Regulation of LHHCG receptor by gonadotropins in rat ovary

Vol.

172,

No.

October

2, 1990

30,

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1990

Pages

REGULATION

OF

LH/HCG

RECEPTOR

IN

Kazuto

Nakamura,

Kaoru

Mi yamotoXXO

of School

mx Nati

of

onal Fujishi

Received

September

GONADOTROPINS

Mi negishi*,

Yoshi

hisa

Obstetrics Medicine,

Yumi

Hasegawa,

Masao

Gunma

Center, ta, Osaka

Sui

Takakura

Yoshi

to

I buk

Gunma 371,

University, Japan

lgarashi

and Gynecology, Maebashi,

Cardiovascular rodai,

6,

BY

OVARY

Takashi

and

Department

RAT

786-792

Research 565,

lnsti

tute,

Japan

1950

To i nvesti gate the regulation of the LH/hCG receptor gene by gonadotropi ns, we examined the effect of PMSG and hCG on the expressi on of LH/hCG receptor i n immature rat ovary. Northern blot analysis of ovar i an RNA revealed a major mRNA of 5400 nucl eoti des and mi nor speci es of 7500.3600,2300 and 1200 nucleotides, and PMSG treatment slightly increased the intensity of all LH/hCG receptor messengers. hCG treatment deSubsequently. creased the number of LH/hCG receptor by day 2 and mRNA I evels by 12h after injection. The level o# mRNA recovered and increased 5-fold of control by day 6. then returned to control levels by day IO, followed by slower decline in LH/hCG receptor in plasma membrane. These studies demonstrate that the effects of PMSG and hCG on the number are closely related to the of LH/hCG receptor actions of these hormones on LH/hCG receptor messenger levels. @1990 Academic PESS, 1nc

Previous

studies

receptor

i n

been

used

have rat

for

pseudopregnant

the

receptor

rats

seem

were

used

library

for

cloning

clarify

the

effect

To

whom

0006-291X/90 Copyright All rights

to

correspondence

determine

of

si

be

the

richest

in

one

of

hormone

of

treatment

purification, to

glycoprotein

of

PMSG-hCG ovary(l,2),

Moreover.

cloning

X

that

pseudopregnant

tors(3,4,5.6).

model

showed

ami

rat

LH/hCG

rice

the

source the

fi

rst the

acid

sequences

ns

be

$1.50

0 1990 by Academic Press, Inc. of reproduction in any form reserved.

this

receptor(7).

gonadotropi

should

and

receptor, no

786

induced

on

addressed

the

LH/hCG

model

of

ovari

has

ovari

es

the

recep-

success

of es

and Thus

LH

of make

we receptor

aimed

of

the this cDNA to mRNA

Vol.

172,

and

No.

protein.

Ovarian

cellular

Treatment

with

alternation

including

LH/hCG

secondary corpus effect both

the

increase

in

hCG

in

from

the

FSH

induces

on

acute

long

term

and by

messenger

aspects

of

mature

Ovulatory

study

receptor

of

morphological

receptor

present

model

the

and

number

The

and

to

LH/hCG

receptor

LH/hCG

a

formation,

of

LH/hCG

COMMUNICATIONS

provides

biochemical

number

the

RESEARCH

undifferentiated

receptor

formation.

of

BIOPHYSICAL

tissue

LH/hCG

decrease

lutea

AND

follicular

development

state.

of

BIOCHEMICAL

2, 1990

doses induced

the

luteinization

and

investigates

the

and

protein

levels

hormone-dependent

ovarian

differentiation

Materials

and

Immature and 20 selected killed

Methods

female rats of Wistar strain of hCG sixty hours later. intervals following hormone by cervical dislocation and liquid nitrogen immediately in receptor binding and Northern

frozen LH/hCG were

minced

line(PBS) for 15 fuged

and using at 30

min for

pended concentrations

in

homogenized glass

600x9 min

PBS

Incubation was carried

of

hCG(Pregnyl) mixture

500 tube, The

~1

amounts

using about

the 30

tion was primers cDNA(7). 2.5 min

amplification, same

a

method(l5),

(1.5-5 by

reverse

performed based PCR at 60

hCG

kb)

and randomly

transcription

using on published reaction "C, and 2

of this

were min

a PCR product cDNA sequences from ovarian total RNA, several clones from and obtained Lambda-Zap sequence kit(TAKARA)

primed total

template

at

30

'C with (600 bp) was a Lambda-Zap using oligo(dT) this

library LHRE. the

787

by

This dideoxy

receptor of

BSA for suction,

50

was 20

an automatic was performed a constant was

specific rat RNA.

