A Uterine Cell Mitogen Distinct from Epidermal Growth Factor in ...

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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.

human epidermoid Cell Biol 93:1-4

Epidermal

growth carcinoma

factor cells

inhibits in

serum-free

growth cell

of

A431

J

culture

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and

the

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in

Experimental

Design.

NY: