Kinase Activity and Protein Synthesis in Testis

BIOLOGY

OF

REPRODUCTION

14,

Follicle

Kinase ANTHONY

54-63

(1976)

Stimulating

Activity

R. MEANS,

Hormone

Regulation

and Protein

Synthesis

JOHN

L. FAKUNDING

of Protein

in Testis

and

J. TINDALL

DONALD

Department of Cell Biology, Baylor College of Medicine, Houston, Texas ABSTRACT Incubation of testis tubules containing predominantly Sertoli cells (SCE) with Follicle Stimulating Hormone results in a rapid activation of cyclic AMP-dependent protein kinase. Multiple forms of soluble protein kinase were shown to exist in Sertoli cell cytosol by DEAE chromatography. Evidence suggests thst only one of the two cyclic AMP-dependent forms is activated by FSH. In addition FSH stimulates testicular protein synthesis and induces the accumulation of a specific Sertoli cell protein, ABP. Kinetic studies reveal this induction to be selective. Moreover, stimulation of ABP can also be accomplished by injection of a derivative of cyclic AMP. These effects are inhibited by prior treatment of the animals with cycloheximide or actinomycin D. Thus, FSH appears to induce de novo synthesis of ABP in a manner which requires prior stimulation of a transcriptional event. Finally, FSH is shown to increase the concentration of Sertoli cell mRNA assayed in a heterologous cell-free protein synthesizing system. Comparison of the time-course of stimulation of mRNA and ABP in response to FSH leads to the possibility that the former is required for the latter. The significance of these events with respect to the biochemical actions of peptide hormones is discussed.

It

is now

well

initiate events

mones chemical to specific

accepted

that

a temporal in a target

peptide

membrane-associated

hormone-receptor

receptors.

interaction

usually

This

the activity of adenylate which results in a change

the intracellular cyclic nucleotide.

concentration It is then

that the of other

cyclic nucleotide modulates biochemical reactions which

or in

be

found

tides

must

be

can

the

stimulate

serve

efficacy

in the

levels

demonstrated

in the specific criteria have for hormones

alteration Such established which

which

to assess changes

of the point

steroid hormone has of the physiologic

peptide

hormones.

mimic

the

stimulation

1972;

Hall,

1970).

Moreover,

manner kinase

responses

time.

in

alter the level of cyclic AMP (Krebs, Bitensky and Gorman, 1973; Soderling et

a number

al., 1973; activation

of

ERC]

as an end point

[cAMPI

+

synthesized

to

creased inactive

binding protein

result

in

an

sociation

and

tory AMP) in

AMP

of

tion. the

will

of steroid synthesis (Gill, In many instances, however,

which

AMP catalytic

In

of be

result

(1) in

in-

subunit of the This allows disinto

the

regula-

(Rcyclic [CI. The

presumably results or structural

enzymes

in an alteration presumably

actions

[CI

results

complex subunit

kinase of

changes

biological

mone

54

holoenzyme

protein

+

AMP

to the regulatory kinase ERCI. the

of

These

hor-

which

The state kinase can

[RcAMPJ

phosphorylation

proteins

been used as an actions of the

in

1974). protein

cyclic

subunit-cyclic and active

activation

Dusecre-

systems

activates investi-

equation:

Newly

the hormone. of cyclic nucleo-

of

Means et al., cytoplasmic

by the

given

by which cyclic AMP has now been thoroughly

of

cyclic

present

mones 1972;

systems

tion end

intracellular

at the

gated

(Catt

In these

popular

a variety ultimately

steroidogenesis

Gill,

Conse-

The protein

1972).

