BIOLOGY
44, 717-724
REPRODUCTION
OF
Ovarian
Surface
(1991)
Epithelium:
NELLY
Autonomous
Extracellular
AUERSPERG,2
Department
IAN
of Anatomy,
Vancouver,
Production
Matrix1
A. MACLAREN,
Faculty
British
and
of Medicine,
Columbia
V6T
of Connective
PATRICIA
Tissue-Type
A. KRUK
University
of British
1W5 Canada
Columbia
ABSTRACT
The ovarian
and
surface
migration
other
extracellular
matrix
and
nature
all three
of
I and
III and
terstitial
out
major
teolytic
produce
was
components
progression
compared
confirmed
199)
by
by
recently
the
the
presence
of collagen
eplthelial/flbroblast
of connective
synthetic
autonomously
of OSE-derived
of keratin
tissue
They
mesothelium
lium, separated
lamina
and
normal
releases
ovarian
lysosomal
breakdown
licle [3,4],
defect
changes
tissue
that
are
connective
tissue
capsule
Similarly,
thought
the stromal
cores
of surface-epithelial
to be derived
from
adjacent
ovarian
However,
rat ovarian
I and
is formed
of collagen
components
banded
may
that
fibrils
of the ovarian
factor
in the
cortical
formation
through
the
line
199)
erty
December
Received
October
This research
of culraised
Cancer
Institute
Research
Council
FAX:
and the National
of Canada.
2Correspondence:
Dr. N. Auersperg,
The Department
of Anatomy,
icine, University
of British Columbia,
2177 Wesbrook
Mall. Vancouver.
Canada.
addition
subpopulations
has
the
to
capacity
to its postulated
stroma.
A capacity
of ovarian
surface
of OSE
paplllae
procells
and
to
In
these
matrix
repair
cells,
present
capacity
of OSE
would
implicate
of the subjacent
study
that
was
acquired
in conjunction
with
and papillae
that histologically
repapillary
cystadenomas
of border-
[7]. The
was
OSE cells
clinically
be
cells
by the
to produce
a col-
this epithelium
in the
connective
tissue.
Furto deposit
significant
less dependent
tissue
cells, and
undertaken
types
1W5
I and
on
might
to determine
their
becontrithere-
whether
formation
is part
it is an abnormal
ROSE
199
ulations.
of cell
An evaluation
density
showed
717
role
ROSE
199,
of keratin,
III by cloned
of the
portive
604-228-2316.
cell line
expression
origin
cell
duced
by both
suggested
that
Faculty of MedBC V6T
crowded,
atypical
immortal
Simultaneous
of interstitial
6, 1990.
by the Medical
or In-
cells
of the
prop-
as part
of the
compared
in (1) freshly
explanted
rat ovarian
surface
epithelium
(ROSE)
cells
in low passage
culture;
(2) an immortal
line, ROSE 239, which resembles
low passage ROSE
cultures
in its monolayered
growth
pattern;
(3) the more
8, 1990.
was supported
types
stromal
preneoplastic
changes
associated
with immortalization.
To
this end, the epithelial
markers
keratin
and laminin
and the
connective
tissue markers
collagen types
I and III were
are
tis-
gen
Accepted
OSE
the capacity
for collagenous
matrix
normal
OSE phenotype,
or whether
cells.
papillae
stromal
When
malignancy
The
repair,
that the
have the capacity
to form
ultrastructurally,
they secontains
as one of its macharacteristic
in
collagen
for
by cloned
that
cause
it would
render
the
butions
by normal
connective
fore facilitate
their
growth.
folcovers
stromal
our recent
study of an immortal
surface
epithelium
(line ROSE
the possibility
that OSE cells
connective
tissue
autonomously:
crete
an extracellular
matrix
jor
III by adjacent
laminin
thermore,
a capacity
of neoplastic
own collagenous
matrix
would
OSE
the
to
[5, 6].
synthesis
sues.
tured
types
epithelium,
lagenous
postovulatory
in
the
the preovulatory
proliferates
and
rupture
and
demonstrate
this
line
[1, 2]. This
important
assumed
that during
postovulatory
deposit
a basement
membrane,
but
cells
underlying
by follicular
that
results
ROSE 239, whose
The epithelial
secreted
characteristic
the matrix,
form
ridges
semble
human
serous
modified
function.
There
is evidence
that
enzymes
that
may contribute
caused
OSE
is the
connective
cyclic
of the stroma
that overlays
and after ovulation,
the OSE
It is generally
the
(OSE)
ovary.