After added min and

at the

gamma with quantiiodinated

cDNA PCR amplificaoligonucleotide LH/hCG I.5

polymerase. to as

and clone

was

ovary

two

Tag used library

ICI The

activity

of rat cycles;

70

using

0.1%

with

sequence run for

of )I

100

hormone

the

hCG

pl

binding, solution. for 12h.

in receptor added to hCG (CR-119)

Purified T

radioiodinated

containing centrifuged removed

radioactivity LH/hCG

non-labeled

Bradford(l4).

solution,

PBS were was

fluid

of fraction.

chloramine yCi/pg,

A size-selected prepared

at

of

Protein of 100

non-specific of standard room temperature

ice-cold mixture for

were

with

hormone

centrifuged and centrithen sus-

decanted

system:

estimate

sawas

preparations(3). the method

by

standard

counted analysis

membrane

receptor preparation

to instead

)I of and the supernatant

phosphate-buffered homogenate was precipitates

supernatant The

following

of

order used incubated

was Scatchard

increasing ty of

receptor the

ice-cold The

used as determined in

was then

was

incubation, to each 3oooxg. precipitate counter.

at

the out 100 1 n

in

homogenizer. and the lO,OOOxg.

and were

'; i %;',"",i,",",'

lapping the isolated clones with

were primed 30 IU of PMSG Animals were sacrificed at treatment. The rats were the ovaries were removed and for subsequent analysis of blot analysis The ovaries

IU

a

min isolate prepared primer,

was

receptor at 95

"C,

After overfrom

sequenced these was sequenced chain termination

We

Vol.

172,

No.

method(l6) LH/hCG ovaries

2, 1990

BIOCHEMICAL

and

found cDNA guanidine-thiocyanate

receptor by

electrophoresis ferred

to

to contain C-8-2560).

through membrane

nylon

described(l8)

to

AND

a

BIOPHYSICAL

the Total

1%

entire RNA method

agarose

film

discrete Ladder(BRL). tometr

at

i c

ol

scanni

Di

ogi

immature

cal

method

obtain

growth

di

fferenti

vo(8,9,

et.al. ture tor

PMSG

11)

and

tor

and

and

Lti/hCG

increase

of of of

from

and

RNA densi

-

the

may

explain, receptor

PMSG

priming,

to

study

to

immature

rats for

i n mRNA

part

of the

levels

788

I

LH/hCG

in

i mmarecep-

LH/hCG

day

-1 -I

recep(data to

which

strain) These

days(221, by LH/hCG

two

0 after

(Wistar

PMSG

is

i n extent studi

es

not i n

receptor

different the

not day

differences

the in

Is

in

the

induced

Therefore,

cated

P.S.LaPoit

in

3

induction

i ndi ccl

day

previous

marked

ar

LH/hCG

of

levels

of

growth

I I cul

respectively.

PMSG period

I i cle

i on

demon-

i ments

in

unchange

strain.

fol

the IU)

fol

induces

in

increase

stayed

have

mRNA

increase

the

studies

recently

LH/hCG

of

injected

granulosa

very

of

was

i nduct

fold

priming

hCG

rat

cover

I2

for

the

hCG

in

priming

(50

to

Wistar

LH/hCG

an by

for

exper

in

with

that

rat

of

th

tated

and

previous

significant

content

linked rat

i n SDS at to Kodak

estimates wI

used

the

after

injection

obligatorily

ze

quanti

ovary

receptors

4

consistent

suggest

as that

exposed

i son

was

including

rise

increase

receptor

results

no

slight

PMSG

trans-

0.1% Si

and

of

hr

mRNA

are

single

52

detected and

pH7.0). and

“C

the the by

(Amersham). successively

prevrous

Mor

is

n

ch

tro(l0).

