Basic fibroblast growth factor in rat corpus luteum stimulates prostaglandin F2-alpha production

Vol.

178,

No.

July

15,

1991

BIOCHEMICAL

1, 1991

AND

BIOPHYSICAL

RESEARCH

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393-399

BASIC FIBROBLAST GROWTHFACTOR IN RAT CORPUS LUTEUM STIMULATES PROSTAGLANDIN FZ-ALPHA PRODUCTION Kazuhiro

Tamura:

Rei Asakai,

and Ryohei

Okamoto

Department of Endocrinology, Medical Research Institute, Tokyo Medical and Dental University, 5-45, Yushima I-chome, Bunkyo-ku Tokyo, 113, Japan Received June 1, 1991 SUMMARY: Rat luteal cells (LC) were incubated with or without basic fibroblast growth factor (bFGF) in a serum-free medium. Prostaglandin F2d (PGFzd) production was stimulated in a dosedependent manner at 0.03~ 1 rig/ml of bFGF. One rig/ml of bFGF caused approximately 5-fold the increment of PGF2d at every stage of LC after 48 hrs of incubation. bFGF also raised progesterone secretion from LC, and this stimulatory effect on progesterone was more distinguishable in an early-, than a middleand a latestage. Additionally, bFGF concentration throughout the luteal phase was assessed using western blot analysis. The protein with typical molecular weight 18 kDa form was in high concentration throughout the luteal phase. These results suggest that bFGF may play a role in the regulation of PGFzd and progesterone production B 1991 Academic as autocrine, but not in mitosis in corpus luteum. Press,

Inc.

Recently, growth factors reported to have possibilities and modulate

the

synthesized in the ovary have been to regulate ovarian steroidogenesis

gonadotropin

actions

Basic fibroblast growth factor (bFGF) fied in the pituitary gland and brain, bovine

corpus

luteum

(CL)

(3).

onto

ovarian

function

which was originally was also isolated

Moreover,

the

mRNA encoding

was expressed in both luteal cells (4) and granulosa These facts prompted us to study the biological role luteal

function.

known as one of function

(6,7,8)

as well

as that

In addition, the

important

and it of

prostaglandin factors may

for be

bFGF

cells (5). of bFGF in

F2d (PGF2d ) is well regulation

has been shown that

uterus-derived

(1,2). identifrom

of

PGFld of

involved

in

luteal

luteal

origin

the

*To whomcorrespondence should be addressed. bFGF, basic fibroblast growth factor; The abbreviations used are Fld; PRG, Progesterone; CL, Corpus luteum; PGFz~ , Prostaglandin PMSG, pregnant mare serum gonadotropin; hCG, LC, Luteal cells; human chorionic gonadotropin.

393

0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, inc. All rights of reproduction in any form reserved.

Vol.

178, No. 1, 1991

luteolysis on the

in rats

BtOCHEMtCAL

(9).

Therefore,

PGF2d and progesterone

MATERIALS

AND BtOPHYStCAL RESEARCH COMMUNtCATtONS

we studied

synthesis

the

by luteal

effects cells

of in

bFGF

vitro.

AND METHODS

In order to obtain a well-defined Animals and preparation of CL: generationof CL, immature female rats of the Sprague-Dawley strain (24b32-day-old) were SC injected at 0800 with 8 IU of by an ip injecpregnant mare serum gonadotropin (PMSG), followed tion with 10 IU human chorionic gonadotropin (hCG) at 1700 2 days after PMSG injection (normal ovulation model). Ovulation took place in the morning of the next day after hCG injection (IO) and were observed in all rats. an average of 13 f 3.2 (n=9) oocytes The day of hCG administration was defined as day-0 and the CL isolated from the rat which was observed ovulating was designated as numbering up to a g-day-old CL a l-day-old CL, with consecutive For the experiment of western (9 days after hCG treatment). blotting, higher doses of 50 IU PMSG and 25 IU hCG were used to obtain a sufficient number of luteal cells. However, it is known that no significant differences were found between the two models with regard to the microscopic appearance of LC or the response to various stimuli (11). Luteal offhag tissue saline fetal After cells times

cell

preparation : All CL removed from ovary was cleaned ovarian tissue under a dissecting microscope. The CL was digested in Cazf- and Mgftfree phosphate buffer containing 0.2 % collagenase, 250 ug/ml DNase and 1 % bovine serum in a shaking water bath at 37 *C for 90 min. cell suspension was filtered through a nylon mesh, luteal (LC) were collected by centrifugation and then washed 3 in an incubation medium.

Cell culture and PGFad and progesterone assay: LC was dispersed -inserum-free culture maurn comprised of 1:l mixture of Eagle's minimum essential medium and Ham'F 12 supplemented with 10 mM Hepes, 2 mM L-glutamine and antibiotics (100 U/ml penicillin, 100 ug/ml streptomycin and 625 rig/ml fungizone). Cells were incubated at a density of 5 x IO4 cells/dish in 500 pl medium at 37 'C with or without recombinant bFGF (human; Takeda) or LH (ovine; NIADDKoLH-26) under a water saturated atmosphere of 5 % CO2 and 95 % The amount of PGF,d was determined using RIA kit (Baxter air. Healthcare Corp.). The content of progesterone (PRG) was measured by RIA with specific antiserum (BioMaker, Israel) as described in detail earlier (12,131. The coefficients of the intraand interassay variations in each RIA were smaller than 10 %. Experimental data are presented as the mean f SE and evaluated by analysis of variance with Bartlett's test followed by Student's t-test. A difference of PcO.05 was considered significant. Western blot analysis: CL tissues (200 mg) of each day-old (3,6 and 9) were homogenized with 2 ml of 25 mM tris-HCl buffer (pH 7.45) containing 3 M NaCl, 1 mM phenylmethylsulfonyl fluoride , 4 uM leupeptin and 2 nM pepstatin at 4 "C using a glass homogenizer. After 10 min., the homogenates were diluted to 0.5 M NaCl with 25 mM tris-HCl containing protease inhibitors and centrifuged twice at 14,000 x g for 15 min at 4 OC. Supernatants were loaded onto 50 pl of heparin-sepharose column. The column was washed with 25 mM tris-HCl containing 50 ml of 1 M NaCl and then resin particles were transferred to an Eppendorf tube and boiled for 3 min in SDSPAGE sample buffer. Buffer containing recombinant bFGF was also 394

Vol.

178, No.,l,

1991

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

performed the same procedure with the column. Proteins were electrophoresed on SDS-15 % polyacrylamide gel and were transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon). The membrane was incubated in 25 mM tris-saline buffer containing 1 % BSA at room temperature with Iodinated [laI]-anti-bFGF monoclonal antibody (5 x 105 cpm/ml, MoAb 98, Takeda) for 5 hr, and then washed and exposed to film (Kodak X-OMAT AR X-ray film) for an autoradiography (-70 'C). RESULTS AND DISCUSSION PGFM production of

bFGF in

under

3-day-old

bFGF stimulated

various

concentrations

LC was measured

(Fig.

1).

(0.03-3

rig/ml)

As shown in fig.

1,

the

PGFZd production between 0.03 and 1 rig/ml of bFGF in a dose-dependent manner. The PGF2d levels significantly increased by treatment with 0.1 rig/ml of bFGF (P
significantly

rig/ml)

8

stimulated

PGFld production

J&!EGd

of

that bFGF by 6 hrs

bFGFllng/mI)

6

i

the

rig/ml

1

LHllOng/ml~

4

/

2

c

6

49

24

Incubation Ihrl

Con1

time

8 1 OControl

~bFGF(lng/ml)

l

A

01

cont.

A03

0.1 bFGF

0.3 i rig/ml

1

3

c/

1

days old:

3

6

Fiq.

1,Dose response effect of bFGF on PGFm production in cultured rat luteal cells. IU, ip) at 57 hrs Immature rats were injected with hCG (10 after PMSG (5 IU, SC) treatment. Luteal cells, which were collected 3 days after hCG treatment, were cultured for 48 hrs in the presence of various doses of bFGF. PGF2(y in the shows the mean f medium was determined by RIA. Each value Similar results were obtained SE of quadruplicate cultures. by three additional experiments.The inset shows thetimecourse of the effect of bFGF on PGFWproduction. A maximal was used. Each point stimulatory dose of bFGF (1 rig/ml) represents the mean + SE of triplicate cultures.

Fiq.

2,Stimulatory effects of bFGF on PGF~Q production in 3, 6 and 9 days after hCG treatment. luteal cells which were collected 3, 6 and 9 days Luteal cells, after hCG treatment,were cultured for 48 hrs in the presence by RIA. of bFGF (1 rig/ml). PGF2a in the medium was measured obtained in a Each value shows the mean f SE of 12 cultures third series of identical experiments. *P
9

Vol.

178,

No.

BIOCHEMICAL

1, 1991

of culture, during the

were

But,

the

similar

though

the

culture

as

tion

with

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

elevated gradually the PGFld levels period. Treatment for 48 hrs with bFGF suggesting that the stimulative number,

and afterwards 48 hrs culture

had no effect on cell effect of bFGF can not number.

AND

to

be attributed

PGFrd that

levels of

PRG levels shown

in

LH tended

the

in

in

the

control

inhibit

an increase

LH (10 group

this group 3. We also

Fig. to

to

the

of

rig/ml)-treated

during

the

were stimulated observed that ability

cell

of

group 48 hrs,

at bFGF

bFGF

to

al-

24 hrs of in combinastimulate

the luteal PGFIa production (data not shown). The stimulatory was also recognized on 6effect of bFGF on the PGFZd p reduction or g-day-old LC as observed on 3-day-old LC (Fig. 2). Levels of the same in each day-old, albFGF-stimulated PGFZd were almost though control compared with rig/ml) old

on luteal LC (Fig.

culture group

levels that of

started, peaked

3).

on g-day-old a 6-day-old.

PRG production PRG levels because

between

12

LC significantly Next, the effect was

were the

and

studied

measured

PRG levels 24 hrs

of

increased of bFGF

on 3-, at

in culture

6-

24 hrs

control as

and

after or

as (1

g-day-

the

LH-treated

previously

re-

-46

-21.5

-14.3

Fiq.

Fig.

3,;;F;;ts

of bFGF on progesterone production in luteal 3, 6 and 9 days after hCG treatment. Luteal cells, which were collected 3, 6 and 9 days after hCG treatment, were cultured for 24 hrs in the presence of bFGF (1 The progesterone level in the medium was rig/ml) or LH (IO rig/ml). measured by RIA. Each value shows the mean f SE of quintuplicate cultures. Similar results were obtained in five additional experiments. *P~0.05,**P<0.01,***P~0.001 vs. control of each control.QP
Vol.

178,

No.

ported

1, 1991

bFGF

(13).

examined,

as

in

human placenta LC (14). The creased as in 3-day-old,

-4

that

thereafter responsibility But, spite

the of

to

PGF1d

Western which

the

18 kDa

prepared

proteins

4). not

also in

the

as reported However,

showed

there in

release is found for

is

of

was why stage

at

as

CL

day-l

to

undetectable

almost

the

decrease

of

PGF2d production of receptors for

to bFGF bFGF.

the

parallel

with

levels of not clear

bFGF-stimuat present.

antibody

revealed

affinity

6-

and

which

were

in while

constant of CL is

bFGF monoclonal

presence

to

vivo.

lack

purified

g-day-old of

of

bFGF

components

of

luteinized

bFGF

levels

indicates luteal reportedly

between

that bFGF of phase. The exissuggested (16,

immunoreactive

detected

bFGF-like

between

discussion

whether

All

of

a signal

the

and

21.5

the

30 kDa

bFGF enhances

translational

sequence

which

via the normal secretory pathway in the extracellular matrix (20,211 its

release

is

may not

not

data).