It is a simple
epitheovarian
structures by a basal
collagenous
undergoes
The
collagen.
epithelium
that overlies
the
from underlying
dense
epithelium
the
surface
ovarian
are
III, keratin,
I and
suggest
which
papillae.
cell types
All three
flbrlls,
line
and
ridges
electron
carcinomas.
INTRODUCTION
The
types
populations.
stroma.
laminin.
banded
activity
in the remodelling
might
be an Important
ovarian
and
polymerized
forms
of
production
microscopy,
the immortal
passage,
which
by proliferation
the
immunofluorescence
in low
199,
cortex
ovarian
We examined
membrane.
using
cells
ROSE
line
of the
repair
a basement
explanted
immortal
highly
production
in mixed
of
of the rat (ROSE),
cells
and
produced
to postovulatory
deposition
by OSE
cultures,
Simultaneous
the lines
of
role, may also express
stromal
components
neoplastic
We
(ROSE
matrix.
an origin
synthesize
types
one
cxtracellular
ruled
in culture
low passage
cell
at least
and
rupture,
incorporation.
resembles
to conthbute
is known
follicular
components
proline
pattern
growth
(OSE)
the site of
over
microscopy,
epithelium
lines
in mixed
described
laminin,
subpopulations
ruled
of collagen
deposition
that the collagenous
neoplastic
out
epithelial/fibroblast
progression.
an
pop-
as a function
matrix
is pro-
recently
explanted
and immortal
modifications
of this matrix
may
in ovarian
above.
and colla-
OSE,
play
and
a sup-
718
AUERSPERG
MATERIALS
Cells and
Culture
Primary
plants
AND
ET AL.
METHODS
Methods
cultures
of
of ovaries
cells
ROSE
from
were
2-3-mo-old
grown
Fischer
from
344
rats
exas de-
scribed
previously
[8]. The origin
of the spontaneously
immortalized
lines ROSE 199 and ROSE 239 has been described
[7,8]. The cells were
grown
in Waymouth’s
(Sigma,
St. Louis,
MO) with gentamicin
plemented
gan,
(25
medium
ig/ml)
752/1
sup-
with 25% fetal bovine
serum
(FBS; Hyclone,
Lofor primary
cultures
and with 10% FBS for estabcultures.
To promote
collagen
secretion,
cultures
were
UT)
lished
maintained
at postconfluence
and
some
were
supple-
mented
with
50 ng/ml
ascorbate
(GIBCO,
Grand
Island,
NY) daily for 1 wk. The cells were
subcultured
as required
with
0.125%
trypsin
(1:250;
GIBCO)
in calcium/magnesium-free
Hanks’
To clone
cells,
Balanced
Salt
Solution
sparsely
seeded
single
and resulting colonies were
recloned
in round-bottomed
further
use.
of lines
To define
ROSE
199
were
rates
ROSE
and
239,
saturation
cells
were
densities
seeded
tial densities
ranging
from 2.0-20.0
X i0
cells/2.0
Cells
in duplicate
wells were
counted
every
2-4
up
to 3 wk
in three
or more
Immunofluorescence
marked,
isolated with cloning cylinders,
96-well
plates,
and grown
for
growth
and
(BSS).
cells
experiments
Microscopy
per
and
at ini2
days
well.
for
line.
Electron
Microscopy
Keratin.
Cells grown
on glass coverslips
were rinsed
in
PBS, fixed, and stored in -20#{176}C
methanol,
postflxed
in -20#{176}C
methanol/acetone
for 5 mm, air-dried for 15 mm, and rehydrated
in PBS/1.0%
bated
with
polyclonal,
BSA for 30
wide-spectrum
against
human
epidermal
for 30 mm; rinsed
with
for 30 mm
with
Laboratories,
vatol [101,
Elkhart,
and examined
Collagen
types
mm.
(callus)
keratin
PBS/i .0% BSA;
FITC-conjugated
IN),
I and
goat
They
rabbit
were
incuantiserum
(RAK) [9] at 37#{176}C
incubated
at 37#{176}C
anti-rabbit
rinsed
again,
by epifluorescence.
mounted
III.
cultures
Confluent
IgG
(Miles
in Gel-
on
glass
coverslips
were
fixed
for 15 mm at room
temperature
in
paraformaldehyde-PBS
(3.7%).
