochondria. X 17,160.
PROGESTERONE
LOCATION
IN THE
a
BOVINE
CORPUS
LUTEUM
1143
QUIRK
1144
Scientific Dominions
Staff of the M.R.C. Trust Demonstrator.
and
D.L.W.
ET AL.
is a Nuffield
microtubules
reprod. Gemmell, R. (1977).
H.
(1974).
Catalase. In: Methods of Enzymatic Analysis. 2nd English ed. (H. U. Bergmeyer, ed). Academic Press, New York. 2, pp. 673-684. Abel, J. H., McClellan, M. C., Sawyer, H. R., Schmitz, M., Niswender, G. D. and Nau, E. (1977). Synthesis and release of a secretory protein from lutein cells. J. Cell. Biol. 75, 374a. Beaufay, H., Amar-Costesec, A., Feytnians, E., ThinesSempoux, D., Wibo, M., Robbi, M. and Berthet, J. (1974). Analytical study of microsomes and isolated subcellular membranes from rat liver. I. Biochemical methods. J. Cell. Biol. 61, 188-200. Belt, W. D., Cavazos, L. F., Anderson, L. L. and Melampy, R. M. (1971). Cytoplasmic granules
and relaxin Endocrinology
levels
in
porcine
corpora
lutea.
89, 1-10. Bergmeyer, H. U., Gawehn, K. and Grassl, M. (1974). Enzymes as biochemical reagents. In: Methods of Enzymatic Analysis. 2nd English ed. (H. U. Bergmeyer, ed.). Academic Press, New York. 1, pp. 495-496. B#{246}hlen, P., Stein, S., Dairman, W. and Udenfriend, S. (1973). Fluorometric assay of proteins in the nanogram
Arch.
range.
Biochem.
Biophys.
155,
2 13-220. Challis, J.R.G., Davies, I. J. and Ryan, K. J. (1973). The concentrations of progesterone, estrone and estradiol-17(3 in the plasma of pregnant rabbits.
Endocrinology Chainley. W. A., (1975).
93, 971-976. Stelmasiak, T.
Plasma
the oestrous 45, 455-461. de Duve,
C.
Enders,
A. Biol.
Essner,
C.
E.
1, pp. M.
ducts
G. by
fractionation:
G. D. during Fert. past
and
20D-50D. of
the
corpus
luteum.
158-182.
Phosphatases. ed).
Hayat,
50,
and Bryant, immunoactivity the ewe. J. Reprod.
Cytology 8,
of Enzymes.
York. Farquar,
Biol.
(1973).
(1973).
of Tissue
Cell.
Reprod.
scopy A.
cycle
(1971).
J.
present.
relaxin
van
In:
Principles Nostrand
Electron
and Methods. Reinhold Co.,
Micro-
(1971).
Processing of
the
anterior
of
secretory pituitary
Mem. Soc. Endocrinol. 19, 79-146. Fishman, W. H. (1974). 13-glucuronidase. In: Methods of Enzymatic Analysis. 2nd English ed. (H. U. Bergmeyer, ed.). Academic Press, New York. 2, pp. 929-943. Fleischer, F. and Fleischer, B. (1967). Removal and binding of polar lipids in mitochondria and other membrane systems. In: Methods Enzymol. (R. W. Estabrook and M. E. Pullman, eds.). Academic Press, New York. 10, pp. 406-433. Gemmell, R. T., Stacy, B. D. and Thorburn, G. D. (1974). Ultrastructural study of secretory granules in the corpus luteum of the sheep during the estrous cycle. Biol. Reprod. 11, 447-462. Gemmell, R. T., Stacy, B. D. and Thorburn, G. D. (1976). Morphology of the regressing corpus luteum in the ewe. Biol. Reprod. 14, 270-279. Gemmell, R. T. and Stacy, B. D. (1977). Effects of colchicine on the ovine corpus luteum: role of
S.
G. and Rubin, R. P. biochemical evidence secretory organelle in the
and
Ultrastructural
gland.
J. Cell.
Biol.
and from
72,
characbovine
799-800. M.T.C., Sheppard, B. L. and Thorburn, G. D. (1975). Evidence in favour of progesterone secretory granules in the bovine corpus luteum. Acta Endocrinol. Abstr. 210. Lacy, P. E., Howell, S. L., Young, D. A. and Fink, C. J. (1968). New hypothesis of insulin secretion. Nature (Lond.) 219, 1177-1179. Laychock, S. G. and Rubin, R. P. (1974). Isolation of
Kramers,
ACTH-induced Steroids 24,
protein
from
adrenal
perfusate.
