Vol. 86, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

February 28, 1979

Pages

INTERFERENCE

OF GONADOTROPIN-RECEPTOR M.R.

INTERACTION

Sairam

and

M.I.

BY SYNTHETIC

1214-1221

ESTROGENS

Berman

Reproduction Research Laboratory Research Institute of Montreal 110 Pine Avenue West Montreal, Quebec H2W lR7, Canada

Clinical

and

Received

January

19,

the

University

of

Montreal

1979 SUMMARY

In in u&o studies, the synthetic estrogens diethylstilbestrol and diethylstilbestrol sodium phosphate inhibited the binding of 1251 ovine lutropin to the rat ovarian receptor and 1251 ovine follitropin to the bovine testicular receptor. As the lutropin binding to receptor is affected to a greater extent, a preferential inhibitory effect may be suggested. Removal of the estrogens from the incubation medium by washing does not restore gonadotropin binding ability,indicating a strong effect. The two compounds were effective in displacing the labeled gonadotropin from the preformed receptor-hormone complex. This effect increased with time of incubation. It appears unlikely that the interference of gonadotropinreceptor interaction may be because of increased hormone and/or receptor degradation by the two compounds. INTRODUCTION Like

many

appears

other to

be

at

the

receptors

for

lutropin

the

and

testis

ovary

been

carried

steroids effects we

out on

of

We have

and

X/79/041

of

the

site

the

cell

and

the

now

that

extended

interfere

studies

gonadotropins

Very

end

products

the

a recent

action

and

gonadotropin-receptor

on

specific

been

1,2).

receptor-hormone

these with

have

During lutropin

the

Accordingly,

(see of

itself.

suspect

of

(FSH)

influence

on

action

membrane.

animals

binding

to

of

follitropin

different

compounds

evidence

the

0006-291

of

(LH)

estrogenic

estrogens

Abbreviations Gonadotropin; T--Testosterone; Diethylstilbestrol

level

hormone

synthetic ovary

a principal

regarding

the

obtained

affected.

Copyright All rights

hormones

demonstrated few

studies such

study rat

on

Leydig

interaction demonstrate

in have

as

the

the

direct

cells

(3)

might here

interaction

be

that in

testis.

used: LH--1utropin; E2--Estradiol 178; A--Androstenedione; sodium phosphate

FSH--Follitropin; HCG--Human Chorionic E2B--Estradiol benzoate; P--Progesterone; DES--Diethylstilbestrol; DES-NaP-(Honvol).

214-08$01.00/O

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

1214

both

Vol.

86,

No.

4, 1979

BIOCHEMICAL

AND

MATERIALS

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

AND METHODS

A crude membrane fraction from pseudopregnant rat ovaries (4) was used as a preparation of highly specific lutropin receptor. Follitropin receptor in the form of a partially purified membrane fraction was prepared from adult bull testis (5) using a modified procedure (6). Both receptor preparations were employed either immediately or after being frozen at -700 for several months, as no difference in binding ability was perceptible between the two forms. Highly purified ovine pituitary lutropin and follitropin isolated in this laboratory and human follitropin supplied by NIH were used for iodination with carrier free 1251 (6) by the lactoperoxidase method. The labeled hormones purified on Sephadex G-100 column had a specific activity of about 50-80 @i/ug and exhibited good binding characteristics. The labeled hormones were used within two weeks of preparation. Each i ncubBinding assays were done in disposable 12 x 75 mm glass tubes. ation mixture contained 100 ~1 of the sample in the assay buffer and 400 1~1 of a suspension of the appropriate receptor plus the labeled hormone added as a single aliquot. The total volume was 0.5 ml in the assay buffer,which was 25 mM Tris-HCl, pH 7.5 containing 10 mM MgCl and 0.1% bovine serum two hours at the end albumin (Sigma). The tubes were incubated at 37 8 C for of which the reaction was terminated by addition of 2 ml cold assay buffer. The tubes were centrifuged at about 2500 x g for lo-15 mins. After aspiration of the supernatant,the bound radioactivity in the pellet was counted in a Beckman autogamma Spectrometer. Tubes designated for non-specific binding contained 1000 fold excess of the unlabeled hormone. The were tion

compounds dissolved of ethanol

to

be evaluated in ethanol. were always

for effects on Suitable controls employed.

