BIOLOGY
OF
REPRODUCTION
A Uterine
38,
1-561
55
(1988)
Cell Mitogen Distinct from Epidermal Growth
Lumi nal Fluids: Characterization
and Partial
ROSALIA
C. M. SIMMEN,3’4
YONG
WILLIAM
F. POPE,3
Department
of Molecular
and
Laboratories
Ohio
KO,3’4
Agricultural
and
XIAO
H. LIU,3’4
FRANK
of Animal
Developmental
Research
Wooster,
Ohio
Factor in Porcine
Purification1
MARK
Uterine
H. WILDE,3
A. SIMMEN2’3’4
Science3
and
Biology,4
Ohio
and Development
44691-4900
State
University
Center
ABSTRACT
Uterine
luminal
DNA
synthesis
purified
200-fold
fluids
(ULF)
in a variety
by heat
from
of cell
treatment,
early
(Days
10
lines.
The major
anion-exchange
and
12)-pregnant
growth
factor
chromatography,
sows
contain
component
in
and
gel
factors
that
stimulate
fluids
has been
using
mouse
these
filtration
derived
AKR-2B
fibro blasts
as an indicator
cell line.
The ULF
mitogen
(ULFM)
is a polypeptide
apparent
molecular
weight
of 4800;
it is extremely
heat stable
and resistant
to treatment
with
urea.
togen
is also present
in ULF from
cycling
sows
but is not detectable
in uterine
cytosolic
extracts
or
isolated
results
from
pigs at Day
in a 50% increase
and
human
does
not
synthesis
epidermal
This
act
for
in
A431
human
purified
mitogen
in
growth
compete
Partially
concert
factor
binding
to
peptide
since
EGF
cells,
stimulates
growth
whereas
is not
factors
inhibited
in regulating
INTRODUCTION
Uterine
lumina!
fluid
(ULF)
contains
vivo
(Knight
synthesis
et al.,
and
1973;
secretion
Kuivanen
of
and
uterine
DeSombre,
to medium
containing
0.5% calf serum
appears
biologically
distinct
from
mouse
inhibited
In
by antibody
addition,
the
EGF
is inhibitory.
in primary
cultures
by
antibody
uterine
with
an
This miin serum
to
growth
factor
pig
of
mouse
and/or
to mouse
ULF
uterine
stromal
Thus
EGF.
EGF
stimulates
ULFM
and
it
DNA
cells.
may
dzfferentiation.
the uptake
and transport
of specific
serum
proteins
by the uterus
(Finlay
et a!., 1981),
and the paracellular
filtration
of plasma
components
across
the
a complex
array
of
molecules,
including
uterine
secretory
proteins
of both
endometrial
and
plasma
origins
(Knight
et al., 1973;
Voss
and
Beato,
1977).
The
protein
composition
of ULF in many
species
is under
endocrine
control.
In particular,
the steriod
hormones
estrogen
and progesterone
are known
to modulate
the
in
is not
receptors.
synthesis
DNA
and
of this factor
cells.
ULFM
its activity
(A431)
carcinoma
also
is dose-dependent
other
The addition
of AKR-2B
(EGF),
human
epidermoid
ULFM
activity
with
12 of pregnancy.
in final
cell density
partially
embryo-
endometrium
Kennedy,
important
(Knight
mediate
proteins
into
1979).
the
uterine
ULF
proteins
roles
in fetal
et a!., 1973;
Buhi
uterine
function
interactions
(Geisert
1984;
Glasser,
1986).
1985),
One
relatively
presence
polypeptide
Accepted
October
5, 1987.
Received
April
13, 1987.
‘This
research
was
supported
in part
by Ohio
State
University
Seed
Grants
to R.C.M.S.
and
F.A.S.
Salaries
and additional
research
support
were
provided
by State
and
Federal
funds
appropriated
to
the
Ohio
Agricultural
Research
and
Development
Center.
Article
No. 76-87.
2 Reprint
requests.
growth
from
factors
extracts
growth
1982;
Ikeda
of
proteins
that
with
respect
in ULF
is that
factors.
A
number
(EGF;
factors
growth
and
play
development
and may also
maternal-fetal
and
Sirbasku,
has received
to possible
comprising
of
has been
identified
in and
of whole
uterine
tissue.
These
epidermal
growth
factor
1984),
colony-stimulating
a!.,
1984),
transforming
551
growth
and
et a!., 1982)
and/or
et a!.,
important
class
little
attention
and
function
lumen
(McRae
are thought
to
peptide
isolated
include
Gonzalez
et a!.,
(CSFs,
Kriegler
et
factors
(TGFs;
552
SIMMEN
Nickel!
derived
et al.,
growth
tumor
basku,
al.,
cells
1984),
1986)
1983),
factor
to
acidic
However,
factors
in
estrogen-inducible
(UDGF)
for rat
(Sirbasku
et a!.,
and a fibroblast
similar
(aFGF).
an
ULF
are
are
of
currently
interested
are transported
they
can act
uterine
growth
and
the
pig
amounts
bryos
Here
and
limited
be
growth
factors
lumen
mediators
obtained
and
of
analyze
sows at
to use
relatively
large
tissues,
and em-
during
development.
concentrated
synthesis
of
tissue
culture
receptor
specific
Research
pCi/pg)
for
media,
reagents,
grade)
and
mEGF
(Bedford,
were purchased
MA).
The
and
and
supplies
from
GIBCO
(Grand
Island,
NY).
epidermal
growth
factor
(mEGF,
rabbit
polyclonal
H] thymidine
[3
from
[125
(6.7
antiserum
Collaborative
I] -mEGF
(176
Ci/mmole)
were
purchased
from
ICN Radiochemicals
(Irvine,
CA) and
New
England
Nuclear
(Boston,
MA),
respectively.
