Vol. 91, No. 1, 1979 November

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

14, 1979

Pages 284-288

STER0ID HORMONES MODULATE PROLACTIN BINDING BY CULTURED PORCINE GRANUL0SA CELLS James M. Hannnond and Elizabeth

Krall

Department of Medicine, Division of Endocrinology The Milton S. Hershey Medical Center The Pennsylvania State University Hershey, Pennsylvania 17033 Received

September

19,

1979

SUMMARY: We have studied the effects of the gonadal steroids - testosterone, 17&estradiol, progesterone, and Sol-dihydrotestosterone on the prolactinbinding activity of porcine granulosa cells maintained in monolayer culture. Testosterone, estradiol, and progesterone all significantly enhanced prolactin binding (55%, 107%, and 112% above control, respectively). In contrast, the non-aromatizable androgen, 5a-dihydrotestosterone, caused an insignificant suppression of prolactin binding. The anti-androgen, cyproterone acetate, did not influence prolactin binding when used alone, and did not inhibit the effects of testosterone. These data suggest that the stimulatory effects of testosterone may require aromatization to estradiol. INTRODUCTION Previous

studies

from

("receptors")

sites

and corpora ical

lutea

for

in vivo,

during

the

reproductive

have

examined activity

binding

to porcine

MATERIALS

since

binding

the

cycle role

by assessing granulosa

have

on freshly

This

a potential

receptor

laboratory

prolactin

(1,2).

control

this

isolated

activity

number

appears

(1,2).

for

ovarian

steroids

the

effect

of these

cells

porcine

of receptors/cell

and pregnancy

maintained

specific granulosa

to be under changes

In the current as regulators hormones

in monolayer

binding cells

physiolog-

substantially study,

we

of prolactin

on prolactin culture.

AND METHODS

Materials: Ovine the Hormone Distribution Schering AG, Berlin. Corp. Tissue culture bovine serum albumin, Schering Corporation; Na[l251] was purchased sterile solution from

prolactin (oPRL) (NIH-Sll and NIH-S12) was provided by Office, NIAMDD. Cyproterone acetate was a gift of All other steroids were purchased from Sigma Chemical medium and fetal calf serum were from Flow Laboratories: fraction V, from Miles Laboratories; gentamicin from the amphotericin B and mycostatin from Squibb. Carrier free from New England Nuclear. Lidocaine was obtained in Astra Pharmaceuticals.

Culture Techniques: Porcine granulosa cells (l-3 mm) porcine follicles as previously described cells were inoculated into 100 mm culture dishes 199 with Hanks salts, a bicarbonate buffer system, gentamicin (SOpg/ml), amphotericin B (2.5 us/ml), 0006-291X/79/210284-05$01.00/0 Copyright All rights

demonstrated

@ 1979 by Academic Press, Inc. in any form reserved.

of reproducti&

284

were isolated from small (1). Approximately lo7 (Falcon) containing Medium 10% fetal calf serum, and mycostatin (50 U/ml).

Vol. 91, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMIJNICATIONS

The cultures were maintained in monolayer for Co2 atmosphere. The medium was changed every added in absolute ethanol (0.5% v/v); control volume of ethanol.

8 days at 37 C in a 95% air-5% 48 hr. Steroid hormones were cultures received an equivalent

oPRL Binding Assay: The binding of oPRL to cultured qranulosa cells was assessed by the methods previously used with freshly isolated qranulosa cells (1) with a few exceptions. Initial studies showed that cells removed from culture plates by a 20-30 min incubation with the local anesthetic lidocaine binding characteristics which were superior to those (3.4 ti) (3), exhibited of cells assayed directly on culture plates or dispersed with trypsin, EDTA, or mechanical scraping. After dispersion with lidocaine, cells from 20-30 control and hormone-treated cultures were pooled, washed twice in medium 199, hepes buffer, pH 7.6, 1% bovine serum albumin, and aliquoted into plastic assay tubes containing the same medium and [ 1251]iodo-oPRL (50,000 cpm or 0.6 nq) in the presence or absence of saturating concentrations of oPPL (2 uq/ml). Preparation and validation of tracer hormone, incubation conditions and computation of specific binding have been described in detail (1). Four to six measurements of specific binding expressed per mq DNA (4) were made for control and hormone-treated cells in each experiment, and the significance of differences computed using Student's t test. The overall significance of a treatment effect was assessed using a chi-squared statistic derived from significance values for a series of individual experiments (5). For comparison, data from different experiments has been normalized as percent control (Table I). RESULTS Data from effect

all

of testosterone

qranulosa

cells

estradiol

appears

testosterone showed are

the experiments

and 17$-estradiol

were

observed

question,

differences

of aromatizing

or after

Sa-dihydrotestosterone.

