Rete testis fluid contains a growth factor for cultured fibroblasts

Vol. 105, No. 2, 1982 March 30, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 391-397

RETE TESTIS FLUID CONTAINS A GROWTH FACTOR FOR CULTURED FIBROBLASTS Kenneth A.R.C.

Institute

Received

January

D. Brown,

Diane M. Blakeley, Brian P'. Setchell

of Animal 27,

Physiology,

Anthony

Babraham,

Henville

Cambridge

and CB2 4AT,

U.K.

1982

A new polypeptide mitogen has been detected at high specific activity The factor, which stimulates DNA in the rete testis fluid of rams (oRTF). has a molecular weight of 45,000 as synthesis in quiescent Swiss 3T3 cells, is heat assessed by gel filtration through Ultrogel AcA 34. The factor stable but is inactivated by proteolytic enzymes and by S-mercaptoethanol. The growth-promoting activity in oRTF does not bind to concanavalin A. INTRODUCTION The Sertoli secreted

cells

of spermatogenesis cultures

without

added

reasons

we considered

namely

it

(4,5). for

proliferate

long

therefore

chosen

collected

in relatively

ABBREVIATIONS:

growth that

previously

Sertoli

periods

(weeks)

be readily medium

to use ovine large

rete

cells

might

to detect

in primary passed.

in that in medium For these

themselves

produce

serum growth

factors;

cultures old

culture,

of mouse 3T3 rats

can be

the cells

do not

large-scale and costly.

(oRTF),

as a source

control

this

Consequently,

fluid

their

investigated

20-day

time-consuming

restis

volumes,

(2,3).

from

in the

and survive

in quiescent cells

with

are unusual

We have

used

are

role

factors

Sertoli

activity.

Although

conditioned

cells spread

attach,

of DNA synthesis

and cannot

harvesting

possible

an assay

stimulation

maintained

will

growth-promoting

through the

cells

together

an important

Sertoli

serum or macromolecular

cell

fibroblasts

for

of the

epithelium,

to play

In addition,

(1).

primary

question

seminiferous

are considered

products,

and secrete

of the

which

of Sertoli

cultures We have

can be cell

secreted

oRTF; ovine rete testis fluid. PBS; phosphate-buffered EGF; epidermal growth factor. IGFl, IGFZ; insulin-like factors 1 and 2. PDGF; platelet-derived growth factor.

saline. growth

0006-291X/82/060391-07$01.00/0 391

Cop.vrrghl 0 1982 bv Academic Press, Inc. .-l/l rights qt‘ reproduction m an! form wserved.