Sephadex
G-50
and
G-200
were
from
Pharmacia
(Piscataway,
NJ),
and
DE52
diethylaminoethy!
(DEAE)
cellulose
was purchased
from Whatman
Ltd.
(England).
All other
reagents
used were
of analytical
grade.
samples
Cell
were
Sows
and
assigned
to
12 and 24
of embryos
h after
in the
sows
not
nonpregnant
allowed
ULF
ULF
pregnant
was
and
flushing
each
Preparation
the
horn
pregnant
to mate
collection.
with
from
group
were
both
animals
30
by
ultrafiltration
through
an
Rockville
as standard.
Centre,
NY) using
The concentrated
at -20#{176}
C until
further
Conditions
Mouse
embryo-derived
(AKR-2B)
were
provided
by Dr. H. L. Moses
versity,
Nashville,
TN).
Madin-Darby
(MDCK)
carcinoma
Type
epithelia!
cells
Culture
three
lines
Eagle’s
calf
were
(0.14
Rockville,
in
(DMEM)
containing
serum
subcultured
m), KC1
(1%),
with
uterine
according
to
Glasser,
1980),
was increased
and
(AKR-2B,
The
(5
using
mM),
ethylene
cells
Unikidney
MD).
Dulbecco’s
A43
10%
1) or fetal
in monoair incuba-
a solution
NaHCO3
diamine
(ATV).
of stromal
tissues
published
cells
from
Day
procedures
contain(7 mM),
tetraacetate
were
established
12 pregnant
(McCormack
except
that the trypsin
to 0.25%
and incubation
and DNAse
I (200
Chemical
Co., St.
mm.
(ATCC,
propagated
(MDCK).
All cells were
grown
at 37#{176}Cin a 5% CO2 -forced
(EDTA,
0.5 mM)
Primary
cultures
30
were
Medium
heat-inactivated
calf serum
layer
culture
fibroblast
(Vanderbilt
canine
cells and human
A43 1 epidermoid
were
obtained
from
the American
Collection
cell
Cells
NaCl
analysis.
pigs
and
concentration
with
trypsin
units/ml,
bovine
pancreatic,
Sigma
Louis,
MO) carried
out at 37#{176}C
for
resulting
cells
were
identified
to
be
stroma!,
based
on their
fibroblastic
appearance
under
the light
microscope.
Cell viability
was estimated
by
trypan
blue
exclusion
and was typically
90%. The
stromal
cells were
plated
in DMEM
containing
10%
heat-inactivated
fetal
calf serum
and grown
as deabove.
Mitogen
assays
utilized
cells
from
3 to
6 passages.
were
mated
the onset
of estrus.
Identification
ULF
confirmed
pregnancy.
Those
collected
nonpregnant
stored
Culture
scribed
Collection
ml
(Bio-Rad
Laboratories,
bovine
gamma
globulin
tor.
ing
AND METHODS
15
(Amicon
Corp.,
Lexington,
MA)
Y2 filter
wt. cut-off
=
1000)
and then
filter-sterilized
a 0.22-pm
filter
(Acrodisc,
Gelman
Sciences,
Ann
Arbor,
MI). The protein
content
of the concentrated
samples
was determined
by the Bradford
assay
dextrose
were
purchased
Purified
mouse
ULF
to
of 2-3
animals
centrifugation,
Amicon
(mol.
through
Modified
Materials
All
The flushings
from
both
horns
pooled,
clarified
by low-speed
All
partial
purification,
a fibroblast
mitogen
from
EGF
that stimulates
DNA
cultures
of uterine
cells in vitro.
MATERIALS
as
To address
have initiated
studies
to
of ULF
obtained
from
states.
We have chosen
readily
iden-
such
development.
we report
the identification,
initial
characterization
of
distinct
primary
factor
estrogen-primed
as an animal
mode!
since
of ULF
proteins,
uterine
can
Siret
the
AL.
tion.
were
mitogenic
to
of
into
the uterine
in ULF
as local
embryo
these
questions,
we
the mitogenic
activity
different
physiological
growth
specific
ULF
in how
UDGF
whether
Ikeda
and
(Brigstock
fibroblast
reports
tification
of UDGF
in the
rats (Le!and
et a!., 1983).
We
1981;
mitogen
uterinemammary
ET
ml
assigned
uterine
during
of
0.9%
to
the
horns
surgery
of
by
saline
solu-
Mitogen
Assay
Dispersed
cells
were
seeded
into
mu!tiwell
(24-
well)
plates
at a density
of 1-5 X 10
cel!s/1.77
cm2
and then
incubated
until
confluent
monolayers
were
formed
(48-72
h). The
media
were
aspirated
and
changed
to 1.0 ml of DMEM
containing
2% calf
serum
for
A43
1 and
AKR-2B
cells
or
2%
fetal
calf
UTERINE
serum
for
the
cells
uterine
become
and MDCK
nonproliferating
cells.
LUMINAL
The majority
of
in these
media
formulations
after
48-72
h due
growth
inhibition
and
depletion
factors.
At this point,
sterile
test
to density-arrested
of serum
growth
samples
(2-200
p1
with
all final
phosphate-buffered
to
were
volumes
saline
wells
containing
depleted
allowed
to proceed
for
with
fixed
changes
(TCA)
of
and
PBS
(0.01
in absolute
cells were
with
each
calf serum.
and
150
G-50
ng/we!!.
incubator
of
EGF
acid
ml).
Optical
the
the
interassay
added
Corp.,
37#{176}Cin the
ULF
at a concentration
of cell
with
using
Buffalo,
number,
ATV
solution,
a hemocytometer
HCI,
pH
column
8.3,
at
in 0.01
4#{176}C.Proteins
M Tris-HC1,
NaCl
from
(0-3
the
night
against
two
resuspended
for
pre-
further
wt.
the
column
nm. Bound
gradient
of
of
PBS,
lyophilized,
and
water.
purification
mol.
and
at 280
a linear
factor
of
cells
and
mitogenic
on
the
and/or
of the
ULF
estimation
mitogen,
of
samples
activity,
the
were
(45 X 2.5 cm) or Sephacolumns
equilibrated
with
and
peak
mitogenic
a flow
tested
fractions
were
pooled,
lyophilized,
and resuspended
in distilled
water.
The
columns
were
calibrated
with
mol.
wt.
standards
comprising
a mixture
of bovine
thyroglobulin
(670,000),
bovine
gamma
globulin
(158,000),
chicken
(17,000),
and
performed
8.3,
on
buffer).
Pools
of fractions
run were
dialyzed
over-
changes
in distilled
loaded
PBS. Proteins
were
eluted
in PBS at 4#{176}C
with
rate of 20 mI/h.
The individual
fractions
were
NY).
were
13.9%.
was
performed
equilibrated
with
(Tris)-
were
pH
M in the same
chromatography
ovalbumin
bovine
vitamin
Growth
insulin
B-12
Characterization
assays
variation
was
Chromatography
For
Assay
radioreceptor
intraassay
variation
Anion-exchange
chromatography
on a DE52
DEAE
cellulose
column
0.01
M tris
(hydroxymethyl)ammnomethane
determined
X 10
cells/605 ml of DMEM
then
incubated
purified
determination
Radioreceptor
EGF
at
partially
fraction)
For
Column
experiments;
and
apparent
seeded
at 3.15
dish containing
The cells were
were
detached
from
plates
suspensions
were
counted
(American
then
separate
6.8%,
was washed
to baseline
absorbance
proteins
were
then
eluted
with
pH 7.4,
0.15
M
rinsed
in several
was
three
was
553
MITOGEN
applied
to Sephadex
G-200
dex G-50
(50 X 2.5 cm)
CO2
absence
(Sephadex
(2 pCi/well)
the cells were
Assay
a humidified
sence
M NaPi,
methanol,
was
cells
counting.
Cell Proliferation
in
H] thymidine
removed
and
H] thymidine
[3
scintillation
AKR-2B
mm2
dish,
plus 0.5%
incubation
20 h. The
distilled
water
and 5% trichloracetic
solubilized
in 0.3 M NaOH
(0.3
Cell-associated
by liquid
[3
200
p1 with
added
to the
media,
and
additional
an
were
then
labeled
with
for 4 h. Media
were
then
washed
NaCI),
corrected
[PBS])
FLUID
(44,000),
(5700),
horse
bombesin
myoglobin
(1600),
(1350).
of the
factor
ULF
sensitivity
Mitogen
to
proteolytic
digestion
confluent
monolayers
of human
A431
epidermoid
carcinoma
cells
in multiwell
(24-well)
plates,
according
to the procedure
of Carpenter
et a!. (1975).
Prior
to binding
of [125 II -EGF,
the cells were washed
with
1.0 ml of binding
buffer
(Dulbecco’s
PBS, pH
was
tested
by incubation
of the partially
purified
factor
(from
Sephadex
G-50
column
fractionation
of
DEAE
Fraction
IV) with
bovine
pancreatic
trypsin
(500
pg/ml;
Sigma
Chemical
Co.)
for 4 h at 37#{176}C.
Trypsin
was then
inactivated
by subsequent
addition
7.4,
and
of
soybean
trypsin
inhibitor
Chemical
Co.)
to the reaction
consisted
of an equivalent
were
taining
containing
0.1%
bovine
serum
5 mM MgCl2).
The
competitive
performed
0.2 ng
and varying
22#{176}
C. The
in 1.0
(70-120,000
amounts
cells were
solubilized
in 0.5
was measured
in
The standards
of receptor
grade
assessed
in the
mEGF.
Al! test
ml
albumin
binding
of binding
cpm)
of
of test samples
rinsed
once with
EBSA]
assays
buffer
conII -mEGF
[125
for 60
binding
mm at
buffer,
ml of 1 M NaOH,
and radioactivity
a gamma
counter.
for the assay were from 5 to 200 ng
mEGF,
and nonspecific
binding
was
presence
of 500
ng of unlabeled
samples
were
assayed
in triplicate
in
trypsin,
the start
K (100
and trypsin
inhibitor
mixed
of the incubation.
Digestion
p g/ml,
Boehringer-Mannheim,
IN) was carried
by heating
at
was compared
proteinase
4-h
with
(500
mixture.
amount
pg/ml;
The
of the
Sigma
control
factor,
together
prior
with proteinase
Indianapolis,
to
out at 37#{176}C
for 4 h, and was followed
100#{176}
C for 5 mm.
Remaining
activity
to that
observed
with
the factor
and
K heated
incubation
at
/3-mercaptoethanol
to
100#{176}
C for
5
mm
prior
37#{176}C.Treatment
of the
(2% v/v) was carried
to the
mitogen
out for
554
SIMMEN
24
h at 4#{176}C;
the
changes
the
of PBS
mitogen
sample
for
then
activity
was
centration
of 6 M
sample
was dialyzed
at
night
control
at
4#{176}C.
The
4#{176}C
for
and
24
was
h and
tested
then
of urea
a final
sample
was
in parallel
for
urea
con-
mitogenic
incubated
with
samples.
AKR-2B
3
on
4#{176}C
for 24 h, after which
the
against
3 changes
of PBS over-
dialyzed
tested
sity-arrested
against
effect
at
i3-mercaptoethanol-treated
were
dialyzed
24 h at 4#{176}C.
