BIOLOGY

44, 889-896

REPRODUCTION

OF

In Vitro

(1991)

Effects of Epidermal Growth Factor, Insulin-Like Growth Factor-I, Fibroblast Growth Factor, and Follicle-Stimulating Hormone on Hamster Follicular Deoxyribonucleic Acid Synthesis and Steroidogenesis1 SHYAMAL

Department University Department

K. ROY2

and

GILBERT

of Obstetrics

& Gynecology

of Nebraska

Medical

of Physiology,3

Ralph

S. GREENWALD3

and

Center,

L. Smith

Research

Kansas

City,

Physiology

Omaha, Center,

Kansas

& Biophysics2

Nebraska

68198

University

of Kansas

Medical

Center

66103

ABSTRACT Preantral 100

studied

by FGF,

using

and

I affected

growth

significantly of the

antral

but

factors

significantly

from

did

bination

of multiple

minates

in the

involved

not

stages

3-10

was

roles on

DNA

after

optimal

that

EGF,

IGF-I,

From

as P4 biosynthesis

that the

results

of hamster

hamster the

and

potential

tors

modulatory

in follicular

granulosa

cells

demonstrated

significantly production production

growth

FSH-induced that is independent

IGF-I cell

monoclonal progesterone

[10].

that

Recently,

IGF-I

causes

depending

has

accumulated

for

protein product theca-interstitial

growth

coworkers factor-I

progesterone of cell antibody production.

fac-

primed [9,101

aromatase

rat

and

have

(IGF-I)

produce receptors

sig-

and estroproliferation.

dramatically Although

Magoffin

et al. [12]

a selective

increase

have

January

8, 1991.

Received

August

16, 1990.

‘Part docrine

of the Society,

data 1990,

has

been Atlanta,

GA.

This

at

work

the

72nd was

Annual supported

Conference by a grant

from

en-

EnNIB

Fax:

to induce ulating

402-559-5015.

889

and FSH

by and

preantral

of events

that that

cul-

EGF

is

of development.

cell

preparation

in

strongly suggest that IGFovarian steroidogen-

Using

bovine

reported

that

demonstrated expression

and

transthe

influence

ovarian bovine

cells, theca

have

Skinner cells

also

demonstrated

also

immuno-

factor (EGF)-like activity cells [16]. EGF consistently

incorporation

as well

in highly EGF-induced

several fold Therefore,

it

DNA

DNA

as proliferation conin-

in the presence of either is obvious that growth regulatory factor

functions. (FGF) [19]

and

steroidogenesis

synthesis

synthesis and

exclustim-

defined culture [3H]thymidine

porcine granulosa cells, whether share a final common intracellular replication

rat cell

factor that specifically binds to EGF bovine granulosa cell proliferation we

follicular DNA

effect.

significant

unaffected

et al. [13] have (TGF) -a gene

growth granulosa

increases TGF[17].

[8, 20]

but

it appears

play important intraovarian EGF [18], fibroblast growth

of bovine tors and

(HD 00596). ‘Correspondence.

Recently,

[3H]thymidine

corporation IGF-I or

demon-

of the

stage

of porcine granulosa cells ditions; however, only the

IGF-I presented

the

[14]. have

reactive epidermal sively in hamster ulates

a of

in immature, diethylstilbestrol-treated cells, and TGF-a attenuates granulosa

a TGF-a-like and stimulates

factors though Accepted

a cascade studies,

Kudlow factor

[15]

in culture.

inFSH

in P45Oac,,

on

activity

Coffey

additive

in rat theca-interstitial

esis, Similarly, forming growth

IGFby

doses

in hamster

involve

previous

expression

both of the

FSH

no were

replication

may

our

synthesis follicles

Using

and

insulin-like

cells

and

induced

of suboptimal had

also IGF-l,

Moreover, the threshold to initiate the S by a single threshold stimulus or a com-

FSH and

EGF,

culture. All these lines of evidence I may fine-tune gonadotropin-induced

stimulates aromatase activity and thus estrogen by rat granulosa cells [11], cellular estrogen increases several fold when IGF-I is added along

FSH

strated

Adashi

and

present

was

of FSH,

accumulation,

and

1 pCi

of EGF,

synthesis

ag/nil)

50,

and

ng/ml)

E2 accumulations

DNA

DNA antral

of polypeptide [6-81.

in culture, that

Moreover, an hibits granulosa

roles

development

nificantly augments gen biosynthesis

with

[51. Evidence

mouse

(5

(E2)

A and

stimulate

factors

follicular

play a central role in ovarian follicular [1] and recent studies have established that

[2-4]

FSH

all stages;

and estradiol-17

exposure;

zyme

FSH, and to some extent LH stimulate in vivo and in vitro of preantral and

for

FGF

of the

DNA

a combination

folliculogenesis. delivered either

growth

INTRODUCTION Gonadotropins development

EGF

doses

inhibited

(FGF) Synergism

(50

doses

suboptimal

1, 5, 10,

factor

control.

a positive

Paradoxically, (A),

and

as

24 h to

growth

Optimal

significantly

synthesis

only

used

ag/nil).

for

exposed

fibroblast

whereas

FGF

replication.

