and FRANK. A. SIMMEN2'3'4. Department .... (6.7. Ci/mmole) were purchased from. ICN Radiochemicals. (Irvine,. CA) and. New. England. Nuclear. (Boston,.
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.
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
REFERENCES Barnes
DW,
1982.
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Epidermal
growth carcinoma
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serum-free
growth cell
of
A431
J
culture
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and
the
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