the

a modest,

anti-androqen,

activity these

equivalent

quantitatively

(6),

the

with

that

exerts

to cultured Although

the

Since

qranulosa

with

testosterone

of binding. Further,

prolactin

testosterone. in this

this

androqen,

was suggested.

effects

could

To address

non-aromatizable

to alter

cells

effect

to show stimulation

failed

than

two hormones

testosterone

effect

17S-

binding

to estradiol. the

failed

acetate,

aromatization also

shown).

or in combination

suggest

after

not

inhibitory

cyproterone

experiments

Progesterone

steroid

insignificant,

when used alone

indirectly,

is

This

binding

comparing

conversion

two experiments

The stimulatory

of prolactin

directly (data

I.

experiments.

stimulator

testosterone

directly

we performed

Instead,

each of several

two experiments

no significant

have been exerted

in

in Table

on prolactin

to be a more potent

overall,

capable

are compiled

binding Collectively,

system

are

exerted

binding

which

to estradiol. a stimulatory to

that

encountered

285

effect

on prolactin with

testosterone

or

estradiol.

Vol. 91, No. 1, 1979

BIOCHEMICAL

TABLE I.

Effect of Steroid Treatment by Cultured Granulosa Cells Specific

Testosterone (500 nq/ml) Estradiol (500 ns/ml) Dihydrotestosterone (500 ns/d) Cyproterone acetate (5 vs/ml) Progesterone

on Prolactin

PRL binding f SEM)

(% control

(500

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Binding

No. of Experiments

*p (treatment vs. control)

155 f 42

10

c.01

207 _+ 87

3

e.01

61 * 27

2

>.5

103 + 64

4

>.9

212 f 73

6

c.01

108 f 73

4

>.9

nq/ml)

Testosterone CypZerone + Testosterone *

acetate

see Materials

and Methods

DISCUSSION Previous receptor tissue

studies

from

this

of

freshly

concentration varies

with

specific

prolactin

declines

with

corpora

lutea

binding

follicular

maturation

receptor

concentration

of luteal

hormonal

control

of prolactin

We have found

dihydrotestosterone, milieu

of granulosa (10)

of putative

In particular, of prolactin knowledge, the modulation

cells

estradiol, receptors the

tissue

high

current of ovarian

in rat studies

in

for

have been

prolactin

FSH, has been cells the

receptors

286

functioning

fluid,

seemed logical

or luteal

--in vivo first

testosterone,

(81,

(13).

--in vitro

by steroid

physiological

androgens

(9),

cells,

and a

to each

implicated

follicles,

suggested

the

assigned

of

in the prolactin-

steroids

estrogens

functions

constitute

in

of 178-estradiol,

in granulosa

granulosa

immature

observations

follicular

and luteal

from

increase

These

described

with

cells The number

again

and ovarian

Receptors

along

ovary.

a further (2).

the prolactin

granulosa

in cells

concentrations

(7).

regulatory

high

occasions

receptors,

have been

of the

and increases

and progesterone

and progestins number

is

shown that

porcine

state

sites/cell

Pregnancy

have

isolated

the physiological

(1,2).

candidates.

laboratory

(11,12).

in the control However,

to our

demonstration

hormones.

of

Vol. 91, No. 1, 1979

Such direct which

--in vitro

regulate

of hepatic

however,

receptors

induces

is

pertinent

it

themselves the

binding

after

explain

sane of the

for

(16),

estrogens

(17).