1973;

exist.

event must be same purpose. The

respective assumed

end point marker. been reported to be such as LH and ACTU

fau,

not

intracellular serve the

cytoplasmic cyclic AMP-depenkinase by peptide hormones is one

of

of the most being measured

target cell is to be elucidated, two criteria must be met. First, a sensitive and accurate marker by which Secondly,

do

markers

another which will

activation dent protein

define the molecular actions of the peptide hormones. If the precise mechanism by which a peptide hormone alters the function of its

must

convenient

quently, sought

triggers

an increase in guanylate cyclase

of the generally

such

horof biobinding

sequence cell by first

lead

attributed

of functo some to

the

of hor-

in question. any

given

cell

a fraction

of

the

protein

FSH

TABLE

1. Activation

response

to

Time after (mm)

of

Sertoli

cell

AND

protein

SERTOLI

kinase

Protein kinase activity ratio (-cAMP/+cAMP)

FSI-I

i.e.,

by

the

activity Follicle

in

FSH.

CELLS

hormone,

of

face

ins,

.200 .235 .275 .360 .390 .420 .500 .500

5 10 15 20 45 60 Tubules (100 mg) from SCE-testis of rats were incubated for the times indicated ng/ml of highly purified oFSH (Papkoff). incubation

protein

absence described

kinase

activity

and presence of 1(T6 M (Means et al., 1974).

cAMP

as

within

will

kinase

assay

ing

a

the

absence

exist

straight to

protein

dent

protein

kinase

but of the

this cyclic

form

then

kinase

activity

subunit)

is only

can

the

cyclic

form.

TABLE

of

when the

activity Thus,

an

increase

in

of

in

obtained

in

by

the

of

excess

of

the

protein

100 yields total protein

in

the

kinase

active cAMP,

and

activity

cyclic

AMP a

AMP

and

ratio.

These

data

of

cyclic

sample. cyclic of of

It

can

are

tissue.

FSI-I

cyclic

incremental

increase

2.

the

levels

same

tissue

levels

of

1 picomole/mg

in in

a

kinase

tissue

initial

intracellular

maximal

mm

in

protein

in

Again,

results

the

the

with

activity

Table

FSH

that

stimulates

exist

intracellular

experiments,

seen

is in

activation

kinase in

measured

be

30

an

in

with

the

cytosol

should

approximately

AMP

after

in

protein

these

were

AMP

reached

AMP

In

of

increases

result

in

by

half

the

in

shown

testis

AMP

percent

that

correlation

increase

ratio.

cyclic

50

suggest

the

with

stimulation

of

increases

are

which activity

kinase

present

kinase,

cyclic

of

after

mm

If physiologic

incremental

activity

catalytic

2

protein

be

protein

percent

Within

then

expressed.

between

the

time-dependent

kinase

time-dependent

divided

(the

A

data

protein

FSH,

a maximum

These

incubation

data

kinase

intracellular

of protein

intracellular

an

cyclic

protein

times

present

re-

The

value

of

is

1

be seen.

being

of

existing

presence

multiplied

is

the

activity

percent

now

equation

of

a

This

that

protein

now

AMP the

2. FSI-I stimulation

FSH

the

(CIRC+C).

ratio)

estimate

as

is

in

In

(RC+C).

cyclic

obtained

kinase

0 5 10 20 30

that of

(C).

AMP-depen-

and 1974;

1. It can

Table

the

in the

reaching

mm.

(or

Means

protein kinase Sertoli cell

FSH

of

percent.

increase

can

occurs 45

all

enzyme

expressed

ratio,

AMP,

kinase

Time after (mm)

be

absence

activity

an

active

ratio

in

raplevel

testis seminiferous tubules Sertoli cells (SCE testis)

of

an

in

shift

kinase

One

assay

ratio

which

Huckins,

of the

absence

cell

AMP

1974;

the

is shown

sur-

I-luckwith

hormone

and

to

mem-

Sertoli

cyclic al.,

in

the

and

of

et

assay

the

the

cyclase

Means

20

incubation

assay

will

cell,

Application

in

to

on

1972;Means

(Means

system that

kinase

presence

target

1972;

ratio

perform-

the in

protein of

activity

This

resulting

[CI.

the

in

binds

present

association

cell

1973).

model

in the

AMP

The

activity

by

cyclic

activity,

AMP.

right

total

time

AMP-dependent the

enzyme

cyclic

the

determine

peated excess

forward

Vaitukaitis,

Means,

seen

previously

enzyme

of

and

contain

active

fraction

of exogenous

order

to

as the

this

testicular

the

FSH, can

the

Vaitukaitis,

day old with 500 Following

measured

Hormone

receptors

1974).

in an increase

result

receptor activates adenylate idly results in an increased

16

was

should

ratio. Stimulating

brane-associated (Means

0 2

55

cyclic

values

vitro.

levels being

Moreover, AMP

the

corresponds

in vitro.

Protein kinase activity ratio (-cAMP/+cAMP)

Cyclic AMP (pmole/mg tissue)

.195 .260 .385 .410 .505

1.14 1.63 2.01 3.51 3.80

Testis tubules (100 mg) from 16 day old SCE-rats kinase was measured as previously described (Means procedure described by Gilman (1970).

were incubated et al.. 1974)

with 500 and cyclic

ng/ml AMP

of oFSU (Papkoff). was assayed by the

Protein binding

56

MEANS

to the

activation first

At

of

the

glance

activates

50

percent

protein

zling. multiple

it of

by on

column.

In

tiple

must protein

order

ated

testis on

linear

dependent 30

mM

and

column

no

activity.

from

of

ly,

peak

puz-

cell

of

that

centrations.

in

existed

are peak

resolved.

both

cyclic

Peak

2 elutes

addition,

Sertoli

at

a

1 elutes

150mM

used

as

KM

and

peak

at

2

histone

for

the These

by

the

in

of

the

of

AMP

maximal

ly

from

cyclic of

AMP

the

ty

that

ATP

the

in the calculated

early

stages

of 1

20

30

40

FRACIION

50

60

70

80

of

of age.

This

the

FIG. 1. DEAE-cellulose chromatography of protein kinase in cytosol of rat testis. Testis from adult SCE-rats was homogenized in 10 volumes of a buffer containing 10 mM Tris (pH 7.8), 1 mM EDTA and 7 mM 3 Mercaptoethanol. Cytosol (300 ml) was applied to a 2.5 X 35 cm DEAE-cellulose column equilibrated in the same buffer. The column was eluted with a linear KCI gradient from 0-400 mM. Aliquots (50 7zl)

in

kinase in the cyclic kinetic

for

1-luckins,

Hall,

1967).

that

FSH

protein

may

were

sence

of

to

(Fig. FSH

that

In

greater ratio

cytosol

al.,

absence demonstrated

2, left panel). was present

suggest

the

Means

and

of

soluble

immature the

the

rat

presence

prepared DEAE

the

with dem-

possibility

other incubation

ab-

or and

chro-

cellulose

of FSH, a profile in Figure 1 was in

days

Means

1 form

was

On

24

1974;

1973;

analytical

the

2. to

previously

Consequently,

on

than

correlates

et

in

the ratio

drops

by about

ratio

activate

only

FSH;

during the

the

Means,

incubated

matographed umns.

the

data

kinase.

testes

was age

(Means

These

data,

sensitivity

1974;

de-

model. It of peak 1 to

ratio

0.85

FSH

protein

rat

development

in

FSH

2

testicular

the

2

in

subunits.

peak

if the

increasing

decline

decline

and

and

from

peak

to

possibility role

into

during

approximately

onstrated

NUMBER

of each fraction were assayed for protein activity as described by Corbin et al. (1973) absence (0-0) and presence (.-----.) of 10M AMP. Peak fractions were pooled for the studies described in Table 3.

a value

AMP-depen-

important

1

of

(KM similari-

to the

kinase

testicular

with

enzyme

cyclic

an

that

2 was peak

the

of activation

Sertoli-enriched

demonstrated

However,

is approximate-

point

quantitated

half

for reaction)

demonstrate of

protein of peak

levels were

cyclic binding

the

maximal

of

play

both

that

concentration

half

and

may

peak

of

This

2 forms

en-

cooperativi-

of

the

results

kinase

velopment was

Kd

function the

protein

The

It

for

These

between

dent

kinase

‘a

needed

AMP).

dissociating

2150

than

for

was next measured 3 shows that the

x 108M.

lower

catalytic

cyclic

X 10-’.

necessary

the

1

10-v

both

for

argues

Table

(or

2-5

10-fold

3.5

and interacting cyclic AMP

concentration

binding

ranges

A

and

ATP.