hormone-receptor receiving

interaction, same concentra-

the

In experiments where the effect on preformed hormone-receptor complex was examined, the receptor preparation was incubated as above with the labeled hormone at 37OC for 2 hrs and washed once with buffer and then resuspended in the original volume of 0.5 ml containing either buffer, ethanol, hormone or the various compounds. They were incubated at 37OC and at specified intervals (up to 24 hrs) triplicate sets were removed, diluted with buffer and the pellet separated ag~ain as above. Radioactivity in the pellet was determined and expressed as percentage of the original binding found during the formation of the first 2 hr receptor-hormone complex. The steroids E2, E2B, St.Louis. The clinically derivative was donated supplied as an aqueous DES-Nap concentrations All

solutions

were

P,

T and A and the DES were obtained used form of DES as its sodium by Horner Laboratories, Montreal. solution was directly diluted with are shown in DES equivalents,

prepared

fresh

just

prior

to

from Sigma, phosphate This compound the assay buffer.

use,

RESULTS Effects

on

binding

Among only binding

the

synthetic of

the

of

the

lz51

different

and

estrogenic

estrogens labeled

lutropin

DES and

gonadotropins

1251

follitropin

compounds ‘DES-Nap to

1215

the

and

to other

significantly receptors

their

receptors

steroids

tested*

interfered as

seen

in

tiith Fig.

1.

.

Vol. 86, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A- I’25-LUTt?OPIN 100

1

80 60

0 N.S.

Q&30)

30-50

m

E2B (20)

10 DES

/(g

l-7

1044

40 20 DES-NaP

10

l-5

VA (30) GTOH

STEROID

B- I’2S-FOLLITROPIN

80

-

60

1

J1

0

N.S. E2[30)

-

40-10030

E2B (201 )(9

20 DES

5-W

P,T;Af30) *ETOH

STEROID

Figure 1. Effect of estrogens on 1251-lutropin (A) and lz51 human follitropin (8) binding to receptor. In each experiment about 1 mg ovarian or testicular membrane protein was incubated with 80-~OO,OOO cpm of labeled hormone (about 1 ng) and the various compounds. The tubes with a total The % volume of 0.5 ml (see methods) were incubated at 37OC for 2 hrs. bound in absence of the unlabeled hormone (zero binding) which was 20% of the added radioactivity is considered as 100%. Non specific binding in this and other experiments represent cpm found in presence of 1 1-19 of highly purified unlabeled hormone. Data in this and subsequent figures represent mean f SEM. In subsequent studies E2, E2B, P, T, A were found to be without influence at 50-100 1-19. EtOH = ethanol control. The DES-Nap also gave a dose dependent inhibition (20-400 pg) in panel B (data not shown).

DES at

approximately

the

ovarian

ding

seen

It Very

thus

0.037

receptor at appears

similar

higher that effects

by

mM reduced about

50%.

concentrations the were

degree observed

the The was

of

binding

of

maximum of

the

inhibition with

1216

lutropin

inhibition order is

DES-Nap.

lz51

of dose The

of 75-80% related inhibitory

to

specific in

bin-

2 hrs.

(Fig.

1A). effect

BIOCHEMICAL

Vol. 86, No. 4, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

50 40-

I 1

0.1 0‘ 2

56

uJ

48

I 10

I 100

ng LUTROPlN B

40 2ojlg

DES 0,

32

'.

8-

1

0.1 0.1

10 10

too too

ng FOlllTROPlN

Figure response represent laboratory.

of

2. of

DES on

that

Influence of varying DES levels A-lutropin and B-follitropin highly purified ovine lutropin

follitropin-receptor

observed

with

The evident

in

Fig.

evaluated 20

pg

with

Effects

in DES,

pellets,

dependent 2A and

ts

preincubat

incubated recovered

effect

of

(Fig.

where

the

DES on

16)

increasing

with

the by

is

was

much

less

than

systems

is

more

clearly

the

the two

slope

and

respective

compounds

centrifugation

assay

for were

1217

of

virtually

estrogens

experiments,

two

concentrations

receptor,

-iI on with

the

radioreceptor

n dose-response

and

these

2B, of

lutrop

follitropin

of

interactions

assay doseSl555BR in this

lutropin.

presence the

In first

dose

on the radioreceptor (human). Sl322AR and and foflitropin isolated

responses

DES.

In

were presence

obliterated, is

reduced

washing

2 hrs washed

whereas

progressively.

on

receptor

gonadotropin preparations

at thrice

of

37OC. with

binding were

The the

membrane assay

Vol. 86, No. 4, 1979

buffer

and

then

follitropin

ug

NaP

not

easily

soluble

effectively of

on Both

3.

at

37’C

With

over

24 (Fig.

unlabeled

lutropin.

of

of the

loss

more

the

stable

led

Fig.

of

the

the

38).

It

estrogens

were

the

was

bound

same DES-

compound

suggest

an

would

effective

membranes.

the

of

and

at to

end

its

be

in

hrs,

was

remains

the

by

unlabeled

that,

again

case

of

the

to

relatively the

after

bound 24

hrs.

follitropin, higher the

the

a fraction

contrast

of

bound

any

enhanced

only In

dissociation

noted

necessary

24

of of

also

complex

still

may

of

absence

presence

receptor.

significant

displaced

the

in

incubation

in

estrogens

follitropin

was

described

hormone by

the

preformed

complex,

increased synthetic

the

experiments

bound

the

amount

estrogens.

by

of

testis

hormone

the

Since

dissociation

shown

bound

Although

bound

the

loss

hormone

a substantial

loosely

receptor-hormone

of

bull

50 1-19 of

procedure.

the

with

complex

as

remained the

(Fig.

on

1251-

receptor

with

results

sites

release

bound

lutropin

3A,

synthetic

to

gonado-

subsequent

lutropin

the

These

lutropin

This

the

occurs,

in

tion

hrs

receptor,

hormone As

ovarian

of

washing

enhanced

Inclusion

labeled

lutropin

the

3A).

of

the

buffers,

complexes

additives

rate

by

DES-Nap

or

the

gonadotropin-receptor

DES and

1251-lutropin

receptor

binding

gonadotropin-receptor figure

of

washing.

gonadotropin

the

inhibition

follitropin

aqueous

by

preformed

The

reversed

in

removed

alteration

the

be

of

preincubation

or

DES-Nap, could

Effects

by

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

presence

2 hrs.~

caused

DES or

is

in

another

binding,

compounds,

be

reincubated

for

tropin 20

BIOCHEMICAL

or

concentra-

follitropin

receptor.

DISCUSSION These

studies

synthetic and

estrogens

follitropin

indicated with

demonstrate can with elsewhere

lutropin

that

binding

structurally

directly

the (3) to

affect

ovarian the the

unrelated

and

the

rat

specific

testicular

estrogenic

1218

such

interaction receptors

compounds

testicular

hormones,

receptor.

also

of

as lutropin

respectively. effectively It

may

the

As interfere

be

noted

that

BIOCHEMICAL

Vol. 86, No. 4, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

o--o DES OJg) b--h

80

DES -NaP($g

)

60 40 2 s

NONE/ETOH

20 N.S.

z

I

I 8

I 4

I 12

I 16

I 20

I 24

HOURS Figure 3. Effect of the synthetic estrogens on the preformed hormoneIncubations were A-lutropin, B-ovine follitropin. receptor complex. After the formation of the initial gonadotropindone as shown in figure 1. the pellet was washed once with the assay buffer receptor complex (2 hrs) volume (0.5 ml) with the indicated and reincubated at 370C in the original At specified intervals triplicates were removed and the reaction substances. centrifuged and radioactivity stopped by addition of 2 ml cold buffer, All values are represented as % of the original in the pellet determined. binding found during the first 2 hr incubation which was 40% and 42% For reasons of clarity the SEM of the added CPM in A and B respectively. bars are omitted as triplicates varied less than 3%. EtOH = ethanol. NS = non specific binding.

the

concentrations

the

ID50

0.037 It

for

mM and has

been

inhibition 1 nM noted

(7)

for of

for

recently

indicating

the

that

in

significantly the

inhibition

125 I lutropin

respectively

can

concentrations, receptors

required

binding

DES and

ovine

rather to

1251-hCG

sensitivity

tissue.