After 3 rinses
in PBS/1.0%
BSA, some of the cultures were treated with 0.1% Triton X100 (Sigma)
for 3 mm at room temperature
and then rinsed
again. The cultures
were incubated
for 15 mm at room
temperature
by 60
(kindly
in 5% preimmune
rabbit
serum
(Sigma)
followed
mm at 37#{176}C
with either
sheep
anti-type
I collagen
donated
by Dr. H.K. Kleinman,
NIH) or with goat
anti-type
III
collagen
(Southern
mingham,
AL). After a second
rabbit
serum,
the cells were
rabbit anti-sheep IgG (Miles
conjugated
rabbit
anti-goat
sissauga,
ON),
respectively,
rations
were
fluorescence.
then
rinsed,
Biotechnologies,
Bur-
incubation
with preimmune
stained
with FITC-conjugated
Scientific,
Naperville,
IL) or FITCIgG (Cooper
Biomedical,
Misat 37#{176}C
for 60 mm. All prepa-
mounted,
and
examined
by epi-
FIG.
1.
Phase
micrographs
of confluent
passage 2, (b) line ROSE 239, and (ci line ROSE
ROSE 239 cells are monolayered
while ROSE
papillae
(see also Fig. 5a). x240.
cultures
of (a) ROSE
cells
in
199. Low passage cells and
199 cells form ridges and
EXTRACELLULAR
EPITHELIAL
Laminin.
Subconfluent
cultures
were
maldhyde-PBS
and treated
with Triton
for collagen. Cultures were incubated
temperature
then
for
60
in 5% preimmune
mm
at 37#{176}C
with
Gaithersburg, MD).
with
ROTC-conjugated
vern,
as described
ROSE
tetroxide
copy
199
and
10
cells were
prepared
goat serum
(Sigma),
rabbit
anti-laminmn
rinsed,
mounted,
and
and
(BRL,
[7].
culture
dishes
ROSE
v
8
z
V
w
6
-J
exam-
0
()
fixed
in glutaraldehyde/osmium
for transmission
electron
methods
LOW PASSAGE
4
z
micros-
2
As.sa
Cells
beled
gland
60 mm,
719
X-100
as described
for 15 mm
at room
above.
by standard
Collagen
in parafor-
Subsequently,
the cells were incubated
goat anti-rabbit
IgG (Cappel,
Mal-
PA) at 37#{176}C
for
ined
fixed
MATRIX
in 35-mm
or 24-well
for 4 h with 25 liCi of [3H]prolmne
Nuclear,
Boston,
MA) per milliliter
cells/pericellular
were
assayed
matrix
separately
et al. [11].
dition
results
of 5 M NaOH
were converted
duction
as described
were
0
la-
dayl
preconfluent
day6
confluent
day7
postconfluent
fraction
and the culture
medium
for labeled
collagenase-sensitive,
trichioroacetic acid-precipitable
Schwarz
plates
(2’,3’-3H;
New Enof medium.
The
material by the method
Incorporation
was
stopped
by the
to a final concentration
of 0.25
into an estimate
of procollagen
by Rowe
and
Schwarz
of
10
ad-
M. The
pro-
[12].
8
z
Lii
0
200
‘0
6
_J
0
x
()
w
4
150
Ct
2.
0
100
0
w
m
day4
preconfluent
day 7
confluent
day
12
15
day
postconfluent
50
z
-J
-J
w
0
10
LINE ROSE
0
0
5
10
15
20
25
DAY
FIG. 2.
Growth
curves
of lines
ROSE 239 (0) and ROSE
onstrating
the higher cell density reached
by crowded
ROSE
plicate counts;
variation
not exceeding
10% of the means.
199
z
199 (.) dem199 cells. Du-
8
V
6
_
0
C)
FIG.
3. Collagen
synthesis
as the proportion
of total I3Hlproline
incorporated
into collagenase-sensitive
over pronase-sensitive
trichloroacetic
acidprecipitable
material.
The percentage
of proline incorporated
was corrected
for the higher proline
content
and partial collagenase
insensitivity
of procollagen
112). Quadruplicate
cultures,
25 sCi/ml
culture
medium
(1 ml/35mm dish or 0.5 mI/i .8-mm well), 4 h incubation;
I3Hlproline:
approx.
3 x
iO cpm/culture;
columns
with wide diagonal
bands, culture medium;
columns with narrow diagonal
bands, cells and pericellular
matrix;
white columns, total % proline
in collagen.
Means
± SD; Comparisons
of first and
last determination:
black arrowheads,
p < 0.05 or less; white arrowheads,
p>
0.05.
z
4
-
2
0
day2
preconfluent
day5
confluent
day8
dayl4
posonfluent
720
AUERSPERG
ET
AL.