that

results

strain

vi

rats,

shown)

the

i n

female

we

65

compar were

PMSG

LH

rapid

protei

Our

induces

very

24

[a3’PldCTP washed

PMSG

cle-ri

number

showed

I

LH.

on,

(20)

However

a

or

mRNA

for Ii

ati The

FSH

bands

with

receptors(l,Z).

vi

fol

treatment

and

by

Moreover,

that

The RNA was and hybridized cDNA probes

screen

i shed c

of from

on

luteinization(l).

strated

that

ographi

assay to

th were citrate. at

intensifying establ

scussi

rat its

with

were Autoradi ng

and bi

for

“C

RNA

Results

In

-70

wi

COMMUNICATIONS

region extracted fractronated

and

Amersham) p-actin

and

were I abeled by nick translation Following hybridization the filters 6x SSC (0.15M NaCl,O.O15M sodium room temperature and O.lxSSC.O.I%SDS XAR

coding was

gel(l7)

( Hybond-N; receptor

LH/hCG

RESEARCH

the of

Vol.

172,

As

No.

shown

LH/hCG

BIOCHEMICAL

2. 1990

Fig

in

receptor day

at

8.

While

experiments

day

saturation

with shown major

Fig

ed(21),

it

the

may

depend

2

istration

caused hr

and

days

remained

after a

of

between

individual

induction

IlhCG

no

of

of

IO-fold

typically

and was

of

increase

maximum

plateaued,

blot

5400

then

apparent

within

declined. change

each

in

groups,

and

from

the

in

intensity

at

RNA of

all

24

hr

receptor (data

molecular

sizes

bands

shown).

reportmarker

messenger not

of

previously

However,

LH/hCG

re-

species

minor

five

administration,

RNA

molecular

those

different

in

ovarian

Although

different

decline

of

nucleotides

PMSG

low

analysis

nucleotides.

on The

gradually

I2

of

are

estimation.

cl25

northern

mRNA

receptor

a

number

reports(l9).

1200

LH/hCG

IO,

peak

there for

2,

7500,3600,2300,and

to

the

studies

previous in

a

for

day

constant

agreement

vealed

and

binding

dissociation

As

8

by

variation

6,

COMMUNICATIONS

the

followed

2,

some

day

RESEARCH

treatment,

increased

was at

hCG

day

and

there

between

the

4,

especially

appeared From

at

day

BIOPHYSICAL

the

decreased

levels

at

following

1,

receptor

AND

for

increased hCG

adminlevel

by Thus.

Fig.1 The effect of PMSG and hCG treatment on the LH/hCG receptor levels. At the indicated time ovaries were harvested for the preparation of plasma membrane and receptor binding was performed with '25i-hCG as described under "Methods". The receptor level each increase(meaniSE) at time point is expressed as the fold Over the day 0 (control). The average receptor concentration before the hCG treatment was 240 f 42 (mean ;t S.E,n=4) f mole/mg of membrane protein.

789

Vol.

172,

No.

2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

4

10

9.57 5,*

4.4--c

a*

C

2 4-

zE 6 b2 45 $E PC +J32 ;z rni, 3

14-

B.

(110 -I

0 2 4

6 8 IO DAY

-1

0

2

6

8

DAY

Fig. 2 The effect of PMSG and hCG treatment on LH/hCG receptor and B-actin mRNA levels in immature rat ovary. From each time poi nt 15 fig of total RNA was prepared and fractionated through a 1% agarose gel and blotted as descri bad under “Methods”, Blots were probed wi th 32P-labeled (A) LH/hCG receptor cDNA or (B) p-actin cDNA. The fi I ters ware exposed to Kodak XRP fi Im. CC), autoradi ographs were quanti tated by densi tometri c scanning. and the increase in LH/hCG mRNA (5400 band) relative to p-acti n IS expressed as arbitrary units. The results shown are representative of 4 experi ments.

administration and

of

sustai

ned

receptor

low

to

agree

we

I

response

to

fol

rates contribute LH

mul

0.

ati

This of

down

2

rapidly

sti

the

this

on

was

0

recovered

to

day

mRNA 10.