Vol. 105, No. 2, 1982 proteins growth

(6,7). factor

We have found which

fibroblasts. growth

BIOCHEMICAL

stimulates

The factor

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

that this fluid contains 1 'H-thymidine incorporation

appears

to be different

a potent into

from

other

polypeptide quiescent

well-characterized

factors. METHODS

Collection and concentration of rete testis fluid. Testicular semen was collected from conscious Clun Forest rams during the breeding season by cannulating the rete testis of each animal under pentobarbitone-halothane anaesthesia as described previously (8). The fluid was kept at 4'C during 24 h collection periods and then the spermatozoa were removed by centrifugation. The supernatant fluid (oRTF) was stored frozen at -2O'C. For use in assay or for biochemical separation, the oRTF was thawed and concentrated ten-fold by pressure ultrafiltration at 4'C using Amicon equipment. Initially DM2 membranes were used but recent experiments have shown that a faster concentration can be made using PM10 membranes with no loss of cell growthpromoting activity. The final protein concentration of the ultrafiltration retentate was 6-8 mg/ml. Cell cultures. Stock cultures of Swiss mouse 3T3 cells were maintained on 9 cm plastic culture dishes (Nun<) andpasaged every 3 days in Dulbecco-Vogt modified Eagle's medium (DME) containing 10% newborn calf serum. Dishes were incubated at 37'C in a humidified atmosphere of 10% CO2/90% air. For use in the 3H-thymidine incorporation assay, 5 x lo4 cells, in 2 ml DME + 10% calf serum, were seeded in 3.5 cm dishes (Nunc) which were incubated for at least 6 days. These cultures were "quiescent" as judged by a low level of 3HAfter a continuous 30 h exposure to 'H-thymidine, thymidine incorporation. less than 1% labelling of cell nuclei is detected by autoradiography (5). Measurement of3H-thymidine incorporation. Aliquots of test agents were directly to the depleted growth medium of quiescent cells. Twenty ul of thymidine were added per dish to give a final concentration of 1 uCi/ml After incubation for 36 h at 37'C, the medium was removed from the dishes the cells were washed twice with cold PBS. The cells were extracted for with 1-2 ml of cold 5% trichloracetic acid, rinsed with ethanol, and air The cells were dissolved in 1 ml of O.lM NaOH and 0.5 ~1 of this solution mixed with 10 ml of acidified scintillation fluid for H counting.

3dded H(1 x loand 30 min dried. were

RESULTS To date, collected similar

from levels

stimulation in Fig.

6 ten-fold three of cell

concentrates

different

serum is thymidine activity half-maximal

a).

included

(activity/unit stimulation

in quiescent

1 (curve to a similar protein) achieved

maximal

of oRTF is

15 times

curve

However that

the is

to newborn

shown calf

serum stimulate the

of calf

at 150 and 2700 ug protein/ml 392

fluid

for

by one preparation

oRTF and calf level.

testis

The dose-response

3T3 cells

Both

rete

have demonstrated

the dose-response b).

from

preparations

activity.

For comparison,

in Fig.

incorporation

rams and all

growth-promoting

of DNA synthesis 1 (curve

have been prepared

specific serum with respectively.

3H-

BIOCHEMICAL

Vol. 105, No. 2, 1982 c In 0 L al a c),o ; a 2 .-: ;; L0 a L 0 .-E

500

-

400

-

300

-

200

-

100

-

m c

AND BIOPHYSICAL

pg

RESEARCH COMMUNICATIONS

protein/ml

Stimulation of 3H-thymidine incorporation in quiescent 333 cells Fig. 1. as a function of the concentration of oRTF (W) or calf serum ( m m). The incorporation of 3H-thymidine was measured as described under METHODS. The values show the means of duplicate determinations which did not vary by more than 5%.

The slight

inhibitory

incorporation

effect

has been observed

oRTF was subjected eluted

with

eluted

in the void

weight

greater AcA

at positions

10 times the

Sertoli

45,000.

to Fig.

We have found

in culture that

not

at a position 28-31

pH 7.4)

The dose-response

activity

has a molecular

the

elution

activity

of 350,000

corresponding (Fig.

stimulatory

material

of mitogenic

weights

and

When oRTF was chromatographed

(PBS,

peaks

G-50 equilibrated the

active

shown).

same conditions

Fractions

All

the

H-thymidine

profile

were detected

and 75,000.

A

to a molecular

2) were

pooled

to this

material

weight

and concentrated is

shown in

2.

has been reported cells

that

3

of oRTF tested.

on Sephadex

to molecular eluted

of oRTF on

preparations

(ptl 7.4).

Minor

by ultrafiltration.

insert It

the

corresponding

of approximately

all

filtration

[results

2 was obtained.

peak of activity

with

indicating

30,000

34 under

concentrations

saline

volume

than

in Fig.

major

to gel

phosphate-buffered

on Ultrogel shown

of higher

that

some

two-thirds

are glycoproteins

approximately

one-third 393

of the proteins which

bind

secreted

to concanavalin

of the protein

in oRTF

by rat A (9).

will

bind

to

Vol. 105, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

20

: L. 2 s

24

: 20

16

0 ; E 4

: .2L :

12

L0 .-,”