These

be fully in the

would

although

Our

the

be possible 391

from

result

cell

with

that the dose of LC in vivo, regard

to

the

immuno-

showed compartthat

the

and extra-

is very low. Therefore, of bFGF used in this

Assuming milieu

whereas the of

imply

the

of

direct

(18,19),

observations

secreted vivo doses

concentrations. concentration in will

understood.

PG and

products

using the bFGF monoclonal antibody bFGF was mainly in intracellular

bFGF concentration to conclude that

are physiological physiological lowing

(2.68-fold g-day-old).

decreased

result,

not in number

the

LC (unpublished

bFGF molecule cellular difficult

this

a question

LC in

histochemical staining that a localization of ments

from

level in

bFGF receptor were

The apparant difference observed. This result exist in CL thoughout forms of bFGF has been

ovary,

bFGF mRNA appear

mechanism

3-,

purified

previously.

PRG synthesis their bFGF

of

heparin

from

day-old

PGFLd production was high receptor in late stage of CL,

using in

was

levels

and

the

all

analysis

at

Figure

receptor

of bFGF-stimulated amount of the

high concentration tence of larger 17).

in

in

every

each control and 1.24-fold

levels

by bFGF addition in CL. The reason

ovaries (Fig. each days was

bFGF which

5-day-old

production

band were

high the

LC of

COMMUNICATIONS

the PRG release from 3 N 4-day-old on PRG production gradually de-

bFGF

decrease

blotting

a main

PGFld.

Considering from in PRG production,

levels a small

PRG production receptor site lated

that

at

(15).

due

of

RESEARCH

from

compared with in 6-day-old,

relative fell

BIOPHYSICAL

PRG secretion

case

observed

and

be

the

AND

also stimulated stimulative effect

suddenly

may

raised

CL aged, as 1.50-fold

We recently aged

BIOCHEMICAL

it study

is

is a the fol-

physiologi-

Vol.

178,

Cal

No.

1, 1991

BIOCHEMICAL

significance

istration (luteolysis)

of

of

al.

recently

rat

ovary

PGF2d has been in many species, reported

PGFrd the

exerts

pseudopregnant

denied luteolytic have

CL, was

PGF26 effect.

a luteotropic

progesterone

role CL. enhanced action

in

production

physiological is required.

role

of

in

old

(before

in

vitro

bFGF-enhanced

old

PGF2d

of

has

because

PGFZd

(24,25).

So,

PGFld

in

a

stimulate of CL may and will

CL might been

can to

CL, of

luteolysis

development possibility age

et the

some

of

day-4

PGFzd-induced

the the

hand,

CL,

CL

In

age

that bFGF markedly than the late-stage in

admin-

of

(9).

in

age

to

by bFGF other

regression

only

young

of bFGF Therefore,

On the

exogenous

luteolysis

refractory

COMMUNICATIONS

the rat (7.8). Olofsson was produced locally in

action

namely

The

cause

functional

Our observation in the early-,

influential of functional that

to

including PGFld which

in

RESEARCH

by bFGF.

shown

luteolytic

rat),

(22,23). Moreover, the PRG production indicate tenance

that

functional

BIOPHYSICAL

enhanced

may be involved

reports, whereas

PGF,d

AND

the mainnot be have

postuated

to

stimulate

understand

LC,

a

further

the study

ACKNOW We are grateful to Dr. K. Kato and Dr. M. Seno of Takeda Chemical Industries, LTD. for the generous gift of recombinant bFGF and its antibody. The authors wish to thank Dr. Y. Sato (Tokyo Women's Medical College) and Dr. H. Kogo (Tokyo College of Pharmacy) for their valuable advise and the National Hormone and Pituitary Program (NIADDK, NIH) for the LH used in this study.

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