239 cells
RESULTS
secreted
variable
material into the medium
ROSE
blestone
i04
X
mained
cells
in primary
monolayers
cells/cm2.
The
culture
and
crowded.
The
but
shed
characteristic
cob-
grew
to a density
239
cells
of 2-3
also re-
(Fig. 1) and
immortal
monolayered
cells/cm2
formed
cells
immortal
reached
into
densities
the
ROSE
ROSE
199
culture
cells
were
of 3-4
X iO
medium
when
monolayered
and morphologically
similar
to the other
two cell types
in
sparse
culture,
but formed
ridges
and papillae
when crowded
and reached
densities
of 7-8
X i05 cells/cm2
(Fig. 2). As
indicated
by proline
incorporation,
collagenase-sensitive
material
in low passage
1.0% of total protein
9.0%
and
of protein
in the
ROSE
secreted
cultures
comprised
into the medium,
cells/ECM
fraction
(Fig.
less
but
3).
than
5.0ROSE
FIG. 4. Collagen
type I (a,c,e) and collagen
type Ill (b,d,f) in the extracellular
line ROSE 199 (e,f). Immunofluorescence
microscopy,
x300.
amounts
of collagenase-sensitive
of total
(0.5-9.5%
protein),
while
the levels
in the cells/ECM
fraction
rose with cell density
from 4.0-5.0%
to over 8.0% of protein.
In the culture
medium
of ROSE 199 cells, collagenase-sensitive
material
usually comprised
less than 1.0% of total protein,
while
in the
cells/ECM
fraction
it rose from
1.5-3.4%
in preconfluent
monolayered
cultures to 3.0-6.5%
in postconfluent
cultures.
lagen
No similar
incorporation
ROSE
cells.
Fluorescence
collagen
types
electron
of ROSE
matrix
relationship
between
cell
into ECM was observed
microscopy
confirmed
the
I and III in all of these
cultures
microscopy
199 cells
deposited
density
and colin low passage
of uncloned
demonstrated
by ROSE
and cloned
the highly
cells in low passage
(a,b),
presence
of
(Fig. 4), and
populations
polymerized,
line ROSE
239 (c,d)
EPITHELIAL
banded
interstitial
connective
tissue
form
of collagen
5). Parallel
cultures
stained
with
demonstrated
this epithelial
basement
antibody
membrane
nent
6). There
(Fig.
atm
in all three
filaments
types
of cultures
(Fig.
of low
passage
in all cells
in varying
proportions
(Fig. 6). The presence
ROSE
of the lines ROSE
of keratin-positive
EXTRACELLUL’R
fibrils
to laminin
compowere
cultures
kerand
239 and ROSE 199
and keratin-neg-
ian
MATRIX
stroma
and
surface
epithelium,
rived
from the mesenchyme
tention
of a degree
of plasticity
stromal
keeping
lability
sume
[13].
in immortal
cells
was due to physiologic
variables.
expression
of all four markers
in clones
of lines ROSE
and ROSE 239 confirmed
that the collagenous
matrix
deposited
by the epithelial
cells (Fig. 7).
enchymal
Very
mesothelial
shapes
Our results
show
crete
an extracellular
components
fibrils
that ovarian
surface
epithelial
cells sematrix
that has as one of its major
of interstitial
collagen.
Although
this study
only examined
matrix
synthesis
by OSE
no reason
to believe
that the cells might
in culture,
there
is
not have the ability
to express
Therefore,
a similar
phenotype
of OSE in the remodelling
be broader
than is generally
thetic
and proteolytic
phase,
in particular,
migrate,
and deposit
play
an
active
derlying
The
functions.
these
cells
a basement
secretory
connective
expression
be related
to the
in vivo.
of the ovarian
cortex
assumed,
involving
role
the
role
appears
to
both
syn-
During
the postovulatory
may not only proliferate,
membrane,
but may also
in the
rebuilding
of the
un-
tissues.
of fibroblastic
close
developmental
relationship
by OSE
may
of ovar-
de-
characteristics
would
be in
of OSE cells,
such as their
and their
propensity
to asand
interconversions
are
ridge.
The recells to express
growth
patterns
have
in culture
occur
in cultured
of epithelio-mes-
also
been
reported
in a
number
of other
epithelia
of mesenchymal
origin,
including the developing
kidney
[15] and, interestingly,
regressing Mullerian
duct [16]. The developing
cornea
and long-
connective-tissue
In cultures
synthesis
were
sity.
