Effect of epidermal growth factor on steroidogenesis by caprinegranulosa cells in culture: interaction with FSH

Rahul Behl*, R.S. PandeyAnimal Biochemistry Division, National Dairy Research Institute, Karnal 132001, Haryana, India

Accepted 23 June 2000

Abstract

Besides gonadotrophins various peptide growth factors have been implicated in the ovarian folliculogenesis. In this study,

the effect of epidermal growth factor (EGF) (0, 0.1, 1.0, 10 ng/ml culture medium) on steroidogenesis by caprine granulosa

cells at various stages of maturation was investigated using serum free culture medium. Caprine granulosa cells were obtained

from ovarian follicles and classi®ed into three classes: small (<3 mm), medium (3±6 mm) and large (>6 mm in diameter). EGF

(10 ng/m culture medium) alone reduced estradiol secretion in granulosa cell from small, medium and large follicles by 62, 48

and 29%, respectively, as compared with control. This inhibition was 50, 36 and 21%, respectively, when EGF (10 ng/ml

culture medium) was applied in combination with FSH (100 ng/ml culture medium). EGF alone stimulated the secretion of

progesterone in granulosa cells from all the three categories of follicles only at highest dose tested (10 ng/ml culture medium).

FSH acted synergistically with EGF in stimulating progesterone secretion by cultured granulosa cells. EGF in combination

with FSH (100 ng/ml culture medium) signi®cantly (P < 0:05) stimulated progesterone secretion by cultured granulosa cells

from all three categories of follicles even at the lowest dose (0.1 ng/ml culture medium) tested. In conclusion, EGF

signi®cantly in¯uences the steroidogenesis by caprine granulosa cells in vitro and may play important role in the follicular

growth and maturation. # 2001 Elsevier Science B.V. All rights reserved.

Keywords: Goat; Granulosa cells; EGF; Steroidogenesis; In vitro

1. Introduction

The peptide growth factors such as epidermal growth

factor (EGF), transforming growth factor-a (TGF-a),

TGF-b and insulin-like growth factor-1 (IGF-1) are

suspected to play a crucial role in ovarian follicular

processes such as cell proliferation, differentiation

and steroidogenesis (Gospodarowicz and Bialecki,

1979; Skinner et al., 1987; May et al., 1988; Adashi,

1992; Mulheron and Schomberg, 1993). The in¯uence

ofEGFonovarian function isuncertain.However, based

onthestudies inmouse(Phippsetal.,1992),human(Das

et al., 1991), porcine (Reed et al., 1993), ovine (Murray

et al., 1993), bovine (Lorenzo et al., 1994) and feline

(Gortiz et al., 1996), there is increasing evidence that

EGF regulates cellular activity of granulosa cells by

stimulation of FSH receptor expression and progester-

one production (Serta and Siebel, 1993; Murray et al.,

1993; Luciano et al., 1994) as well as inhibition of FSH-

induced LH receptors, inhibin secretion and aromatase

activity (Dunkel et al., 1994). In this regard, nothing

is known about the role of EGF in caprine ovary.

The present study has been undertaken to establish a

Small Ruminant Research 40 (2001) 57±62

* Corresponding author. Present address: Animal Genetics

Division, National Bureau of Animal Genetic Resources, P.B.

No. 129, Makrampur Campus, GT By-pass Road, Karnal 132001,

Haryana, India.

0921-4488/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved.

PII: S 0 9 2 1 - 4 4 8 8 ( 0 0 ) 0 0 2 1 4 - 5

role of EGF in caprine ovarian follicular growth

and maturation by evaluating the effect of the EGF

on steroidogenesis by goat ovarian granulosa cells

obtained from follicles of different sizes.

2. Materials and methods

Cell culture medium 199 (containing Earle's salts

and 30 mM hepes/l, without glutamine), fetal calf

serum (FCS), ovine follicle stimulating hormone

(FSH), human recombinant EGF, estradiol-17b, tes-

tosterone and estradiol and progesterone antisera were

obtained from Sigma Chemical Co., St. Louis, MO,

USA. Tritiated (1,2,6,7-3H) progesterone and (6,7-3H)

estradiol were obtained from Radio Chemical Center,

Amersham, England.

All other reagents used in the present study were of

the analytical grade.