8 x

of

of

peak

calculated

holoenayme

the

absence

cyclic

a

a KM

activator

hand,

for

coefficient

en-

with

other

a KM

enzymes contain multiple AMP binding sites. The capacity

a

Hill

the

KM’s

the

is greater than 1.5 data suggest positive

AMP

zymes.

On

2 exhibits

peak

kin-

histone

for

is different

1 exhibits

con-

2 protein

similar

ATP.

Sertoli

3. When

substrate

AMP

2. Peak

the

exhibit

and

cyclic

in the

Table

exogenous

Final-

equal

of

complexes

However,

ty

KCI.

sh contained peak 1 and peak

properties

an

to

the

present

approximately

in

peaks

that both complex.

2 are in

are shown

both

cyclic

AMP-

peak

kinetic

whereas

was fractionthat by using

of

is

indicating holoenzyme

rats

ase enzymes

respect

rats

peaks

and

in

1 and

zymes

mul-

AMP [Rd

adult

Some

the

cytosol exchange

whether

SCE

two

the ion

1 shows

flow-through In

be

be emphasized kinase exist

kinase

gradient

cyclic

might

determine

Figure

activity KCI

to

cytosol

bind cyclic represent an

only

can be demonstrated by at low ionic strength

protein

DEAE. salt

The

of

FSH

cytoplasmic

the

chromatography a DEAE-cellulose

forms

cells,

that

kinase

cytosol. These forms preparation of tissue followed fraction

kinase.

fact

of

AMP-dependent However, forms

protein

the

ET AL.

col-

identical obtained

hand,

when medium

FSH

TABLE

3. Kinetic

properties

of testicular

AND

protein

SERTOLI

CELLS

kinases.

Peak

Histone

I

Peak

II

Phosphorylation

KM

Histone

KM

57

(mg/mI)

0.75

0.70

3.0 1.7 8.0 1.7

3.0 1.2 3.5 1.6

4.6 1.2

2.0 0.9

ATP (X 105M) cAMP +cAMP, 106M

KM Hill

cAMP (X Coefficient,

Cyclic

AMP

10’M) cAMP

Binding

Kd (X 10 M) Hill Coefficient

Peak I and Peak II testicular protein kinase were isolated by DEAE ion exchange column chromatography of rat testis cytosol. Histone phosphorylation was assayed by the procedure of Wastila et al. (1971), using approximately 150 g of Peak I and 80 pg of Peak II. Cyclic AMP binding was measured by a modification of the Gilman (1970) procedure in which 20 mM potassium phosphate, pH 6.5 was substituted for 50 mM sodium acetate pH 4.0. The amounts of Peak I and Peak II used were similar to those used for histone phosphorylation.

peak

1 activity

peak

2 remains

In

is considerably

addition,

a small

pendent

activity

through

of

the

These

of

data

suggest

activation

of

this

would

the total catalytically Thus,

uric,

This

that

explain

1

why

FSH

behaves

cyclic

subunit.

al.,

activity

may

result

only

in

If

so,

kinase.