177-184. Leclerq-Meyer, V., Le Marchand, J. and Malaisse, W. J. (1974). Possible role of a microtubular-microfilamentous system in glucagon secretion. Diabetologia 10, 2 15-224. Leymarie, P. and Gueriguian, J. L. (1970). Progesterone-binding by the soluble fraction of corpus luteum from the pregnant cow. Res. Steroids 4, 239-246. Lowry. 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurementwith the Folin phenol reagent. J. Biol. Chem. 193, 265-27 5. Novikoff, A. B. and Goldfischer, S. (1969). Visualization of peroxisomes (microbodies) and mitochondria with diaminobenzidine. J. Histochem.
Cytochem. Novikoff,
A.
B.
peroxisomes.
17, 675-680. and Novikoff,
J.
P. M.
(1973).
Cytochem.
Histochem.
Micro21,
963-966.
progland.
J.
5050-5056. Microperoxisomes in the late pregnancy corpus luteum of rhesus monkeys (Macaca mulatta). J. Histochem. Cytochem. 23, 259-368. HUbscher, G. and West, G. R. (1965). Specific assays of some phosphatases in subcellular fractions of small intestinal mucosa. Nature (Lond.) 205,
44-76. cells
Laychock,
of progesterone.
209-215. Gospodarowicz, D. (1973). Preparation terization of plasma membranes corpus luteum. J. Biol. Chem. 248, Gulyas, B. J. and Yuan, L. C. (1975).
(M.
New
T.,
in the secretion 49, 115-117.
for a steroid-containing perfused cat adrenal
REFERENCES Aebi,
Fert.
Novikoff, A. B., Novikoff, P. M., Quintana, N. and Davis, C. (1973). Studies on microperoxisomes. IV. Interactions of microperoxisomes, endoplasmic reticulum and lipifuscin granules. J. Histochem. Cytochem. 21, 1010-1020. Pennington, R. J. (1961). Biochemistry of dystrophicmuscle. Mitochondrial succinate-tetrazolium reductase and adenosine triphosphatase. Biochem. J. 80, 649-654. Reddy, J. K. (1973). Possible properties of microbodies (peroxisomes). Microbody proliferation and hypolipidermic drugs. J. Histochem. Cytochem. 21, 969-971. Reynold, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-213.
Rubin,
R. P., Sheid, B., Mccauley, S. G. (1974). ACTH-induced the perfused cat adrenal
exocytosis?
Endocrinology
R.
protein gland:
and
Laychock, release from evidence for
96, 370-378.
PROGESTERONE
Sottacasa,
G.
L.,
Kuylenstierna,
B., Ernster,
L
IN THE
and
An electron-transport system with the outer membrane of liver mitochondria. A biochemical and morphological study. J. Cell Biol. 32, 415-438. Stacy, B. D., Gemmell, R. T. and Thorburn, G. D. (1976). Morphology of the corpus luteum in the sheep during regression induced by prostaglandin F20. Biol. Reprod. 14, 280-291. Steck, T. L. (1972). Membrane Isolation. In: Membrane Molecular Biology (C. F. Fox, and A. D. Keith, eds.). Sinauer Associates Inc., Stamford. pp. 76-114. Stephens, R. E. and Edds, K. T. (1976). Microtubules: Bergstrand,
A. associated
(1967).
LOCATION
BOVINE
CORPUS
structure,
LUTEUM
chemistry
and
1145
function.
Physiological
Reviews 56, 709-777. Trotman, C.N.A., Fiddes, I.J.S., Grund, E. R., McHanwell, S., Sanders, D. J., Sanderson, C. and Shaw, B. (1976). lmmunoreactive gastrin in differential and density gradient fractions of rat gastric mucosa. J. Endocrinol. 68, 5-12.
RECOMMENDED
Enders, Biol.
A.
C. (1973).
Reprod.
Savard, K. (1973). Iuteum. Biol.
Cytology
REVIEWS
of the corpus
luteum.
8, 158-182.
The biochemistry Reprod. 8, 183-202.
of
the
corpus
OF
REPRODUCTION
Subcellular
20,
1133-1145
Location
of Progesterone
A Biochemical, SUSAN
J.
(1979)
Morphological
QUIRK1,
Nuffield
DAVID GEOFFREY
Department
in the
Bovine
Corpus
and Cytochemical L. WILLCOX, DILYS D. THORBURN’ of Obstetrics
and
Luteum:
Investigation M.
PARRY2
and
Gynaecology
and
Nuffield
Institute
John
for
Radcliffe
Medical
Hospital,
Headington,
Research,
Headley
Oxford,
Way
U.K.