The
the
All
activity
at
urea
samples
using
Values
are
den-
cells.
mean
paired
±
t-test
SD.
Data
(Winer,
ULF
by
were
compared
using
12
pregnant
and tested
sows
for
(Carpenter
and
Cohen,
1976;
Klagsbrun,
1978;
Brown
and Blakeley,
1984).
The crude
ULF
sample
exhibited
mitogenic
activity
towards
both
AKR-2B
fibroblasts
and MDCK
epithelial
cells,
but failed
to
of
A43
1 epidermoid
car-
Since
AKR-2B
cells exhibited
the maximal
mitogenic
response
to crude
ULF,
these cells were used to
further
study
the resident
mitogenic
components.
To
characterize
ity, uterine
Sephadex
the factor(s)
responsible
fluids
were
subjected
G-200
columns.
Aliquots
were
tested
then
for
mitogenic
activity
togenic
elution
standards.
activity
eluted
as a single
peak
between
the
positions
of the 1350
and 17,000
mol.
wt.
Rechromatography
of the pooled
active
on
a Sephadex
into
DNA
a ) shows
by monitoring
of
fractions
H] thymidine
Figure
1 (Panel
for ULF
activto gel filtration
in
of each fraction
uptake
fibroblasts.
[3
G-50
column
of
that
AKR-2B
the mi-
indicated
determined.
the presence
in both
precisely
the
other
and
ULF
or serum
significant
from
was
possible
present
to
in
these
data
factor(s)
is not
unique
to
activity
from
was not
uterine
evident
tissue
in
of
sows.
presence
of similar
extracts
prepared
2c)
No
it was not
amounts
secretions,
mitogenic
of pregnant
activity
fraction
a and b) demonstrates
of mitogenic
activity
nonpregnant
the
mitogenic
column
ULF samples.
Although
quantify
the
relative
that
(Fig.
2d).
and
every
Figure
2 (Panels
of similar
amounts
pregnant
the
columns,
of
from
ULF
Day
activity
was
12 pregnant
comparable
apparent
sows
to
in
(Fig.
that
fractionated
obtained
from
Day
10
(Day
12 of estrus)
sows
pregnant
and
were
fractionated
To
determine
ULF
logue,
mEGF
ULF
(50
identified
in
corresponds
to porcine
EGF
or an EGF
rabbit
immunoglobulin
G (IgG)
specific
was tested
for inhibition
of unfractionated
whether
the
mitogen(s)
anafor
activity.
Figure
3a shows
that
pg)
inhibited
the
stimulatory
unfractionated
ULF
fibroblasts.
The
at the antibody
did
12
pregnant)
not
total
used)
effect
was
concentration
be antibody-specific,
control
antibody
protein)
(Day
since
not
TABLE
1. Mitogenic
from
Day 12 pregnant
anti-mEGF
effect
of
(IgG
an
raised
reduce
the
on
activity
of pooled
sows using different
AKR-2B
(60% inhibition
but appeared
equivalent
against
[3
IgG
crude
HI thymidine
uterine
indicator
incor-
luminal
fluid
cell Iines.*
(ULF)
Cell
line
Amount
(Mg protein)a
Relative
l3Hlthymidine
incorporationb
AKR-2B
50
200
50
200
50
100
7.9
10.7
2.2
3.7
1.0
1.0
MDCK
A431
to
amount
of
an unrelated
an
approximate
native
mol.
wt.
of 4800
(Fig.
lb).
Other
ULF
samples
from
pregnant
and nonpregnant
sows
were
examined
for the presence
of low
mol.
wt.
mitogenic
activity
to correlate
such
expression
with
the physiological
state
of the animal.
ULFs
cycling
G-200
aliquots
uterine
cytosols,
whereas
mitogenic
activity
in sow
serum
was confined
to the high mol. wt.
region
of
the columns.
from
Day
ultrafiltration
DNA
synthesis
cells (Table
1).
of
obtained
1977).
mitogenic
activity
by its ability
to stimulate
[3 HI thymidine
uptake
into DNA of several
well-characterized
indicator
cell lines.
This
assay
previously
has been
shown
to correlate
with
the cell proliferation
activity
of known
growth
factors
in several
other
systems
enhance
cinoma
Sephadex
The
cytosolic
RESULTS
Unfractionated
was
concentrated
AL.
suggest
Statistics
Student’s
ET
from
in
aProteil,
(Bio-Rad
bResul
concentrations
Laboratories).
are expressed
into
the DNA
that
incorporated
phosphate-buffered
5AKR28
Darby
moid
canine
carcinoma
of
=
were
as the
quiescent
cells
by cells
which
saline
(n
3).
mouse
kidney
cells.
determined
ratio
in
Bradford
A431
cells,
=
human
assay
incorporated
of added
equivalent
fibroblast
cells,
the
[3Hlthymidine
the presence
received
an
embryo-derived
epithelial
of
using
ULF
volume
MDCK
A431
=
over
of
Madinepider-
UTERINE
LUMINAL
a.
FLUID
effect
670
58
4417
II
II U
0
240
0
1.0
200
2
x
C
I’
20
0
C
80
C
E
=
N,
IC
30
Fraction
40
50
The
by
anti-EGF
IV
and
so
-
.
40
described
in more
then
activity
examined
be
the
the
the
activities
Sephadex
different
(50 g
Materials
and
in the following
purified
effect
purified
distinct
of
G-50
in the
per well).
puriby
of partially
for
anti-
partial
undertaken
of
Figure
3a demonstrates
that
ULF activity
was significantly
IgG,
of
might
under
detail
added
whereas
reduced
DEAE
Fraction
fraction
were
not
presence
or absence
This demonstrated
4800
mol.
from
mEGF.
0.2
Fraction
wt.