in

the

only

lS

0

0000101

PPOTIIO

515211

PEAK

I

1973;

of of

intracellular

22

other

activation

of

quent the

kinase

that

this

protein

for

glycogen In

the

testis

1974; Ii

00

0

II

0

2

00

IS

20

05

50

FIG. 2. Effect of FSH on soluble protein kinase of rat testis. Tubules (1.5 gm) from 16 day old SCE testes were incubated ± FSH (5 pg/mI) for 30 minutes. Cytosol was prepared as described in Figure 1 and analyzed on 0.7 X 10cm DEAE columns. Elution s with a 0-400 mM linear gradient of KCI. Fractions were assayed for protein kinase (Wastila et al., 1971) plus (.---.) or minus (0-0) 106M cyclic AMP.

end

Means, point

has

actions

been

in

Means,

1974; Means,

1969; however,

sought

of

FSH

the

to

sensitive is in

testis Means,

the

no

with

(Means, the

Sertoli

years

bio-

cell. that

FSH

translational

and Means

has

en-

proteins

assess

the

and

It

an

a different

to

1975;

can

2

in

fraction

(Means

cyclic

known

P

multiple

some

1974). link

lipase

res#{231}onse to

results

on

for

in

provided

Consequently,

be

altered

respectively. there

transcriptional

events

fat

subcellular

known

stimulates

enzyme

been

FSH

1975). must

chemical

or

has

of

every

in

present

and

protein

are labeled

that

One that

an

cell

if cells

cell.

protein

phosphorylated

phosphorylation

hanced

target

is to show

cells,

Sertoli

protein

FSH. Indeed, be demonstrated

0

fat

the

subse-

in

and

and

FSH

how

facilitates

results

information

muscle

for is

a specific

synthetase

in skeletal

specific

question

events

structural

Such

activation

as a sensitive of FSH at the

in the

these

Means,

The

kinase

events

the

of

et

hormone

phosphorylates

question.

(Means

1974;

next

phosphorylation

function

20

The

investigate

to

of

kinase

1975).

peptide

biochemical

way

form

can be used the effects

level.

any

al.,

the

soluble

Huckins,

et

kinase determine

intracellular

and stimulates

a specific protein

and

adenylate

increased

AMP,

of

Means

activates

an

cyclic

Fakunding

percent

#{163}50PEAK

to

AMP-dependent 1974;

or

testis,

leads of

presence 50

the

in

activity

of protein index to

protein kinase holoenzymes become active. FSH binds to membrane receptors on

or

cells which

catalytic

flow-

the

Sertoli cyclase accumulation

AMP-inde-

in

protein

even

of

panel).

catalytic

FSH

peak

concentrations

cyclic

apparent

isolated

while

2, right

of

now

column.

the

high

(Fig.

peak

is

as that

precisely

diminished

unchanged

been

Hall,

1967;

and Hall, difficult,

nucleotide-mediated

It

58

MEANS

events on

with

the

and

RNA

effects

strates the of immature

effect

from

testes

of

time

with

of

of

rats.

rats

treated

incubated It

1 h protein 60 percent

tion

the

maximal

incubated

in

4-valine,

1 h

for

vitro whereas,

with

FSH

Following

labeling

of

obvious

a variety

sponse

1973;

to

deter-

ticular

androgen

the

results

to

FSH.

each

and

the

These

stimulation

data of

rats

tissues

were

However, critical

of C’4 a

relatively

protein

synthesis

and

therefore, out.

This

selective led

changes

us to

begin

by

could

not

a search

for

(ABP)

has

the

been

cell 1974;

Sertoli

Tindall

et

al.,

Tindall, 1975). hypophysecchronic

adminis-

et al., 1974; Hansson et et at., 1974; Sanborn et at., we are interested to deter-

of

be used

could

as a specific

Data

are per

5 shows

that

of FSH to demonstrable

the

expressed mg

of

toward that

as

cytosol

a single

immature increase

30 mm. This protein a maximal value by

301

for

bound

on was

developed quanti-

system (1974)

ABP.