DNA

It is possible

(5

replication,

androstenedione

and

was

FSH

as intraovarian factors regulating the total magnitude of stimulation

duplication.

as well

DNA ineffective.

(P4),

indicate

ag/mI)

and

totally

stimulated

observed

(100

were

hamsters

(IGF-I),

factor-I

fofficular

was

< 0.05)

proestrous

ag/mI)

IGF-1-mduced

stimuli.

of DNA

in proliferation

FGF

affect

results

subthreshold

initiation

enhanced

(p

These

FSH

(1

factors

and

from growth

replication.

progesterone

follicular

factors.

growth

of DNA

stages,

of EGF

fofficles

insulin-like

of growth

significantly

follicles, and may play of the cell cycle depends

phase

rate

preantral

stimulated

P4 accumulation

(EGF),

doses FSH

7-10)

(stages

factor

the

ng/ml

a few

dose

all three

any

antral

suboptimal

100

only

suboptimal

FSH

and growth

to determine

(3H]thymidine

and

1-6)

(stages

of epidermal

ng/ml

and

steroidogenesis

their

Aland

growth facpathway roles in

in regpreantral

890

AND GREENWALD

ROY

follicles assessed

are yet to be determined. the roles of EGF, IGF-I,

rately or roidogenesis primary that

combined-on by using

follicular intact proestrous

to preovulatory

supports

stages

follicular

Receptor

grade

murine

pure,

which

95%

with

ED

of 2.2

[21]

in a serum-free

METHODS

EGF,

basic

FGF

(final

Collaborative

Research

conc:

(Cambridge,

nant human IGF-I (with ED native IGF-I from its receptors) (Terre Haute, IN); Dulbecco’s and antibiotics methyl-[3H]thymidine

bovine

6.25

pg

in-

MA);

recombi-

Louis,

purchased

MO).

All other

from

various

analytical

grade

commercial

(Lenexa, was

at 0900 h proestrus (Day serum FSH and LH were

Ovaries Ringer

were collected solution with

chemicals

10 pg streptomycin Follicles at stages 6-10 7-8 layers of granulosa follicles with licles of various

KS); from

were

cells

and

dissociated and no theca; initial

1 = estrus) levels [23].

8-10 = at stages

of thecal

cells

[21]).

=

7

+

10. Follicles were 0.12% NaHCO3 +

pg streptomycin to 1 ml fresh Cambridge,

+

medium MA),

the following ment under

rinsed 0.5%

sulin fore, dium.

and

cultured

protocols 5% CO2

long-term

resulted insulin

+

i03

35 pg fungizone in a 24-well at in

[3H]thymidine/ml. The fered with neither the induced [3H]thymidine However,

in DMEM BSA

num-

culture

with 15 mM Hepes penicillin G + 10

and ITS, transferred culture plate (Costar,

for

24 h [22]

according

37#{176}C in

air

in

presence basal nor incorporation

in follicular was an essential

U

a humidified the presence

(including

24

h)

were

Amersham).

As positive exposed stimulates

to

incubated

for

without

in-

24 h [22]. Thereof the culture me-

24 h with

to 100 DNA

factors

trations inducing

to determine follicular

any synergistic or additive DNA synthesis. In the present

suboptimal

to be

dose

for only definition

consistent 1 ng/ml

and

FSH

different

of growth

as the

one

a few stages of suboptimal

but smaller response. of growth factors and

combi-

at suboptimal

that

barely

conceneffects study, induces

in we DNA

of follicles, unlike the condose is one that elicits a For all practical 5 ng/ml of FSH

purposes, were found

suboptimal.

3

assess

whether

(optimal

medium stenedione After processed content

was

growth

factors

affect

follicular

ste-

follicles at all stages were incubated for 24 h (optimal dose) EGF, IGF-I, or FGF, or 100 dose)

saved (A), and

the culture to measure as described

FSH,

and

to measure estradiol-173

1 pCi

[3H]thymidine/ml;

progesterone (E2).