The
of
that
ULFM
inability
filtration
chromatography
12 pregnant
sows.
(a.)
of
ULF
uterine
luminal
fluid
(22.5
mg) was chroma-
tographed
on a Sephadex
G-200
column
(45
X 2.5 cm)
equilibrated
with
phosphate-buffered
saline
(PBS).
The flow
rate was 20 mI/h,
and
3-mi fractions were collected. Aliquots
(200 MI) of every other fraction
were than tested in triplicate
for mitogenic
activity,
as described
in
Materials
and Methods
(b.) Fractions
20-30
from
the Sephadex
G-200
run were
concentrated
with
an Amicon
Y2 filter,
re-applied
to a Sephadex
G-50
column
(36
X 3 cm),
and
processed
as in (a.) Arrows
indicate
the
elution
position
of column
standards.
(.),
A2e0;
(h),
activity
profile
on mouse
embryo-derived
fibroblast
cells.
these results.
of the ULF
heat-treated
at 100#{176}Cfor
remove
denatured/precipitated
was
found
the ULF
approximately
factor
not
the
eluted
factor,
was
of
7 mm.
to reduce
The
buffer.
and centrifuged
proteins.
Heat
the
mitogen
4a
ionic
at high
salt
corresponding
to
strength
concentration.
the
major
was
pH 8.3, and
equilibrated
demonstrates
buffer
of
it removed
in crude
ULF
fraction
resident
mitogenic
activity
quantitatively
bound
by
in low
to
treat-
activity
M Tris-HCL,
column
Figure
to
3b)
unfractionated
supernatant
against
0.01
an anion-exchange
IV
(Fig.
10 or Day 12 pregby
ultrafiltration,
(see Table
3), although
20% of the total protein
same
of the
was
changer
not
shown).
then
dialyzed
loaded
onto
that
in heat-treated
the
anion
and
The
mitogenic
ex-
subsequently
DEAE
fraction
activity
(Frac-
tion IV) was dialyzed
against
PBS, further
chromatographed
on a Sephadix
G-50 gel filtration
column,
and
aliquots
of the fractions
were tested
for the ability
to
stimulate
DNA
synthesis.
Almost
all of the activity
was
found
(17,000
mol.
as previously
by cells
receiving
added
ULF
(data
not
In addition,
increasing
the anti-EGF
anti100 g did not further
increase
its inhibitory
DEAE
Fraction
to A431
cells
collected
from
either
Day
sows
were
concentrated
76%
ULF
Na
of
binding
ULFs
nant
with
30
concentrations
-mEGF
supported
purification
(data
I0
[125!]
further
For
ment
poration
shown).
body
to
(Fig.
mitogenic
was
increasing
displace
60
Gel
Day
that
1.1
70
FIG.
1.
(ULF)
from
suggested
the
latter
possibility,
mitogenic
activity
was
the
partially
antigenically
Ii
factor(s)
to mEGF.
examine
of the
significantly
the antibody
Na
data
1)
anti-EGF
IgG.
unfractionated
40
These
4800
mol.
wt.
genically
related
ULFM
>..
02
shown).
EGF
analogue
that
reduction
in its
IgG and that
the
sections.
0
04
not
using
the
protocol
Methods
and discussed
0
0.6
(data
crude
ULF
contained
EGF or an
was
responsible
for
the
observed
mitogenic
activity
with
anti-EGF
To
fication
E
555
MITOGEN
to
elute
in the
wt.) and
shown
size-correspondence
DEAE
Fraction
identified
(Figs.
IV
1,
region
between
myoglobin
Vitamin
B-12 (1350
mol. wt.),
in Figure
1, demonstrating
of
and
4b).
the
mitogenic
the ULF
factor
More
accurate
activity
in
previously
size deter-
556
SIMMEN
Day 0 prlgnant
620
70
ET
IJLF
58
44
AL.
b. Day
12
.3
7
nonpregnant
670
168
ULF
44
70
17
a
a
0
0
60
60
#{149}
-
‘0
#{149}0
S
‘C
E
So
C
C
40
0
0
0
0
30
C
C
C
60
E
>..
C
>..
C
10
Fraction
c. Day
2 pr.nant
170
158
ut.rin.
No.
Day
cylosol
12 prqnant
670
L3
4417
156
s.rum
44
‘.3
7
A540
a
a
‘I-
so
1.0
Lo.
‘9
S
K
E
E
40
08
40
C
0
0.6
3.0
0
C
0.4
C
E
oa
.
I.0
N)
Fraction
FIG.
2. Mitogenic
activities
of pregnant
mouse
embryo-derived
fibroblast
cells.
ULF
extract
from
the uteri
of Day
12 pregnant
matographed
on a Sephadex
G-200
column
every
other
fraction
were
assayed
in triplicate
mination
using
additional
and nonpregnant
sow uterine
luminal
fluids
(ULFs),
uterine
cytosolic
extract
and maternal
serum
on
from
Day
10 pregnant
(38 mg, Panel
a), and Day
12 nonpregnant
(25.6
mg, Panel
b) saws,
cytosolic
saws
(40 mg, Panel
c), and serum
from
Day
12 pregnant
saws
(36 mg, Panel
d) were
separately
chro(45 X 2.5 cm) using
phosphate-buffered
saline
as eluent
at a flow rate of 20 mI/h.
Aliquots
(200 Ml) of
for mitogenic
activity
(h).
Arrows
indicate
the elution
positions
of mol. wt. markers.
(.),A250.
standards
(bovine
the
insulin
and bombesin,
5700
and 1600 mol. wt., respectively)
confirmed
a mol. wt. of 4800
for the factor.
Figure
5
shows
the stimulatory
effects
of unfractionated
ULF
and the partially
purified
fractions
on AKR-2B
cells
as a function
of dose.