6

.-s PE 2 ,I

4

0

5

10

15

25

20

30

35

40

fraction

Fig. 2. Concentrated oRTF (4.5 ml) was applied to a 2.5 x 70 cm column of Ultrogel ACA 34 previously equilibrated with PBS (pi-I = 7.4). The column was eluted at 4’C with PBS at a flow rate of 20 ml/hr. Fractions (10 ml) were collected, the absorbance at 280 nm was measured (a-O), and aliquots (ZOO ~1) were tested for ability to stimulate 3H-thymidine incorporation in quiescent 3T3 cells (A-A). The void volume (Vo) and the total volume (Vt) of the column were determined with blue dextran and potassium dichromate respectively. The elution positions of bovine serum albumin (67,000) and ovalbumin (43‘00 ) are also marked. The results show the mean of duplicate determinations of 9 Hthymidine incorporation. In this experiment, addition of 200 ~1 of concentrated oRTF or 200 nl of calf serum produced incorporation values of 74 x lo3 and 86 x lo3 cpm per dish respectively. Fractions 28-31 were pooled and reconcentrated four-fold b ultrafiltration (PM10 membrane). The dose-response of this material in the 3T3 Y H-thymidine incorporation assay was measured (insert).

a column

of

activity

bound

not

concanavalin to

the

lectin

However

(Table

1)

none

of

suggesting

that

the

growth-promoting

the

active

material

is

a glycoprotein. The

growth

disulfide

factor

linkage.

complete

.oss

of

heat-inactivated

However, min)

A-Sepharose.

.

in

oRTF

Treatment

growth

Incubation

(2%)

resulted

urea

(6M)

or

did

oRTF

a loss

guanidine

oRTF

activity not

promoting of

in

of

stimulatory enzymes

the

appears

cause

24

be

with

a polypeptide

h at

of

97%

biological

HCl

(4M)

caused

4’C

treatment

loss

any in

oRTF in

is the

394

16%

of

oRTF

of activity heat

stable of

Similar and

30%

contains

a

resulted

in

with

(Table

presence

activity. only

that

trypsin/chymotrypsin

whereas

activity for

to

losses

previously

11).

(100°C

for

20

(Lmercaptoethanol incubations of

ac

with vity

a

BIOCHEMICAL

Vol. 105, No. 2, 1982

AND BIOPHYSICAL TABLE

Concanavalin

RESEARCH COMMUNICATIONS

1

A-Sepharose fractionation bound and unbound

of oRTF: fractions

stimulatory

activity

of -

‘H-thymidine (cpm

Additions

control,

100 ul

incorporation x lo-i/dish) 4.3

PBS

oRTF,

unfractionated,

100 ~1

oRTF,

conA

bound,

oRTF,

conA

non-bound,

192.5

100 yl

12.5 123.3

100 ul

A column containing 2 ml of concanavalin A-Sepharose was equilibrated with O.lM ammonium acetate buffer (pH = 6.0) which contained 1M NaCl, 10m3M CaCl?, and 10e3M MgC12. A 0.5 ml sample of concentrated oRTF was applied to the column. The unbound proteins were washed from the column with acetate buffer and collected as a single fraction. When the protein concentration of the eluate had returned to zero, the bound glycoproteins were eluted from the column with acetate buffer containing 0.1M o-methyl-D-mannoside. Both collected samples (unbound and bound proteins) were dialysed against 0.02M ammonium formate (pH = 6) at 4’C, lyophylized, reconstituted in 0.5 ml of PBS and tested in the 3T3 cell 3tlthymidine incorporation assay. The values show the mean of two determinations of 3H-thymidine incorporation which did not vary by more than 5%.

respectively

(Table

different

11).

samples

of

Similar

results

(not

shown)

were

obtained

with

severa

oRTF.

DISCUSSION Our

results

peptide

show

growth

We suspect since

it

cross

the

recent

factor.

that is

we have

the

homogenates

prepuberal

observed

3T3

cells.