days

regulation

in to

day

then

slowly

6,

LH/hCG

decreasec adat

i on

I eve1

of of

hCG

treatment.

that

is

decayed the

ant

Subse-

intensity

found

by

receptor. the

in

expected

degr

after

Throughout

790

down

due

to The

6

usually

pseudopregnant

decline

own

been loss

groups

be

of

data

has

the

messenger

I ater. 4

in by

could

regulation

at

in

several

hCG

days

These

1’2hr LH/hCG

receptor

The

levels

increase day

of

i ncreased

mRNA at

increased

maximal

the

receptor

treatment

of

cause

lowing

hr. It

and

elucidated.

and/or

to

quently,

be

of

48

tizatron

correlation

by

number

etal.(20).

occupancy(ll,l2).

I evels

transcription

I evel

to

within

LaPolt

the

than

mRNA

bands

be

the

0)

desensi

to

remained

receptor

day

appear

other

hCG

of

Although

processes

to

on

decreased

of

levels

messenger

(day

report

i nducti

were

and

one control

the

hCG

receptor

day the

ovary(l2).

LH/hCG

may

of

with

that

decreased

at

20%

reported

rat

hCG

al

I

five ant

5 back

experi

prior

fold

ot to

menta:

th

Vol.

172,

No.

period

there

Thus

was

the

IO

equivalent

fold

returned

the

other

hand,

and

maintained receptor

followed

by in

a

treatment.

In

our

rise

process

tively

time

receptor.

and

on result

of

receptor

a

recep-

of

levels

increased

the

prior discrepancy

to

hCG

is

not

between

there

two

in in

to

a

receptor

be of

of

LH/hCG

direct

and

well

messenger

levels~

Thus, may in

also

expected

the

were

change

treatment

regulation variety

ovaries

stable

that

ap-

Although

appeared.

more

marked the

membrane.

luteinized LH

a

preceded

regulation

LH/hCG can

was

which

the

gradually

lead the

up

was

levels

difference

transcriptional

it

the

the

days

LH/hCG

observed

for

in

changes

of

that

IO

of

communication

mRNA

mRNA,

levels

production.

LH/hCG

to

return

receptors

luteinization,

change

Since

steroids

due

receptor

of

type

long

reason

after

LH/hCG

these

this

the

the

of

LH/hCG

of

plateau

after

receptor

On

10.

above.

similar

of

of

be

level

with

that

The

30%

day

reached

number

of

LH/hCG

receptor

the

associated

nisms

change

described

of

the

levels.

maintained

by

values of

not

were

control

to

mRNA

were

showed

levels.

attributed

control

data(21,22).

even

high

of

the

as

mRNA

data

only

be

receptor

the

decline

mRNA

can

levels

to

partly

rat

of

changes

These

regard.

may

levels

pearance

days.

reach

this

studies,

in

receptor

levels

to

In

of

of

previous

later

strains

levels

reported

days

but

the

The

with

clear,

receptor

slower

et.al.(20)

three

LH/hCG

COMMUNICATIONS

p-actin

LH/hCG

of

membrane.

well

LaPolt

more

in

state

LH/hCG

mRNA

the

agree

the 6-10

LH/hCG

tor

to

RESEARCH

receptors

steady

levels

they

of

in

BIOPHYSICAL

change

increase

elevated

and

AND

appreciable

no

increase

These

the

BIOCHEMICAL

2, 1990

increase the

receptor indirect

mecha-

take

rela-

the

LH/hCG ovarian

net in

the

vivo

effect

of

will

influences.

Acknowledgments The the

authors

NIAMDD

wish Hormone

to

express

their

Distribution

gratitude Program,

791

to and

National

Dr.A.F.

Parlow, Pituitary

be

Vol.

172,

Agency

No.

I

This

work

for

Science

2, 1990

for

BIOCHEMICAL

of

SUPPLY

was

BIOPHYSICAL

radioimmunoassay

supported and

AND

by

Culture

a

kit grant

of

RESEARCH

from

for the

COMMUNICATIONS

rat

gonadotropins

Ministry

Japan(02670732)

of

and

Education

Uehara

Memorial

Foundation

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