Since
unfractionated
ULF
stimulated
maximal
DNA synthesis
at a concentration
of 100 pg/ml,
and the DEAE
Fraction
IV and Sephadex
G-50
fraction,
exhibited
maximal
mitogenic
activities
at
approximately
6 pg/rn!
and 0.5 pg/rn!,
respectively,
approximately
the
chromatography
20-fold
and
No.
steps
200-fold
resulted
purification
in
of
ULFM.
DNA
synthesis
in A43 1 cells
concentrations
of EGF
(Barnes,
al.,
1983;
Table
ULFM
(Sephadex
concentrations,
2).
In
G-50
stimulated
is inhibited
by ng/m!
1982;
Kawamoto
et
contrast,
partially
purified
fraction)
DNA
added
at
synthesis
different
in these
cells (Table
MDCK
cells,
2). ULFM,
however,
is not mitogenic
with no response
elicited
in these
cells
doses
as high
Figure
6
as 500
shows
ULFM
AKR-2B
(Sephadex
fibroblastic
ng/ml
(Table
the effects
2).
of partially
G-50
fraction)
cells plated
on the
at a low
to
at
purified
growth
density
of
in
UTERINE
LUMINAL
FLUID
MITOGEN
557
a.
‘I
10
-#{231}
E‘-8
C
x
N)
N
N
N
N
N
N
0
C
0
N
N
E
N
N
N
N
‘72
N
N
N
N
N
0
U.
mEIF
ULF
0.10
DEAE
F1a64168
LI
Z5.S
6.10
SompI.
F,OC$168
0.19
q
Protil.
sdd,d
1.0
0.8
0.6
0.4
0.2
‘0
n_I EGF-Standardcurv.(#{149})
ut
LJLF
(DEAE
FIG.
unfractionated
Fraction
antibody
DNA
of
(DEAE
carcinoma
DMEM
presence
a density
medium
number
mitog.n
(a)
00
--
0.1
000
I
10
traction)
3. Evidence
that
the uterine
luminal
fluid
(ULF)
mitogen
is distinct
from
epidermal
growth
factor
(EGF).
(a.) The mitogenic
activities
of
ULF
and of the partially
purified
mitogen
(from
DEAE
ion-exchange
chromatography
and from
sephadex
G-50
filtration
of DEAE
IV) were
assayed
in the presence
(diagonally
striped
bars)
and absence
(white
bars)
of anti-mouse
EGF
(lgG,
50 Mg). The effect
of the
on mEGF
(100
ng) is presented
as positive
control.
Results
are expressed
as relative-fold
stimulation
of (3 HI thymidine
incorporation
into
mouse
embryo-derived
fibroblast
cells over
phosphate-buffered
saline.
(b.) EGF-receptor-binding
activity
of the partially
purified
mitogen
Fraction
cells
IV)
(A).
containing
of the
at various
concentrations
Purified
mouse
EGF was
0.5% calf
factor
(150
was determined
as standard
by
competition
with
[‘2511-EGF
for
specific
binding
on
A431
human
epidermoid
(.).
serum.
Cells grown
in the
ng total
protein)
grew to
50% greater
than
those
0.5%
calf serum
alone.
was statistically
significant
+
used
in the presence
of
The increase
in cell
(p<0.01)
and was
observed
in three
separate
experiments.
Table
3 presents
a summary
of the
effects
of
proteolytic
enzymes,
denaturing
conditions,
reducing
agents,
and
heat
on the mitogenic
activity
of the
partially
purified
4800
mol.
wt.
ULFM
(Sephadex
G-50
fraction).
This mitogen
appeared
to be a polypeptide
since
its activity
was abolished
by incubation
with
trypsin
and
proteinase
K. The
mitogen
extremely
stable
to heat treatment,
maintaining
of its activity
after
7 mm
at 100#{176}
C. Finally,
was
100%
the
activity
sociating
reduction
disupon
The
cultures
initial
in vivo.
fraction)
stroma!
of the
factor
was
unaffected
agent
urea
but was totally
by mercaptoethanol.
mitogen
of
step
activity
uterine
towards
of the
Partially
purified
was
added
at
cells
prepared
ULF
cells was
determining
from
factor
(Sephadex
concentrations
of Day
the
on primary
next
examined
its physiological
ULFM
varying
uteri
by
abolished
as
an
role
G-50
to
12 pregnant
558
SIMMEN
ET
AL.
TABLE
cinoma
stimulated
(ULFM).
2. I3HlThymidine
cells
and
Madin-Darby
by the
indicated
Cells
Sample
A431
ULFM
(Sephadex
uptake
by human
canine
kidney
doses
of uterine
A431
cells
luminal
Conc.
(ng/ml)
[3 Hi thymidine
incorporated3
60
G-50
fraction)
120
652,821
(275,262
±
737,129
(275,262
±
MDCK
C
50
G-50
150,277
(362,064
±
500
CO
aVI
‘C
are
ceiving
only
in parentheses.
bmEGF
4O
N)
sows.
counts
an
per
SD
±
volume
mouse
epidermal
Stimulation
of
=
min
equivalent
(n
of
growth
=
3).
The
34, 221
39,745)
5721
39,745)
1635
±
±
fraction)
2.
±
(275,262
250
ULFM
(Sephadex
±
946,564
250
mEGFb
epidermoid
car(MDCK)
when
fluid
mitogen
39,745)
965
±
3779
(384.6
±
4478
(3846
±
19,465)
50
329)
±
252
329)
±
values
for
phosphate-buffered
cells
saline
factor.