ABSTRACT
The subcellular biochemical, homogenates this particulate
location
of progesterone
in the bovine
cytochemical up to one-third of banded discretely
morphological showed that progesterone
and
corpus
luteum has been investigated by Differential centrifugation of tissue the total progesterone could be sedimented. Most of at low density on sucrose gradients. Marker enzyme techniques.
studies showed that it was not associated with either the microperoxisomes, lysosomes or mitochondria nor appreciably with the vesiculated subcellular components such as plasma membrane and microsomes. Morphological examination of tissue sections showed that the luteinized cells of the bovine corpus luteum contain electron dense granules. Three types of granules were present. Some of these granules were identified as microperoxisomes and lysosomes by cytochemical techniques, but a third type of granules was also present. Morphological and cytochemical studies of different regions of the sucrose density gradient confirmed the distribution of subcellular organelles as judged by marker enzyme techniques. Electron dense granules were observed in material fixed from the progesterone peak, while microperoxisomes and lysosomes were found in denser regions of the gradient and separate from the progesterone
band.
These results support the hypothesis that some of the progesterone is sequestered within electron dense granules.
INTRODUCTION
are electron
The secretion of steroids from steroidogenic tissue is usually assumed to occur by diffusion from the site of synthesis through the plasma
number
membrane
related
to the
into port
the blood, the steroids 1973). In the
extracellular
space
phase, creases.
is when High
Accepted Received ‘Present
Department Queensland 2Present rics and
Headington,
maximal
and
thence
the
these
to
the
regression
(PGF2y),
luteolytic
progesterone biosynthesis derates of progesterone biosynthesis
September 25, 1978. June 6, 1978. address: University of Physiology, St.
into of
bovine
presence some of
extracellular
by
of
of
occurs
and
granule
(Gemmell
et al.,
1976;
Stacy
tissue corpus When F2a
progesterone
ceases
secretion et al.,
granule
colchicine
is
the
prostaglandin
decrease
and
The
exocytosed
activity
induced
by
space.
being
secretion
decreased
of many which are
as regression of the (Gemmell et al., 1974).
marked
progesterone
luteum
corpus
granules
secretory
is a
the
with the granules,
and decreases luteum proceeds
between the progesterone the
associated dense
expelled
where binding proteins transto their site of action (Enders, corpus luteum, marked differ-
ences in morphology are observed midluteal phase of the cycle when biosynthesis
and
in
1976).
Both
production
are
to ewes
administration
(Gemmell and Stacy, 1977). These results suggest that the secretion of progesterone from the luteal cell might occur by exocytosis of hormone containing granules. Polypeptide hormones hormones from the
Queensland, Lucia, Brisbane. of
4067, Australia. address: Nuffield Department Gynaecology, John Radcliffe Oxford, U.K.
of ObstetHospital,
1133
such as insulin, glucagon and the of the anterior pituitary are secreted cell by this mechanism. Intracellular
transport
of
granules
mediated
by
microtubules;
chicine,
which
interfere
to
the
cell
drugs, with
periphery such
is
as col-
microtubular
1134
QUIRK
also
function
interfere
these 1971;
hormones Leclerq-Meyer
Edds, In
1976). this
and
et
study
fractionation,
we
that
the
of
the
presence
al.,
al.,
1974;
of
Farquar, Stephens and
used
centrifugation studies
sedimentable
luteum
is
to
proges-
associated
with
of granules.
MATERIALS
chemicals
All
AND
were p-nitrophenol HEPES
13-D-glucuronide, N’-2-ethane-sulphonic
4,
METHODS
DCPIP
Analar
grade.
Trizma
phosphate,
Ficoll
was
Hopkins
and
Centre,
from
Williams
obtained Pharmacia,
and
Ci/mmol)
Amersham.
3’, and
were purchased
(80-110
Radiochemical (4-phenylspiro was obtained reagent from
(3,
tetrahydrochloride)
(2,6-dichlorophenol-indophenol)
l’FII-progesterone
7.0,
p-nitrophenol-
(N-2-hydroxyethylpiperazineacid), diaminobenzidine
4’-tetraaminobiphenyl
from Sigma. sucrose from
S,. was retained. In some experiments aliquots of the cytosol were loaded on to sucrose density gradients as described below.
1, 2,
6,
from
7. the
Fluorescamine
lfuran-2 (3), l’-phthalanl-3, from Roche and Folin-Ciocalteau Fison Scientific.
3’-dione) phenol
Density Centrifugation
After evaluation of several different density gradients, linear 20-40% w/w buffered sucrose gradients were prepared using an ISCO Model 570 gradient former. The gradients were of 36 ml capacity with a 1.5 ml underlay of 50% w/w sucrose. After
loading,
Chemicals p-nitrophenol,
AL.
Sucrose Gradient
subcellular
cytochemical
the
corpus
secretion
1968;
gradient and
demonstrate
the
et
have
density
morphological
terone
with
(Lacy
ET
the
gradients
were
centrifuged
in a Beckman
SW-27 rotor for 2.25 h at 25,000 rpm (82,500 X gay), and eluted in 2 ml fractions by upward displacement with 60% w/w sucrose using an ISCO Model 184 tube-holding and piercing mechanism coupled to a nonperistaltic pump. The sucrose concentration or density (20#{176}14#{176}) of individual fractions was determined by refractometry (Bellingham and Stanley refractometer, Tunbridge Wells, U.K.). Fractions were stored at -20#{176}Cuntil further analysis was carried out. In some experiments, fractions of identical density from more than one gradient were pooled. Some pooled gradient fractions were fixed directly by glutaraldehyde (see below) for morphological and cytochemical examination. The fixed material was collected by centrifugation and treated as described below for tissue sections.