3H-DHT

ABP levels decline Figure 5 also reveals

C

and

Tes-

1973).

protein of

is

the ducts,

(French

Ritzen,

following

ABP

nous injection results in a within reaching

and

to assess the acute effects of FSH cell. Androgen binding protein

Figure

protein.

high been

(Tindall

whether

point Sertoli

tative assay picomoles of

time half

and

a product al., 1975;

FSH

with has

epididymis

binding

be et

be

epididymis

passes through the efferent

assayed by an electrophoretic by Ritzen and coworkers

counts

a general

caput

reappears

of

mine

re-

constant

suggested

but

end the

exami-

to H3

protein

testis, into

French

al., 1973; Vernon 1975). Therefore,

no in

However, a choice of a 1 h labeling not select for proteins with a short

be ruled

tomy tration

com-

appear

more

ratio

testicular

for

H3-valine.

could

in

Tindall et al., 1975; Means and This protein disappears following

of

treated

identified

binding

the tubules

the

enters

shown to (Fakunding

Cytosol was then polyacrylamide gel peaks can be seen demonstrating the

proteins.

reveals then

presence

with

Moreover,

fraction

value.

the

differences

by plotting

nation

FSH. would

of

qualitative

from

Ritzen,

from

the

androgen

observed.

incubated

incubation

termed secreted

is

still

that

3I-i-dihydrotestosterone 10-’#{176}M). This protein

seminiferous

1 h in

tissue

was

binds (Kd

affinity

proteins

by FSH.

has been

protein

has been stimu4 h after incuba-

response

bined and homogenized. prepared and analyzed by electrophoresis. Radioactive throughout the gel length

life

of

in

ment

in

periods

conducted this

turning over stimulated

which

Figure 4 were obtained. In this experitestis tubules from untreated rats were

shown

c’

were of

A

isolated

for 1 h in the be seen that

can

response

selectivity

synthesis

were

various

synthesis and that

experiments

the

mine

3 demon-

protein

for

and

by

When

on

rapidly selectively

gonadotrophin

Figure

Tubules

C’4-valine.

lated

within

this

FSH

SCE FSH

presence

of

synthesis.

protein

ET AL.

intraveSCE rats in ABP

continued to rise 2 h. Subsequently, the

the

E-lI#{149}----.I

I

::

control injection

-

FSH

I

160

level. of the

nsrnl

r6

CONTROL

1 b

75-I

‘-3

‘2000 IO

2

u

l000 5

0

a

2

(I,

TIME

3

4 0

(HR)

FIG. 3. Stimulation of testicular protein synthesis by FSH. Rats (16 day old SCE) were injected intravenously with 50 pg FSH and killed at various times thereafter. Testis tubules were incubated for 60 mm with C’ 4-valine as previously described (Means and Hall, 1967). Following incubation tubules were homogenized in trichloroacetic acid and radioactivity incorporated into protein measured by liquid scintillation counting (Means and Hall, 1967).

,

05-

? FRACTION

FIG. 4. response to and reference:

NUMBER

Synthesis of testis cytosol FSH. Methods are described Means (1974).

proteins in the

in text

FSH

AND

SERTOLI

CELLS

It

2.

59

has

been

difficult

tabolism C

C,

E

effects

on

data

of

the me-

RNA

and

revealed

that

is most

ABP of

was

link

nucleotide

interest

likely

to

deter-

mine whether this effect could be mimicked by injection of cyclic AMP. For these studies, a cyclic AMP derivative which readily enters cells and activates protein kinase lyzed by phosphodiesterase

U,

a) 0

E

et al.,

unding

Muneyama 1974). This

Ac

It 3 I HR I

I

TIME AFTER HORMONE

4

was

5. Effects

of a single

injection

of

or LH

on androgen binding protein levels in testes of 16 day old SCE rats. Hormones (200 pg) were administered intravenously in 0.1 ml saline. At the times indicated rats were killed, cytosol was prepared from testis and A.BP quantitated as described by Ritzen et a!. (1974).