(P4),

andro-

period, follicles were recovered and [3H]thymidine incorporation and DNA previously [2], and the results were

expressed as pmol [3H]thymidine and E2 in the spent medium

pg were

DNA’ assayed

24 h. by RIAs

P4, A, using

to P4 [24], A (Dr. J. Resko, Oregon Regional Primate Center, Beaverton, OR), and E2 (Dr. D. Collins, University, Atlanta, GA) as previously described

[25, 26]; the results a more meaningful

were unit

expressed to describe

as pg steroid/follicle follicular steroidogen-

as

esis

than steriod/p.g DNA. All experiments were repeated at least three times with four replicates from different animals per treatment group. Statistical analysis consisted of two-way ANOVA and Duncan’s multiple range tests, and Student’s t-tests with the level of significance at 5%.

environof 1 pCi

of 6.25 pg insulin interFSH- and growth factorand steroidogenesis.

death within ingredient

in vitro

2

antisera Research Emory

bers of follicles at stages 1-10 used in the studies were as follows: 80 for stage 1, 40 for stages 2-4, 30 for stage 5, 10 for stage 6, 8 for stage 7, 4 for stage 8, and 2 for stage 9 and

act. 20 Ci/mmol,

nations

ng/ml

antral fol1-5 were

The

Experiment

roidogenesis, with 50 ng/ml

(stages 1-4 = 1-4 layers of granstage 5 = 5-6 layers of granulosa

appearance

can stimulate

[4].

To

Madwere

35 pg fungizone/ml, pH 7.2-7.4. were dissected by hand (stage 6 cells and early thecal layer; stage

+

(sp.

E4eriment

in sterile Krebs’ i03 U penicillin +

incipient antrum; stages sizes [21] and follicles

=

enzymatically ulosa cells

4 of cycle; Day at low baseline

at room temperature 15 mM Hepes and

factors

synthesis

synthesis ventional

sources.

Adult, female golden hamsters (90-100 g; Harlan, ison, WI) with at least three consecutive estrous cycles killed when

growth

follicles at stages 1-10 were also FSH (NIH-17), which consistently

define

Amersham Corporation (Arlington Heights, IL); calf thymus DNA, bisbenzimide (Hoeschet dye 33528), collagenase type Ia, and deoxyribonuclease I were from Sigma Chemical Co. (St.

which

controls, ng ovine

Follicles

of 1.1 ng/ml in displacing was obtained from Imcera modified Eagle’s medium

were from Hazelton (sp. act. 20 Ci/mmol)

(DMEM)

To determine

proliferation

sulin, 6.25 .tg transferrin, 6.25 pg selenium, 1.25 mg BSA, and 5.35 .ig lipoic acid/ml of medium) were purchased from

1

follicular DNA synthesis and their minimum effective doses, follicles were cultured for 24 h in the absence or presence of 1, 10, 50, and 100 ng of EGF, IGF-I, and FGF, and 1 pCi

culture

(from

fIbroblast

ITS

Experiment

[3H]thymidine

AND

and

we sepa-

[22].

stimulates

ng/ml)

study,

DNA synthesis and stehamster follicles from

development

MATERIALS brain,

In the present and FGF-either

RESULTS Effects of Optimal and Suboptimal Doses of Growth Factors on DNA Sthesis (Figs. 1 and 2) In the

absence

of FSH

or growth

factors,

follicular

synthesis remained at baseline levels as determined [3H]thymidine incorporation in the acid-insoluble

DNA from fraction.

GRO’FH

FACTORS

AND

FUNCTIONS

FOLLICULR

891

6-

#{149}Control #{149} FSH-100

#{149}EGF-5Ong

5.11

f

FGF-50

o

IGF-I-5Ong

ng

4-

::L 3U

2-

1

2

3

4

Stages

5

6

7

8

9

10

of development

FIG. 1. In vitro effects of suboptimal doses of growth factors on DNA synthesis of proestrous hamster follicles. Results are mean ± SEM and there were four replicates per group. Note that for stage 9 follicles, FSH or IGF-l had no effect; however, EGF and FGF could induce some response. The rate of (3Hlthymidine incorporation was higher for stages 1-3 compared to larger stages, suggesting that these follicles contained actively proliferating cell

population.

No significant follicles observed

changes

in acid-soluble

between controls (data not shown),

pools

and experimental suggesting that

for the

of [3H]thymidine was saturated irrespective regimens. Consistent with our previous results incubation for 2 h [4] or h culture significantly (p incorporation into follicles 1). Enhanced stages was

incorporation observed with

stage

groups internal

1-10

shown);

was pool

and 1-7,

of treatment obtained with

8 h [27], FSH (100 ng/ml) in 24