binding

Using

monolayer

binding

activity,

synergistically (veldhuis changes

suggests

Granulosa

have been

to those

and Hammond, unpublished). concentrations

thecal

exerts

These described

invoked

ovary

a direct

to

--in vivo

effect

to study

prolactin

interact

by porcine

here

prolactin

to synthesize

here

observations

Results

in 17ci-hydroxy-

and prolactin

secretion

alter

to be accounted

elements

employed

estradiol

progesterone

rat

are deficient

probably

cultures

to stimulate

in receptor

from

cell.

that

in the

are unlikely

by the granulosa

we have noted

administered

on the

of

cells,

pathways

cells

progesterone

similar

cultured

increases

testosterone

induction secretion

testosterone

Similar

derived

of the

steroids

of progesterone

androgens

estrogen

As discussed that

of exogenous

Consequently,

on prolactin

the

interactions

the

Even with

metabolites.

effects

hormonal

stimulation

(14).

whether

to estrogens.

and require

involves

to estradiol.

actions

complex

For example,

own receptor

evidence

the

by conversion

--in vivo

active

conversion

avoid

--in vivo.

to inquire

available

In contrast,

lase

its

or via

section,

(15).

binding

which

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

observations

prolactin

lactogen

prolactin

binding

BIOCHEMICAL

granulosa

suggest

that

cells the

may have physiological

relevance. ACKNOWLEDGEMENTS The authors appreciate the provision of ovine prolactin by the Hormone Distribution Program, NIAMDD, and of cyproterone acetate by Scheriny, AG. We also thank Dr. Marshall Jones, Department of Behavioral Science, The Milton S. Hershey Medical Center, for valuable statistical advice, Dr. Johannes Veldhuis for reviewing the manuscript, and Mrs. Marlene Thompson for secretarial assistance. This

work

was supported

by NIH Grant

No.HDlOl22.

REFERENCES 1.

Rolland,

R.,

2.

ROllaId, R., 1083-1091.

3.

Rabinovitch,

and Hammond, J.M. GUnSalUS,

M.,

G.,

(1975)

Endocrin.

and Hammond, J.M.

and DeStefano,

M.J.

287

(1975)

Res. (1976)

--In Vitro

Commun. 2, 281-298. Endocrinology 11,

379-381.

98,

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K.

BIOCHEMICAL

(1956)

4.

Burton,

5.

Winer, B.J. (1971) Statistical Principles pp. 49-50, McGraw-Hill, New York.

6.

Anderson, L.D., Schaerf, F.W., and Channing, C.P. and Corpus Luteum Function, ed. C.P. Channing, J. pp. 187-197, Plenum Press, New York.

7.

Veldhuis, press.

J.D.,

Demers,

8.

Richards,

J.S.

(1975)

9.

Schreiber, 597.

J.,

10.

Schrieber,

J.R.

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Schomberg, D.W. (1979) in Ovarian Follicular eds. Channing, C-P., Marsh, J., and Sadler, Press, New York.

12.

Randell,

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Richards,

14.

Kelly, P.A., 1408-1415.

15.

Gibori, 162-169.

16.

Bjersing, 639-640.

17.

Armstrong, D.T., Gaff, A.K., and Dorrington, J.H. Follicular Development and Function, ed. Midgely, pp. 169-181, Raven Press, New York.

Reid,

P.

Posner,

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315-322.

R.,

and Ross, Program

Biol.

P.L.,

G.T.

10,

J.J.

2nd.

ed.,

in Ovarian Follicular and W.A. Sadler, Endocrinology,

Endocrinology

Endocrine

in

Society,

99,

590-

Abstract

#694.

and Corpus Luteum Function, W.A., pp. 155-168, Plenum

199-215. (1976)

and Richards, H.

(1979) Marsh, (1979)

(1976)

and Friesen,

and Carstensen,

J.M.

Design,

8, 1174-1184.

of the

Reprod.

B.I.,

in Experimental

and Hammond,

Endocrinology

and Williams,

Keyes, L.,

J.

L.M.,

(1979)

(1974)

J.S.,

G.,

Biochem.

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Biochim.

99, 1571-1581. Endocrinology

Endocrinology Biophys.

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97, 103, 86,

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Steroid hormones modulate prolactin binding by cultured porcine granulosa cells.

Vol. 91, No. 1, 1979 November BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 14, 1979 Pages 284-288 STER0ID HORMONES MODULATE PROLACTIN BIN...
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