Assays Tissue
Manipulations
Midluteal phase bovine corpora lutea were obtained from 2 local abattoirs. The material was placed on ice within 10 mm of death of the cows. All subsequent manipulations were performed at 0-4#{176}C. In the laboratory, some 60-90 mm later, the corpora Iutea were decapsulated, minced using a Climpex tissue mincer (fitted with a stainless steel grid perforated by 1.7 mm diameter holes) and weighed. All sucrose solutions were buffered with either 0.01 M Trizma 7.0 (pH 7.5; 5#{176}C) or 0.01 M HEPES, pH 7.4. A 10% w/v homogenate in buffered 0.25 M sucrose was obtained (H,) with 3-5 strokes of a Potter-Elvehjem homogenizer (Thomas, Philadelphia, PA; No. C44491, 0.177 mm clearance) driven by an MSE homogenizer at quarter speed. The homogenate (H,) was centrifuged at 600 X umax for 5 mm (MSE, Mistral 6L) to remove nuclei, large pieces of cell debris and incompletely homogenized and unbroken cells. After centrifugation, the supernatant (5,) was decanted and recentrifuged at 10,000 X av for 30 mm (Beckman Spinco L2-65B). Floating lipid was removed with a clean dry glass rod. The supernatant (S2) was decanted and the pellet resuspended in buffered 0.25 M sucrose (H2, 10,000 X g pellet) by 3 handstrokes of a Potter-Elvehjem homogenizer (No. A8427, 0.127 mm clearance) to a total volume in ml equivalent to the number of g of original minced corpus luteum tissue (after allowing for sampling). Between 1.0 and 1.5 ml of this 10,000 X av pellet (H2) was loaded on to sucrose density gradients as described below. A microsomal fraction (H,, 100,000 X g pellet) was obtained by centrifuging supernatant 2 at 96,300 for 30 mm and resuspending the pellet in buffered sucrose. The supernatant or cytosol fraction,
Progesterone was measured by radioimmunoassay et al., 1973). Pregnenolone, 17-hydroxyprogesterone, 20n-hydroxy-pregn-4-en-3-one, cortisol and estradiol-17j3 showed less than 1.5% cross reaction with the progesterone antiserum. The solvent blanks were routinely equivalent to 1.116). Progesterone levels were
(2) (1)
The
assayed.
recovered
progesterone,
negligible
dehydrogenase
activity
5%
13-glucuronidase,
alkaline phosphatase, reductase and inosine The latter 3 enzyme
acid
of
were
catalase
of
the and
than phosphatase,
and
pletely terone
tube
less
NADH-cytochrome c diphosphatase activities. activities were spread
at
8
low
100%.
onwards
from
tube
8
highest
from
tube
resolved from the peak, while a small
onwards
14
the
lysosomes, were com-
particulate proportion
progesof the
microsomes and plasma membrane overlapped in this region. Compared with the homogenate, the enrichment (relative specific activity) of progesterone was
greatest
ment of experiment
in
tubes
7-8-fold shown
experiments ranged from
Maximum
the enrichment 7-17-fold. The
2, which entered
enrich4-6 in the In 4 other this other
were obtained and inosine
enzyme is also of mitochondria.
located Tubes
progesterone showed
gradient,
at
densities
progesterone mum
peak,
enrichment
position endoplasmic
greater
of
and usually
maximal reticulum
than the
position
corresponded
on 1 had
that a much
enrichment owing to the presence of protein which did not enter the gradient. All marker enzymes showed higher ments
region signi-
microsomal marker enwas, however, less than
contained the
in only
in this region c reductase
the enrichment
diphosphatase, zymes. This
and
3-8.
occurred in tubes in Figs. 4-6.
ficant enrichments for NADH-cytochrome
not
density
50%
percent
set
onwards, whereas acid phosphatase, j3-glucuronidase, catalase and succinate dehydrogenase
2-fold. The former the outer membrane
organelles.
sucrose, about
succinate
of
about
13-glucuronidase
presumably
activity,
enzyme
=
tubes
acid
some
100,000
(sucrose density >1.146). Thus mitochondria and microperoxisomes
Gradients
Figs.
centrif-
or activity
mass
Pellet
(cytosol)
activities Distribution
5.7 0.9 0.7 7.0 4.7
6.1 5.1
X g pellet
significant
containing
chosen
density
100,000
(not
10,000
differential
or enzyme activity in each fraction is expressed relative to the homogenate, mean ± SD; the number of experiments is indicated in parentheses.