1219

the

lutropin

hypophysectomized reduce

greater

are

rats, binding of

the

For

high. ovarian

receptor

(Figs.

1 and

DES at to

instance,

the

lower testicular

hormone-deprived

is 2).

BIOCHEMICAL

Vol. 86, No. 4, 1979

In

all

testicular

the

receptor

increase

the in

labeled

hCG

(cited

of

this

hormone

on

lutropin these

binding

ability

dislodging

by not

or

DES-Nap

It

is

aware

unlikely and/or

be

as

without

follitropin

at

equivalent

noted

that

50 ug

to con-

preincubation

prevented

rat,

the

DES

subsequent treatment

significantly

&

ViVO

altering

the that the

fail

to

and

the

cells

produce

in

frequently

thus

of

apparently

effect

the

been

number

in of

present

studies

bind

the

the

utilized

follitropin

cannot

progesterone

preferential noted

v-LAXO

DES,has

Such

8,~).

of

action

the

of

DES or induce

the

gonadotropin

showed

that

studies

presence

DES and might

may

estrogens

bind

to

DES-Nap

explain

synthetic

from

This

the

estrogens

the

preformed

membrane

which

have

estrogens

shown

a direct

or

peptide,protein

could

be enhancing

resulting supposition

with

in

decreased

is

supported in complex

the

estrogens

gonadotropin binding

binding. of hormone

the

com-

effective

hormone-receptor

subsequent

receptor

unknown.

(receptor)

were

preparations

blocked

remains

membrane

changes

receptors. the

gonadotropin’ the

synthetic

conformational

hormone of

the

DES-Nap

effectively of

estrogen

observations.

labeled

washing

to

may

male

cells

direct

Preincubation

followed

hormone

which the

3).

We are

and

The

viva

of

the

(see

subsequently

data

of

degree

such

DES,

granulosa

10)

in

that

and

(Fig.

in (11).

possible

of

it

receptor

of

ovary

mechanism

ponents

the

of

the

in

The

lower

hypophysectomized

action

receptor

of

a much

binding

(7).

number

sites

dose

the

the

However,

lutropin

mitogenic

binding

by

In

here,

to

3).

a large

binding

is

affected

affects

The

shown

2 and

binding.

follitropin

It

1, with

preferentially

some

was

(Figs.

follitropin

to

experiments

receptor

centrations of

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

degradation

labeled

DES

receptors. of

the

receptor. ACKNOWLEDGMENTS

This investigation was supported (Honvol) was generously supplied FSH (NIH-HS-1) was supplied by Bethesda.

by by the

grants Horner Hormone

1220

from the Laboratories, Distribution

MRC

of Canada. Montreal. Officer at

DES-Nap The human NIAMDD,

Vol. 86, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES 1.

Ryan,

R.J.

and

Lee,

2.

Catt,

K.J.

and

Dufau,

3.

Sairam,

4.

Lee, 228-233.

5.

Cheng,

6.

Sairam,

7.

Hsueh, 103,

M.R. C.Y.

and

and

C.Y. M.L.

Berman,

Ryan,

K.W.

A.J.W.,

(1976)

Clin.

M.L.,

16-29. 14,

(Submitted

J.

Clin.

l-15. for

Endocrinol.

Endocrinol. in

14,

Reprod.

Steroids

Endocrinol., Dufau,

Reprod. Biol.

(1975)

J.

J.

Biol.

M.I.

R.J.

(1575)

M.R.

(1976)

Metab.

publication). Metab.

41,

40,

581-589.

press.

and

Catt,

K.J.

(1878)

Endocrinology

1

log6-1102.

8.

Goldenberg, Endocrinology

9.

Louvet,

10.

Hillier, 100,

11.

Lucky, Ross,

R.K., 90, J.P.

and

S.G., 1539-1549. A.W., G.T.

Vaitukaitis, 1492-1498.

J.L.,

Vaitukaitis, Knazek,

Schreiber, (1977)

and

J.L. R.A.,

and

J.R., Endocrinology

(1976)

G.T.

Endocrinology

Ross,

G.T.

100,

S.G., 128-133.

Hillier,

1221

Ross,

(1977)

Schulman,

(1972).

99:

758-764.

Endocrinology

J.D.,

and