DNA
synthesis
was
then
moni-
tored
by
the
relative
increase
in [3 H] thymidine
uptake
over PBS. Figure
7 demonstrates
that the incorporation
of [3 H] thymidine
in quiescent
confluent
cultures
of stromal
cells was increased
maximally
by
‘0
‘C
E
C
approximately
3-fold
upon
response
was dose-dependent
by anti-mEG
F IgG. These
mitogenic
fashion
to EGF
2.
C
C
E
range)
>.
under
our
assay
addition
of ULFM.
The
and was not inhibited
cells also responded
in a
and human
IGF-1
(ng/ml
conditions
(Fig.
7).
C
N)
Fraction
FIG.
tionated
pregnant
exchange
No
4. Ion-exchange
and
gel-filtration
chromatography.
Unfracuterine
luminal
fluids
(ULFs)
pooled
from
Day
10 and
12
saws
were
subjected
to ultrafiltration,
heat
treatment,
anionchromatography
and
gel filtration;
purification
was
moni-
tored
by stimulation
of DNA
synthesis
in quiescent
mouse
embryoderived
fibroblast
(AKR-2B)
cells.
(a.) Unfractionated
ULFs
(20 mg)
were
prepared,
and the DEAE-cellulose
anion-exchange
column
(14
X
1.7 cm)
was
developed
as described
in Materials
and Methods.
The
indicated
fractions
were
pooled,
concentrated,
and
tested
for
their
ability
to stimulate
3
Hi thymidine
uptake.
Values
indicated
are the
percentage
of total
ULF
activity
for each fraction.
(b.) DEAE
Fraction
IV (65 Mg) was lyophilized,
resuspended
in phosphate-buffered
saline,
and
dialyzed
against
3 changes
of the same
buffer
prior
to chromatography
on a Sephadex
G-50
column
(50 X 2.5 cm).
Fractions
were
collected
and monitored
for absorbance
at 280 nm (.).
Aliquocs
(100
ul) of
ability
alternate
to stimulate
fractions
DNA
were
synthesis
then
examined
in AKR-2B
in
cells
triplicate
(A).
for
reare
their
TABLE
mitogen.
3. Characterization
of
the
porcine
uterine
luminal
fluid
(ULF)
Activity
Expt.
Treatmenta
1
2
Trypsin,37#{176}C,4h
Trypsin
+ trypsin
inhibitor,
Proteinase
K, 37#{176}C,4 h
3
4
5
#{243}Murea,4#{176}C,24h
2% (v/v) mercaptoethanol,
100#{176}C,7min
aThe
ULF
Sephadex
remaining
G-50
11%
100%
0%
100%
0%
100%
37#{176}C,4 h
4#{176}C,24 h
fraction
to the above
protocols
as described
Results are expressed
as the activity
concentration
of appropriate
controls.
(0.67
under
remaining
(%)
Mg) was
treated
Materials
relative
according
and
to
Methods.
the
same
UTERINE
LUMINAL
FLUID
559
MITOGEN
,#{231}’
00
0
K
E
80
C
0
6 50 fraction
60
C
.
DEAE
40
traction
ULF
Cuntractionafed)
E
>..
C
20
N)
1.0
ai
FIG.
5. Response
of mouse
embryo-derived
fluid
(ULF).
The
activities
of unfractionated
pooled
fraction
(o) were
tested
at the indicated
three
determinations
for each
dose.
fibroblast
(AKR-2B)
ULF
(Day
12 pregnant
doses
for this ability
-
10.0
-
100.0
cells to increasing
doses
of unfractionated
saws)
and of the corresponding
DEAF
to stimulate
DNA
synthesis
in AKR-2B
and partially
Fraction
IV
cells.
Values
purifed
(A) and
represent
uterine
luminal
Sephadex
G-50
the mean
of
20
C
0
4
‘9
I0
a
I0
vs
b
‘I
C
x
C
0
L
E
2
E
>..
‘C
I0
4-
z
I
>
#{149}0
C.)
antI-
ULFM
nq Sample
30
60
mEGF
240
kIGF-i
mEGF
200
60
100
ULFI
240
1-)
FIG.
2
3
4
5
Days
FIG.
6.
Effect
of
uterine
luminal
fluid
(ULF)
mitogen
on
mouse
embryo-derived
fibroblast
cell growth.
Cells
(3.15
X 10’)
were
seeded
per 60 mm2
dish
in 5 ml of DMEM
containing
0.5%
calf
serum
and
incubated
at 37#{176}Cin 5% CO2 /95%
air. Additions:
none
(.),
150
ng
ULF
mitogen
(G-50
fraction,A).
At indicated
times,
duplicate
dishes
were
removed
and
the
cell
numbers
were
determined.
Each
point
represents
mean
cell counts
± SD (n = 3).
midine
Uterine
Materials
resents
added
7. Effect
of uterine
incorporation
stromal
cells
into
were
luminal
DNA
cultured
and
Methods.
Relative
the
ratio
of radioactivity
ULFM,
murine
epidermal
fluid
of
mitogen
(ULFM)
porcine
uterine
and
labeled
as
1+)
on
stromal
described
[3
HI thycells.
under
f3Hlthymidine
incorporation
repuptake
by cells in the
presence
of
growth
factor
(EGF,
Collaborative
Research
Inc.,
Bedford,
MA)
or human
insulin-like
growth
factor-i
(IGF-1,
AMGEN
Biologicals.
Thousand
Oaks,
CA)
over
that
incorporated
by
cells
receiving
an equivalent
volume
of phosphatebuffered
saline.
Anti-mouse
EGF
IgG (50 Mg/mI)
was added
to cells
alone
or in the presence
of ULFM.
Values
are the mean
± SD
of 3
culture
wells.
SIMMEN
560
ET AL.