a
enrichment
terone
by
obtained
c reductase
insensitive)
Inosine
activities
enzyme
homogenate.a
lutea
lower soluble enrich-
that
of of
the
maxito
the
enzyme activity. The and plasma membrane
PROGESTERONE
markers and
thus
This ation
were
spread
throughout
not
heavily
enriched
distribution in vesicle
during elements
homogenization of the cell.
separate
lysosomes,
the
denser
the
gradient
medium,
fraction.
terone
because density of
No
of the variwhich arises
these
membranous was made
attempt
and
the enrichment of the gradient
was less than if the gradient to effect a separation. Prolonged centrifugation
had
of the to
micro-
been
h)
of
Linear
as
gradients
Ficoll range 0.25
above
were
also
2.25,
18
or
h,
at the
top
gradients
nor
of
the
use
of
the
midcycle
extensive
endoplasmic
corpus
network
reticulum
of
characteristic
secretory tissues and an abundance of dense granules ranging in size from
30
75
20
50
10
25
Ficoll
1
4
7
10 TUBE
13
16
19
NO
Thus,
gradient.
centrifugation the
of an
very similar gradients.
considerable alkaline and acid phosactivities remained with the progesprolonged
cells
the
and
However, phatase
peak
Cytochemistry
in
progesterone obtained
terone
proges-
20-
resulting to those
neither
of
evaluated
20.25
profiles were with sucrose
separation
Luteum
contained
of steroid electron
0-20% w/w and 5-25% w/w M sucrose. They were centrifuged
for
and
luteal
agranular
extended
(17.5
The
the
enzymes.
marker
Corpus
1137
LUTEUM
improved from
luteum
obtained (2-4-fold)
CORPUS
Morphology
40% w/w sucrose gradients gave maximal enrichments of progesterone and marker enzymes at the same densities as found for the shorter centrifugation time. Thus, by 2.25 h in a 20-40% w/w sucrose gradient, equilibrium had been attained. over the buffered
BOVINE
in any
mitochondria
so that regions
peroxisomes, in
occurs size and
IN THE
LOCATION
of
sucrose
as
density
>
FIGS. 1-3. The distribution of progesterone. enzyme activities and protein on 20-40% w/w sucrose density gradients. Aliquots of the 10,000 X av pellet, prepared from an homogenate of bovine corpora lutea, were loaded on to 20-40% w/w sucrose gradients. Centrifugation was carried out for 2.25 h at 82,500 X
0
20
gay.
The from
abscissa the
denotes
gradient. each tube
the
On the is plotted
tube ordinate,
number the
as eluted mass of
in as a percentage mass or activity recovered from the gradient. The percentage of the original homogenate mass or activity in the 10,000 X g pellet and the mass or enzyme activities in each aliquot applied to the gradient were, respectively: progesterone, 26.3%, 6.06 Lg; protein, 21.4%, 20.05 mg; succinate dehydrogenase, 5 8.8%, 25.0; acid phosphatase, 29.8%, 0.819; i3-glucuronidase, 39.3%, 0.206; catalase, 19.2%, 0.767; alkaline phosphatase, 23.3%, 0.318; NADH-cytochrome c reductase, 40.8%, 7.64; inosine diphosphatase, 28.5%, 0.534. The percentage recovery of the applied mass or activities from the gradient ranged from 80-120%. 1) #{149}, progesterone; 0, protein; 0, % sucrose, w/w. 2) #{149}, acid phosphatase; 0, catalase; #{149}.P-glucuronidase; 0, succinate dehydrogenase. 3) #{149}, inosine diphosphatase; A, NADH-cytochrome reductase; A, alkaline phosphatase. activity
10 TUBE
or the
total
13
16
19
NO
30
20
>>
10 C
0
20 I 1
4
I
I 7
I
I
10
TUBE
I
II
13
NU
I
16
19
QUIRK
1138
ET AL.
0.1-0.4
pm,
in
Many
shape.
cell
some
periphery
of
being
(Fig.
of
of
the
and
appeared
expelled
the
and
in
>
ing
C
4
these
C
phatase
(size
Some
I
I
I
I
I
I
I
10
7
TUBE
I 13
I
I
I 16
>5
granules
were
C
The in
In
may 1
7
4
10
TUBE
16
13
19
NO
FIGS. 4-6. The relative specific activity of progesterone and marker enzymes on 20-40% w/w sucrose density gradients. Details of the experimental procedures are given in the legend to Figs. 1-3. The results shown are from the same representative experiment. The abscissa denotes the tube number as eluted from the gradient; the ordinate value is the relative specific activity or enrichment. The relative specific activity is the percentage of mass or activity recovered in each tube divided by the percentage of protein covered in that tube (H1 = 100%). 4) ., progesterone; 0, % sucrose, w/w. 5) S acid phosphatase; 0, catalase; 0, succinate dehydrogenase; #{149}, #{216}-glucuronidase. 6) #{149},inosine diphosphatase; A, NADH-cytochrome c reductase; A, alkaline phosphatase.