2.1. Granulosa cell culture

Granulosa cells were cultured in serum free med-

ium as described by Gong et al. (1994) with some

modi®cations.

Culture medium was medium 199 supplemented

with glutamine (20 nmol/l), sodium pyruvate (10

mmol/l), sodium bicarbonate (10 mmol/l), testoster-

one (70 nmol/l) and penicillin/streptomycin solution

(containing 10 000 IU penicillin and 10 mg strepto-

mycin/ml) (10 ml/l).

The goat ovaries were obtained from the local

slaughter house, put into thermos ¯ask containing

medium 199, 1% (v/v) penicillin/streptomycin solu-

tion and 0.1% FCS and transported to the laboratory

within 2 h of animal slaughter. Ovaries were cleaned

of adhering tissue and washed repeatedly with Hank's

balanced salt solution (HBSS). Follicles were isolated

mechanically and grouped as small (<3 mm), medium

(3±6 mm) and large sized (>6 mm). The isolated

follicles were observed under microscope for growing

or atretic stages based on the transparency as

described by Nicosia et al. (1975). The granulosa cells

were isolated from these categorized growing follicles

and checked for viability by trypan blue exclusion test

as described earlier (Behl and Pandey, 1999). The

viability ranged between 30 and 40%.

The granulosa cells were plated (approximately

5� 105 viable cells per well) in 12 well disposable

culture plates containing 2 ml culture medium with

10% FCS per well. After 24 h left over medium was

aspirated. The red blood corpuscles were removed

with the medium. The cells were washed ®rst with

HBSS (Hank's balance salt solution) and then with

plain medium to remove the non-viable cells. The

granulosa cells were then incubated in culture medium

without FCS and supplemented either with EGF (0,

0.1, 1.0 and 10 ng/ml culture medium) or in combina-

tion with FSH (100 ng/ml culture medium) for another

24 h. At the end of the culture period, spent medium

was stored at ÿ208C for hormone estimation.

2.2. Hormone assay

Concentrations of estradiol and progesterone in

spent medium were measured by radioimmunoassay

as described by Arora and Pandey (1982).

2.3. Estradiol-17b

The sensitivity of the assay was 2.5 pg per tube. The

inter- and intra-assay coef®cient of variation (CV)

were 10.9 and 7.5%, respectively.

2.4. Progesterone

The sensitivity of the assay was 10 pg per tube. The

inter-assayCVwas14.3%andtheintra-assayCV11.9%.

Both estradiol and progesterone were undetectable

in medium 199 and culture medium (medium 199 with

supplements).

2.5. Statistical analysis

All the values represent the mean� S:E:M: (n � 5).

Statistical difference between treatments was ana-

lyzed by two-way analysis of variance (ANOVA)

(Snedecor and Cochran, 1981).

3. Results

3.1. Effect of EGF on estradiol-17b

EGF (0.1±10 ng/ml) treatment signi®cantly

(P < 0:05) inhibited the estradiol secretion by gran-

ulosa cells from all the three categories of follicles

(Fig. 1). The response pro®les were similar between

58 R. Behl, R.S. Pandey / Small Ruminant Research 40 (2001) 57±62

granulosa cells from small and medium sized follicles,

showing signi®cant (P < 0:05) inhibition even at

minimum dose tested for EGF of 0.1 ng/ml. The

inhibitory effects continued to increase upto the high-

est dose of EGF (10 ng/ml culture medium). The

minimum effective dose that could inhibit estradiol

secretion by granulosa cells from large follicles was

1.0 ng/ml culture medium.

As shown in Fig. 2, EGF (10 ng/ml culture medium)

signi®cantly (P < 0:05) inhibited estradiol secretion

even in the presence of FSH (100 ng/ml) by granulosa

cells from all three categories of follicles. However,

the minimum effective dose of EGF that could sig-

ni®cantly (P < 0:05) inhibit FSH stimulated estradiol

secretion by granulosa cells from small and medium

sized follicles was 1.0 ng/ml.

3.2. Effect of EGF on progesterone secretion

As shown in Fig. 3, EGF (0.1±10 ng/ml) treatment

stimulated signi®cantly (P < 0:05) the secretion of

progesterone from all three classes of follicles only at

the highest dose (10 ng/ml culture medium).