DISCUSSION
as well
This
initial
report
describes
characterization
the partial
of a major
purification
growth
and
factor
as primary
not
to
graphic
cultures
component
in uterine
luminal
fluids
of early-pregnant
and
nonpregnant
sows.
This
uterine
luminal
fluid
mitogen
(ULFM)
is a polypeptide
with
an apparent
mol.
wt. of 4800.
This
factor,
like EGF,
stimulates
(25,000),
platelet-derived
3 1,000)
18,000).
and
fibroblast
ULFM
also
mitogenesis
cells,
is
A431
EGF
receptor
represent
the free
stable.
basis
of
in fibroblastic
sma!l
mol.
of
(AKR-2B,
wt.,
and
However,
ULFM
immunological
uterine
stromal)
is extremely
heat
is distinct
from
and biochemical
EGF
on
analyses
the
of
its activity.
First,
ULFM
activity
is not neutralized
by the addition
of antibody
specific
for murine
EGF,
indicating
immunological
unrelatedness
of ULFM
and
the
growth
for the
receptors,
factor.
Second,
binding
of
suggesting
in target
cells.
synthesis
in A431
dose-dependent
ULFM
is
moting
regarding
activity
the
ULFM
does
I] -mEGF
to
distinct
receptors
EGF
cells
does
that
fashion.
most
likely
in sow
inhibitory
unfractionated
ULF
EGF
or EGF-related
not
the
uterine
effect
that
DNA
ULFM
in a
growth-pro-
secretions.
of anti-EGF
mitogenic
molecules
exhibit
to
only
activity
are also
of ULF.
In addition,
anion-exchange
of
heat-treated
ULF
demonstrated
fractions
stimulate
respond
not
compete
A43 1 cell EGF
for the ULFM
[125
Third,
not
Our data
IgG on
suggest
that
components
activity
towards
AKR-2B
cells.
These
fractions
account
for approximately
38%
of ULF
growth
factor
activity.
The
absence
of distinct
AKR-2B
mitogen
peaks
representing
these
activities
when
(unfractionated)
ULF is
chromatographed
on Sephadex
G-200
gel filtration
columns
may
indicate
similar
mol.
wts.
of these
factors
and ULFM.
Finally,
partially
purified
ULFM
cannot
account
ULF
on MDCK
these
additional
for the mitogenic
activity
of crude
(epithelial)
cells. Further
studies
on
factors
present
in ULF are currently
underway.
In this
the
ULF
stimulate
This
respect
was
study,
detected
[3H]
presence
of mitogenic
on
basis
thymidine
the
uptake
assay,
although
relatively
to growth
factor
type,
of
by
its
cells
to demonstrate
being
stimulatory
(IGF)-1
since
AKR-2B
examined
mol.
wt.
binding.
form
of
the
latter
ULF
raises
or
mechanism
Sirbasku,
the
is
(28,000-
is noncompetitive
in
ULFM
does not
growth
factor
nonmitogenic
in
our
and
EGF
its
from
of
tissue
UDGF
other
ULFM
origins
(Ikeda
(DiAugustine
to
and!
and
et
a!.,
1985;
Teng
et al., 1985)
are probably
synthesized
locally
by
uterine
cells.
By analogy,
these
ULF
mitogen(s)
may
also be of uterine
origin.
Alternatively,
they
mediated)
rived growth
may
or
result
from
paracellular
factors.
In this
transcellular
transfer
regard,
strated
mEGF
(receptor-
of
plasma-deULF is known
of both
plasma
and DeSombre,
The
physiological
function
of
activities
in sow uterine
secretions
G. Stancel
(personal
communication)
and
1985).
to
uterine
growth-promoting
is also unknown.
has demon-
that
intraluminal
administration
antibody
can
inhibit
growth
of
of
the
antimouse
uterine
epithelium,
suggesting
EGF
involvement
in uterine
growth
in vivo.
It is possible
that
ULFM
may
play
a similar
role,
either
autocrine
or paracrine,
such
that
mal
in concert
with
known
growth
as EGF
and
IFG-I.
To date,
we
ULFM
stimulates
mitogenesis
in
cells
in vitro
but
have
yet
to
similar
effects
towards
these
in its
1 cells
factor
Finally,
insulin-like
question
of
of
transport.
1984)
in
nonspecific
with
has been
shown
to
it
but
factors
(16,000be distinct
from
that
ULFM
is a mitogen
distinct
peptide-growth
factors.
apparent
specific
accumulation
to
cell type
specificity
to AKR-2B
and A43
since
cells
its chromatofrom
TGF-j3
assay
and
exists
in all biological
fluids
to date (except
human
milk)
bound
to high
(150,000;
50,000)
carrier
proteins.
We
ability
correlate
with
cell proliferation
activity
of known
growth
factors
in other
systems.
Indeed,
this factor
can promote
the proliferation
of fibroblastic
(AKR2B) cells, confirming
its identity
as a mitogen.
ULFM
appears
activity,
(5700-6000),
activity
in culture.
stromal
growth
growth
appears
to
contain
other
proteins
tissue
origins
(Kuivanen
chromatography
several
other
mitogenic
TGF-ct
suggest
known
The
of uterine
MDCK
cells.
On the basis of
properties,
ULFM
is distinct
demonstration
fibroblastic
thelial-like
latory
effects
the uterus.
towards
ends
uterine
are
epithelial
in progress.
stimulators
have
shown
uterine
strodemonstrate
cells.
Studies
Nevertheless,
our
of
ULFM
activity
towards
both
(AKR-2B,
uterine
stromal)
and
epi(A43 1)
cells
suggests
possible
reguof this factor
on both
kinds
of cells in
ACKNOWLEDGMENTS
We thank
Cindy
Fisher
for typing
the
Coy
for
manuscript.
expert
techinical
assistance
and
Beverly
UTERINE
LUMINAL
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