FIG.
7.
A luteal
cell
from
a cow
at
Day
13
of
the
estrous
present, some of which are irregular in shape (arrows) and cell (double arrows). The cytoplasm is packed with smooth secreting Golgi regions can be seen (G). X 7400.
lu-
the
range
Golgi
and
been
enrichment
caused
shape
of
irregular.
with in
by
various
the
usually
increase
also
of
sections,
consistent
The
where
fractions
vesicles
was
dense
5-6
These
tissue
sucrose
electron
tubes
gradient
was
from
that
10).
to the
sections.
have
in
fractions
membrane
contrast
size
were corpus
observed
in
(Fig.
from
tissue
phos-
acid
(lysosomes)
fixed
present
many
sizes.
of
exocytosis
Few
concentration
contained
generally
bovine
showed
maximal
granules
and
8).
was
of
gradients
were
exo-
were
9).
density
>2
pm) (Fig. granules
progesterone
C4
any
luteum. (size
undergoing
midluteal
Examination
6
other In 24 0.1-0.2
from
granules
staining
(Fig.
cisterni
19
NO
a
were stained with alkaline and thus were identified as None of the positive stain-
0.2-0.4
the
(Reddy, Gulyas
1973;
them
observed
in
been
have
al., 1976) and out to identify
observed
positive
teum. 4
process
exocytosis
lysosomes
corpus granules
nonstaining
was
observed Y
these
were
The
larger >
1
at the
the
by
Novikoff,
and
granules
cytosis.
Il
in
cell
1975; Gemmell et study was carried
pm) a fifth of diaminobenzidine microperoxisomes.
6
irregular seen
tissues
these and to distinguish granules in the bovine fields containing 400
NO
be
steroidogenic
Novikoff
1973;
and Yuan, cytochemical
10
to
from
Microperoxisomes
TUBE
were were
7).
identified >
which
granules
that
found
pleomorphism
the
experimental
manipulations. Electron dense granules were observed in material from tubes 11-12 and tubes 17-18 of the gradient. These granules were not associated with the presence of progesterone and were subsequently shown by cytochemical peroxisomes
techniques or lysosomes.
contained with
endoplasmic
consisted
mainly
lysosomes
and
cycle.
11-12
also
that
Large
Tubes
mitochondria
17-18
some
plus
microperoxisomes.
of
studies very
numbers
few
of
of
the the
densly
are in the process of being surfaced endoplasmic reticulum
many
micro-
mainly
reticulum. of
Cytochemical showed
be
Tubes
many membrane vesicles consistent presence of plasma membrane and
the
agranular
to
fixed granules
staining
fractions in
granules
frac-
are
expelled from the and many actively
PROGESTERONE
IN THE
BOVINE
CORPUS
LUTEUM
1139
of a luteal cell from a cow at Day 13 of the estrous cycle incubated with alkaline diaminoto demonstrate microperoxisomes. The section is not counterstained and the dense osmiophilic reaction product can be seen over a microperoxisome (MP). Nonreacting granules can also be seen (G) some in the process of exocytosis (arrows). X 11,247. FIG.
benzidine
8. Portion
LOCATION
QUIRK
1140
tions
5
and
6
stained
DAB (not contained
shown). substantial
positively
staining
sional 11).
In
observed
the
vast
tubes
17-18,
of the gradient. observed only
the
densest
maximal
the
in
sample zone at the top of any progesterone entering
in this
dense were
granules
were
(tubes
distri-
tubes
where
located
were
also
observed.
nor
The
microperoxi-
the
Our results Gemmell We
(1975).
et
have
of the total is particulate, centrifugation.
Since
have
homogenization.
the
some
and some
proges-
total
present enrichment
in
sucrose,
d
=
up to of liver
We
observed which
(1971)
has
progesterone When
subjected
most
was
shown
density fraction
lysosomes It could
similarly
containing to density
of the
distorted
a
released
granules. gradient
sedimentable
in
well
centrif-
progesterone
entered the gradient. The portion found in the sample zone at the top of the gradient, along with some of the lysosomal and micro-
was low
was that
associated with were visible in of
the
the
progesterone-
dense
these
granules irregular
enrichment were in
of the progesterone-enriched was low compared with
Granules
gradients.