It

in is

polypeptides

including

Our

together

results, in

vitro,

well

of

be

this

of

not

shown)

culture

also

that

androgen

secrete

binding

those

of

molecular

support

Feig

a polypeptide

395

this

isolated

from

medium

growth-promoting Sertoli

cells

protein

(13,14)

et

al. growth

(12)

be

determined.

by

Sertoli (45,000)

secrete and

factor.

could is

the (12).

In

addition,

harvested

from for

a variety transferrin that

the

seminiferous

activity

suggest

cells,

conclusion

mitogen

conditioned

a poly-

weight

of

a polypeptide that

to

probably

cells

contains

established

with

In

activity,

remains

this

Sertoli

contain

specific

locally, of

(10,ll).

high

factor

produced

mice

(results

cells

at

a polypeptide

that of

and

may

barrier

Sertoli

vivo

factor that

contains,

source

blood-testis

rat

in -~

oRTF The

unlikely

report

epithelium

that

mollse of (15).

these

cells,

Vol. 105, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL TABLE

Susceptibility

of conditions,

the

RESEARCH COMMUNICATIONS

11

growth-promoting reduction, heat

activity in and proteolytic 3

oRTF to denaturing enzymes

Expt.

Additions

I

control, PBS oRTF, PBS, 4’C, 24 h ORTF, 6M urea, 4’c, 24 h 24 h oRTF, 4M guanidine.HCl, 4”C, 4”C, 24 h ORTF, 2% B-mercaptoethanol, oRTF, control 10% calf serum

10.4 246.2 207.0 175.8 18.3 232.2 370.6

II

control, PBS oRTF + PBS oRTF + T/C in PBS oRTF + heat-inactivated 10% calf serum

9.4 87.2 5.6 105.1 93.4

T/C

in

H-thymidine incorporation (cpm x lo-3/dish)

PBS

Samples of concentrated oRTF were treated with dissociative or reducing agents for 24 h at 4’C as indicated. The samples were subsequently dialysed against PBS for 48 h at 4°C prior to analysis for growth-promoting activity. In a separate experiment, concentrated oRTF was treated with an equa1 volume of 1) PBS, 2) PBS containing trypsin and chymotrypsin (T/C), 3) PBS containing heat-inactivated T/C. Trypsin and chymotrypsin were present at a final concentration of 1 mg/ml. The samples were incubated at 37°C for 4 h and then heated at 1OO’C for 20 min prior to use in the 3T3 3H-thymidine incorporation assay. The values show the mean of two determinations of 3H-thymidine incorporation which did not vary by more than 5%.

The

than

molecular

the molecular

IGFZ) with

of

weight

of other

mitogenic

was carried

weight However,

the

G-50 or Ultrogel

activity

in fractions

containing

is bound

to a carrier

strong. promoting

The relationship activity

to be determined. is

inactivated

reduction.

3T3 cells

present

AcA34

protein,

between

in homogenates

In contrast

to lOO"C,

but

the oRTF-derived

is

is not

factor 396

weight

of mouse

material

that

of oRTF

on

Thus,

if

to be very

in oRTF and the growthSertoli

cells

of a lower

inactivated is

a

stimulator-y

material. appears

and

the and

of significant

factor

higher

chromatography

factor

profiles

the association

the growth

gel

a growth

elution

molecular

somewhat

is possible

of

showed no evidence lower

Since it

a complex

is

EGF, FGF, PDGF, IGFl

(16,17).

chromatographic

The mouse-derived by heating

(e.g.

conditions

represents

factor

growth

polypeptides

for

Sephadex

the factor

oRTF-derived

non-dissociating

molecular protein.

the

activity

out under

determined binding

weight

heat

(12)

remains

molecular

weight,

by sulfhydryl-bond stable,

but

is

BIOCHEMICAL

Vol. 105, No. 2, 1982 inactivated not

after

identical.