There was signi®cant interaction (P < 0:05)

between EGF and FSH (100 ng/ml) as EGF syner-

gistically stimulated FSH-induced progesterone

secretion by granulosa cells obtained from all three

categories of follicles (Fig. 4). When added in

combination of FSH (100 ng/ml), EGF was effective

in stimulating progesterone secretion signi®cantly

even at minimum dose tested (0.1 ng/ml culture

medium).

4. Discussion

To our knowledge, this is the ®rst description of the

role of EGF in caprine ovaries. This study demon-

strated that EGF inhibits estradiol secretion by cul-

tured goat granulosa cells. Results show that EGF even

at low concentrations (1.0 ng/ml) can suppress the

stimulatory in¯uence of FSH. Similar observations

Fig. 1. Effect (mean� S:E:, n � 5 cultures) of EGF on the secretion (ng/5� 105 cells/24 h) of estradiol-17b by goat granulosa cells at various

stages of maturation, cultured under serum free conditions. *P < 0:05 compared with the respective controls.

R. Behl, R.S. Pandey / Small Ruminant Research 40 (2001) 57±62 59

Fig. 2. Interactions (mean� S:E:, n � 5 cultures) between FSH (100 ng/ml culture medium) and EGF on the secretion (ng/5� 105 cells/24 h)

of estradiol-17b by goat granulosa cells at various stages of maturation, cultured under serum free conditions. *P < 0:05 compared with the

respective controls.

Fig. 3. Effect (mean� S:E:, n � 5 cultures) of EGF on the secretion (ng/5� 105 cells/24 h) of progesterone by goat granulosa cells at various

stages of maturation, cultured under serum free conditions. *P < 0:05 compared with the respective controls.

have also been made in primary cultures of rat gran-

ulosa cells (Dorrington et al., 1987). Therefore, if at

any stage of follicular development, FSH and EGF are

present in follicular ¯uid, it could be predicted that

estradiol synthesis would be suppressed, or if already

activated, estradiol synthesis would be inhibited.

As this study was conducted on granulosa cells at

different developmental stages, it was demonstrated

that inhibitory effect on estradiol synthesis was more

profound in granulosa cells from small and medium

sized follicles than in granulosa cells from large

follicles. Follicular development can be divided into

two stages: in ®rst phase, there is growth of granulosa

cells and aromatase activity is maintained at low level;

in the second stage, both growth and differentiation

occur (Garzo and Dorrington, 1984). More pro-

nounced inhibition of estradiol secretion by EGF in

granulosa cells from smaller follicles suggests that

EGF acting as autocrine and paracrine regulator may

be involved in de®ning these stages of follicular

development.

Whereas EGF inhibited the estradiol synthesis by

cultured goat granulosa cells, this was not the case

when progesterone secretion was examined. EGF

stimulated the progesterone secretion by granulosa

cells from all three categories of follicles. The stimu-

latory effect of EGF on progesterone secretion by

cultured granulosa cells was enhanced by the presence

of FSH. In the presence of FSH, EGF was effective at

concentration as low as 0.1 ng/ml culture medium in

all three categories. Recent studies proposed that EGF

regulates cellular activity of granulosa cells by stimu-

lation of progesterone production (Serta and Siebel,

1993). Tilly et al. (1992) and Luciano et al. (1994)

based on their studies on rat ovarian granulosa cells

proposed that EGF by enhancing progesterone pro-

duction by cultured granulosa cells prevents granulosa

cell apoptosis. EGF may be involved in preventing

Fig. 4. Interaction (mean� S:E:, n � 5 cultures) between FSH (100 ng/ml culture medium) and EGF on the secretion (ng/5� 105 cells/24 h)

of progesterone by goat granulosa cells at various stages of maturation, cultured under serum free conditions. *P < 0:05 compared with the

respective controls.

R. Behl, R.S. Pandey / Small Ruminant Research 40 (2001) 57±62 61

atresia by stimulating progesterone secretion in goat

ovarian follicles.

Although it is dif®cult to explain the inhibitory

effect of EGF on estradiol secretion by cultured goat

granulosa cell vis a vis enhanced progesterone pro-

duction, it is evident from these results that EGF plays

a de®nite role in the goat ovarian follicular dynamics.

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