For
of
mucosa (Trotman
band
intestinal 1.17-1.18 we
which higher
at
example,
of usually
shape. The granular that of other
contain
which with these
polypeptide
density the
in
granules
a high
rather
granules. hormones
the the
gradient.
electron
and
that
in
observed in The endowere in fact
cell organelles. The low density at progesterone banded would be compatible the sequestration of progesterone within
ties
some
reticulum. enzymes were and although
fraction, the large endoplasmic reticulum in was responsible for the
contained
progesterone;
during
homogenizer
15% of the homogenate.
progesterone
that
of
fractions
Duve
from
apart
contamination
micrographs peak
activities
experi-
density,
to data were
amounts and showed higher at higher densities (3 1-36% w/w 1.132-1.156). Thus, it is unlikely
organelle our
lower
up
greater
minor
probably
enrichment a narrow density d = 1.094-1.117)
considerable vesicle contamination the progesterone-enriched peak. plasmic reticulum marker enzymes
enzyme
during
sequestered
was considerable, and morphological other cell organelles
microsomal
cytosol,
most
over sucrose,
the progesterone-enriched volume of agranular the corpus luteum
enriched
lysosomal
been
progesterone
that progesterone membrane vesicles
tissue
have
the gradient. the sucrose
with the endoplasmic the microsomal marker throughout the gradient,
marker
occurred De
damage preparation
ugation,
a third
large
use of a Potter-Elvehjem
can
from
contain
progesterone
must
the
and
of
this
remained
also
or membrane.
enrichment Enzymatic that no
electron
both
procedures,
that
cells
would
which
least
x g centrifugation
600
unbroken
in
mental
be
the
at
the corpus luteum be sedimented by is probably an
in
can figure
since
recovered
damage
that
This
microperoxisomal
were
extend the findings and Kramers et al.
shown
progesterone since it
underestimate step removed fragments terone.
confirm and al. (1974)
must
peak
at
overlap However, spread
found,
DISCUSSION
and
an organelle
occurred reproducibly range (23-28% w/w
somes.
of
gradient
within
enzyme
those
lysosomes
density
and the 17-fold. showed
levels
neither
the
was presumably of the 10,000 We estab-
centrifugation.
cytosolic
The
activity from
cytochemical
marker in
progesterone
latter
(Fig.
Thus
gradient
the
and
Furthermore,
electron
progesterone
of
present
morphological
confirmed
butions.
that
because
Acid phosphatase material obtained of
lished
microperoxisomes
probably
in
region
occa-
12).
the
findings
observed
of mitochondria
part was
Thus
less
enzymes, resuspension
(which many an
AL.
peroxisomal marker leached out during x g pellet or during
alkaline
only
were
field,
per
Fig.
and
granule,
number
17-18,
with
11-12 activity),
granules
nonstaining
were
positively
In tubes catalase
ET
gastrin
in
sucrose
granules
band in sucrose at d et al., 1976). The densi-
found
for
the
other
cell
=
organelles
FIG. 9. Portion of a luteal cell from a cow at Day 15 of the estrous cycle incubated to demonstrate the presence of acid phosphatase activity. The section is not counterstained and reaction product can be seen in the Golgi cisternae and in lysosomes (L). Other granules (G) can be seen which are not positively stained. X 17,368. FIG.
(G,
arrows)
10.
Fixed pellet from are present, together
tubes
with
5-6
vesicles
of a 20-40%
of various
w/w
sucrose
sizes and other
density
gradient.
membranous
Dense
components.
staining granules X 27,388.
PROGESTERONE
LOCATION
IN THE
BOVINE
CORPUS
LUTEUM
1141
QUIRK
1142
of
the
bovine
those
corpus
found
gradients
strated
in
of
sections
of
Both
and these
cellular
in
whereas
space, granules
are
(Parry,
vitro
are
which
secrete
released
into
adrenal,
ACTH
the
tion
and
(Rubin
Rubin,
1974;
authors
also
secreted
fixed
In
suggested
that
a protein
in
parts
of
corresponded tive
to
marker
that
contain
for
enzymes
electron
dense
microperoxisomes
or
chemical
data
electron
dense
addition,
FIG.
support
11.
Fixed
is
tissue
likely
pellet
the
per-
and These were
gradient
other Their the
these
organelles. which did
lysosomes. the
that
from
did
granule
the is
production
also contain cyto-
that
these
progesterone. a protein
11-12
the
biochemical
and
also
might cytochemical
contain results
support the concept that progesterone is localized within electron dense granules which are neither microperoxisomes nor lysosomes. Further confirmation of the hypothesis that steroid
hormone
secretion
occurs
via exocytosis
ACKNOWLEDGMENTS The
These
tubes
levels
cycle of that relaxin
than
respec-
not
contention contain
of
location
for
granules,
relaxin
of steroid containing granules must await the purification of granules in quantities sufficient to investigate their composition more fully.
sucrose density and lysosomes
fractions,
granules it
the
form.
observed
concomitant
However, that high
gradient
1977).
peak.