We have

ram testis

contains

reduction

with

a similar

growth

but into

the the

could

(12)

Sertoli

These

part

that

suggested

control

which

is not

inactivated

that

a Sertoli

speculations

primary

of the epididymis,

of

by

result

suggests

both

of mouse and sheep

that

of secretion

cell-derived

of spermatogenesis,

is

homogenates

This

in the course

perhaps

or by synchronizing

factor its

that

clearly

testis.

remain

of growth-promoting

cell-derived

the possibility first

have

shown)

modified

and rete

are

not

in the testes

may be chemically

in the

finding

activity

may be present

of spermatogonia

our

(results

or dithiothreitol.

tubules

spermatocytes.

addition,

the

factor

a role

proliferation

that

factor

et al.

found

growth-promoting

seminiferous

play

recently

RESEARCH COMMUNICATIONS The two factors

by B-mercaptoethanol.

8-mercaptoethanol

growth

Feig

tene

reduction

AND BIOPHYSICAL

exciting

the

in prelepto-

possibilities.

In

in rete

testis

fluid

out of the

testis

and raises

may be to regulate

cell

transported

where

factor

by regulating

DNA synthesis

activity

function

growth

the mitotic

rate

is normally

suggests

division high

in (18,191

REFERENCES 1. 2. _3 . 4. 5. 6. I. 8. 9. 10.

11. 12. 13. 14.

1.5. 16. 17. 18. 19.

Means, A.R., Fakunding, J.L., Huckins, C., Tindall, D.J., and Vitale, R. (1976) Recent Prog. Horm. Res. 32, 477-527. Tung, P.S., Dorrington, J.H., and Fritz, I.B. (1975) Proc. natn. Acad. Sci. U.S.A. 72, 1838-1842. Verhoeven, C., Cailleau, J., and de Moor, P. (1980) Molec. cell. Endocrinol. 20, 113-126. Halley, R.W., and Kiernan, J.A. (1974) Proc. natn. Acad. Sci. U.S.A. 71, 2908-2911. Brown, K.D., and Halley, R.W. (1979) J. cell. Physiol. 100, 139-146. Setchell, B.P. (1974) J. Reprod. Fertil. 37, 165-177. Waites, G.M.H. (1977) In: The Testis (eds A.D. Johnson and W.R. Gomes) Vol. IV, pp. 91-123. Academic Press, New York. Suominen, J., and Setchell, B.P. (1972) J. Reprod. Fertil. 30, 235-245. Wilson, R.M., and Griswold, M.D. (1979) Expl. Cell Res. 123, 127-135. Setchell, B.P., and Waites, G.M.H. (1975) In: Handbook of Physiology [eds D.W. Hamilton and R.O. Greep) Sect. 7, Vol. V, pp. 143-172. American Physiological Society, Washington DC. Setchell, B.P. (1980) J. Androl. 1, 3-10. Feig, L.A., Bellvi, A.R., Erickson, N.H., and Klagsbrun, M. (1980) Proc. natn. Acad. Sci. U.S.A. 77, 4774-4778. French, F.S., and Ritz&r, E.M. (1973) Endocrinology 93, 88-95. Hagenas, L., Ritz&i, E.M., Ploen, L., Hansson, V., French, F.S., and Nayfeh, S.N. (1975) Molec. cell. Endocrinol. 1, 339-350. Skinner, M.K., and Griswold, M.D. (1980) J. biol. Chem. 255, 9523-9525. Gospodarowicz, D., and Moran, J.S. (1976) kRev. Biochem. 45, 531-558. Bradshaw, R.A., and Rubin, J.S. (1980) J. Supramolec. Struct. 14, 183-199. Nicander, L. (1957) Acta Morph. Neerl.-Stand. 1, 99-118. Nicander, I,. (1958) Acta Morph. Neerl.-Stand. 1, 337-362. 391