The
immortal
199 cells,
periods
of rapid
with cell density
ROSE
passage
cells.
a collagenous
intestinal
cells
trix.
epithelium
produce
cell
lines,
represent
a stroma
It would
matrix
It is likely
pattern
particularly
produced
significantly
growth,
though
this
to a level approaching
appear,
is part
type, and that the immortal
that led to an altered
secretion
growth
that
forms
of collagen
[17, 18].
of low passage
ROSE cells, levels
relatively
high and independent
that
these
of the
It is particularly
characteristics
of which
similar
phenotypic
modulations
cells
[14]. Various
degrees
term
cultures
of rat
examples
of epithelial
DISCUSSION
both
of the gonadal
that permits
as well
as epithelial
with other
properties
of keratin
expression
fibroblast-like
ative cells in cloned
sublines
of ROSE 199 and ROSE
239
indicated
that the diversity
in the expression
of this marker
The
199
was
721
cultures
form
ridges
monolayers
separated
interesting
of collagen
of cell denmore
atypical
less collagen
during
production
increased
that of normal,
low
therefore,
that deposition
of
of the normal
ROSE
phenocells had undergone
and distribution
changes
ROSE
the
other
rich in
are
199
cells.
that
ROSE
related
changes
of this ma-
to the
199 cells
atypical
in crowded
and papillae,
as well as multiple
by layers
of collagenous
matrix
I’
FIG. 5. (a) Vertical
plastic section
through
a ridge in a crowded
ROSE i9
culture,
showing
the cells (dark staining)
overlying
matrix (light staining).
Toluidine
blue stain, x400.
(b) Electron
micrograph
through
the surface
of a ridge, as shown
in (a). Note
typical of epithelial
cells, and abundant
f’ibrils in the underlying
stroma.
x18,000.
(cI Higher magnification
of the extracellular
matrix
the typical striated appearance
of interstitial
type collagen
fibrils. x56,000.
cell
[19]
#{149}
s #{149}
the core of extracellular
the intercellular
(unction
shown in (b), illustrating
722
199
FIG. 6. Laminin
in the extracellular
matrix (a,c,e)
(e,f). Immunofluorescence
microscopy,
x300.
Thus,
increasing
epithelial
numbers
cells
per
unit
commodated
through
extracellular
matrix.
papillary
outgrowths
the
rat and
human
of
area
and keratin
proliferating,
of plastic
a modest
in cultures
monolayered
substratum
a continuous
increase
In contrast
to some
other
of normal
OSE in vivo
[13, 21]. Only
(b,d,f)
ac-
in available
species
[20],
are scarce
in
increase
that are benign
greatly
amplified
Our
results
derived
or of borderline
and forms
papillae
support
from
atypical
the
hypothesis
immortal
malignancy,
with stromal
that
OSE
in these
the OSE is
cores [25].
extracellular
cells
may
of ROSE
a means
are
structures
has been
reported
to occur
at the site of follicular
rupture
and in postmenopausal
women
[5, 22-24].
However,
in a high proportion
of serous
ovarian
tumors
trix
ET AL.
AUERSPERG
maprovide
cells
in low
to circumvent
growth
numbers
inhibition
of OSE
ovarian
surface
vivo might
papillae
that
capacity
of
tonomously
contributed
therefore
)a,b),
the
line ROSE
restraints
239 (c,d) and line ROSE
of density-dependent
in culture.
Similarly,
cells are accommodated
through
basis of our results,
tonomous
deposition
gression.
passage
the
formation
in vivo, increased
on the limiting
of papillae.
it is tempting
of an
to speculate
extracellular
matrix
On
the
that the auby OSE in
at least in part provide
the stromal
cores
for
occur
physiologically
and pathologically.
The
ovarian
cells to form
a collagenous
matrix
auwould
render
the cells less dependent
on stroma
by normal connective tissue cells, and might
be an important
factor
in ovarian
neoplastic
pro-
EXTRACELLULAR
EPITHELIAL
FIG. 7. Coexpression
of epithelial
and stromal
characteristics
(c) in clone E1i/A4
of line ROSE 199. Laminin
(dl and collagen
immunofluorescence
microscopy,
x300;
C: electron
microscopy,
in cloned ROSE population.
type III (e) in the extracellular
x85,000.
ACKNOWLEDGMENTS
We
thank Carolyn
Schwarz
Burr
8. Adams
Dr. Jie Pan for excellent
and
for the gift of highly
purified
MATRIX
collagenase,
and
assistance,
technical
Dr. P.B. Clement
Dr.
R
for valuable
advice.
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