progesterone-enriched
In
the
the estrous therefore
by relaxin
that
regions relaxin. Our
glucocorticoids
progesterone-enriched
and
during unlikely
porcine
observed
cat
increase
an
into
fractions from microperoxisomes
the
of
perfused
Laychock
in granule
location
the electron dense granules in the progesterone-enriched
the
produc-
al.,
in
de-
(1970),
possibility that which we observed
within
et
granules
pregnancy were who suggested
progesterone
the
that
to
Gueriguian
was
ewe, and
progesterone
protein
similar
a
corpus
a protein the
1974;
Gemmell
located
the
of al.,
bovine
within these granules. al. (1975) reported
not coincide sheep. It is
for
fell
that
glucocorticoid
et
al.
yet-investigated the on density gradients.
dense
to
et
Abel
in
and
corpus luteum of Belt et al. (1971)
related
the
by
and progesterone secretion observed by Gemmell et al. (1974). Since an assay for bovine relaxin is not available, we cannot exclude the
caused
granules
release
with
gradients,
In
increased
the
fusate
were
incubate.
of
an
space.
rose
stimulation
number with
along
also
the of
not
circulating
luteum,
reported
concomitantly
the
shown
pericellular
we have component
them
is
evidence
data)
Leymarie
render
candidate
preliminary protein
was contained Chamley et
electron
progesterone,
decrease
or
pen-
unpulished
exocytosed
that
and
the
corpus
from
granules
have
Electron
These
of
have
but this
Gulyas
Willcox,
(1977)
We
by
also
the
al.
increase
an
secretion
in
et
(1977).
to
possible described
(unpublished
We have
secreting
protein
scribed
1976).
group
secreted
granules
A
luteum
(Reddy,
ovine
preparations of
number with
of
into
Abel
similar
and
slices
granules
Recently, in
Quirk
that actively
observations)
a third
the
dense.
luteum.
1973;
secreted.
within
present. reported
into
present electron
progesterone-binding
been
secreted
AL.
granules
tissues
al.,
et
never
demon-
are
Novikoff,
Gemmell
are
dense
corpus
have
and
1975;
organelles
bovine lysosomes
steroidogenic
Novikoff
Yuan,
dense
organelles
in
1973;
electron of
Microperoxisomes
previously
with sucrose
we have
studies,
3 types
present
broadly
1973).
cytochemical that
and
agree
discontinuous
(Gospodarowicz,
In our are
luteum
using
ET
must
be
of
a 20-40%
We are indebted for expert technical assistance to Misses Georgina Pooley and Elaine Johnston and to Mr. Ian Cookson. We are very grateful to the staff of R. A. Rixon Ltd. and F.M.C. (Thame) Ltd. for material. The supply of dated bovine corpora lutea from Dr. S. Dixon, A.R.C., Compton is gratefully acknowledged. We thank Dr. G. Jenkin for helpful discussions and Mrs. Jill Iredale for her help in preparation of the typescript. G.D.T. was a member of the External
w/w
sucrose
density
gradient,
after
incubation
with
alkaline diaminobenzidine to demonstrate microperoxisomes. The section is not counterstained and the dense osmiophilic reaction product can be seen over microperoxisomes (arrows). An occasi9nal nonstaining granule can be seen (G, double arrows). Membrane vesicles constitute the remainder of the field. X 34,650. FIG. 12. Fixed pellet from tubes 17-18 of a 20-40% w/w sucrose density gradient incubated to demonstrate the presence of acid phosphatase activity. The section is not counterstained. The dense reaction product identifies a lysosome (L); some nonbackground stain is observed. The rest of the field is comprised mainly of mitochondria. X 17,160.
PROGESTERONE
LOCATION
IN THE
a
BOVINE
CORPUS
LUTEUM
1143
QUIRK
1144
Scientific Dominions
Staff of the M.R.C. Trust Demonstrator.
and
D.L.W.
ET AL.
is a Nuffield
microtubules
reprod. Gemmell, R. (1977).
H.
(1974).
Catalase. In: Methods of Enzymatic Analysis. 2nd English ed. (H. U. Bergmeyer, ed). Academic Press, New York. 2, pp. 673-684. Abel, J. H., McClellan, M. C., Sawyer, H. R., Schmitz, M., Niswender, G. D. and Nau, E. (1977). Synthesis and release of a secretory protein from lutein cells. J. Cell. Biol. 75, 374a. Beaufay, H., Amar-Costesec, A., Feytnians, E., ThinesSempoux, D., Wibo, M., Robbi, M. and Berthet, J. (1974). Analytical study of microsomes and isolated subcellular membranes from rat liver. I. Biochemical methods. J. Cell. Biol. 61, 188-200. Belt, W. D., Cavazos, L. F., Anderson, L. L. and Melampy, R. M. (1971). Cytoplasmic granules
and relaxin Endocrinology
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Rubin,
R. P., Sheid, B., Mccauley, S. G. (1974). ACTH-induced the perfused cat adrenal
exocytosis?
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L
IN THE
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structure,
LUTEUM
chemistry
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Enders, Biol.
A.
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