curve

and

was

similar

to

istration

of

of

amount

stimulation

of

specificity

of this

As

to be strable

LH

ABP

by total

in

indicating

very

the

response. previously,

mentioned

induced under

results

if the

FSH and yet steady-state

little

hormone

protein

was

not be demonlabeling condi-

tions, the half life time of the response or the total concentration of the protein would have to be minimal. Indeed, the half life time of the response

of

binding total sents

the

effect

of On

nature

required

us

induction

to

of the

mine

whether

tions

of

would

of

other

response was

this

necessary timed

injection

the

after maximal

level

least 4 h after Tindall, 1975). inducible

offered might

of

first,

FSH

ABP for

at

is administered

levels least

continue

an

the initial This type enzymes

in

many

of gene

function.

systems

production

Huckins,

1975; cyclic back.

Means,

still is known

result the

in

the

as well

release

of

In

order

rats.

by

route

are

the

and

that FSH involving

peak and

1974;

These

shown

in

AMP

FSH.

out

due cyclic

al.,

to

this other

AMP

was

hypophysecto-

data

administering

was reduced These values

mm

rule

into

response response.

at

syntheal.,

hormones

to

8-bromo

These if a

90

FSH

et

trophic

as complications

effects, SCE

et

Fakunding

intratesticularly

obtained

protein

(Means

be

of

Intravenous injection of suffers from a serious drawthat exogenous cyclic AMP

pituitary.

peripheral

would

effects

and

maxito 2

1975).

AMP

can

stimulation

data

1974;

cyclic than

2 h (at

additional

and

from

These

established

Means

the

temporal

8-bromo stimulation

injection) (Means and of response is typical

the first suggestive evidence influence ABP by a mechanism

induction

such

AMP

admin-

1975;Means

FSH.

the

pro-

injecor

as

previously

response

rapid reaching the injection compared

cyclic AMP of ABP. At

multiple

and

cyclic

It

the

following

However,

8-bromo stimulation

deter-

of ABP.

the testicular concentration experiments proved positive

FSH a true

to

potentiate

either

long

second

to

was

1975;

intravenous

of

observed

to

with

on

mized

a single

magnitude

1975).

sis

total

the

the

response

injected

the

that

to be more 1 h following

possibility

the

Tindall,

FSH(Fakundingetal.,

compatible

repre-

hand,

of ABP

that

It

androgen

testis

percent

the

carefully

FSH

in the

demonstrate

process.

on

h. Moreover,

1

ABP

0.03

protein.

transient

FSH

is around

protein

concentration approximately

testicular

of

and

hydro(Fak-

1971; Muneyama et al., is 8-bromo cyclic AMP.

those

Tindall,

appeared mal level h in

same

Means

is not chosen

compound resulted in a stimuof androgen binding protein in the cell. Moreover, the shape of the induc-

tion

and

but was

this

of

lation

FSH

1975;

et al., compound

demonstrated

injection

Sertoli FIG.

the

directly

cyclic our

the de novo synthesis stimulated by FSH it

0

of

on

with subsequent synthesis. Since

protein

a)

to

effects

FSH-mediated

along FSH

Figure

6.

data

the

same

Again,

and FSI-I 1 h following resulted However,

with

via

both

result in injection in

a by

compared to are consistent

greater 2 h the

the

FSH with a

response of 8-bromo cyclic AMP at 1 h a peak response of FSH at 2 h. Thus, there

appears

to

be a direct

effect

of

8-bromo

cyclic

AMP on ABP synthesis in the Sertoli cell. These data offer the first suggestion that ABP synthesis may be mediated through a prior stimulation of the The the

a

intracellular next

increase

level

problem in the

of cyclic was

ABP

AMP.

to establish

assayed

by the

whether binding

60

MEANS

ET AL.

STIMULATION OF ABP BY FSH AND 8 BROMOCYCLIC AMP

-

I

Protein

I

Synthesis

11

z

AS P

.0

.7

a. C

0

0

}

I5

>-

E

E

.6

>10

0. =

a

.5

C

a)

ft-fin

0.

C,

E

FSH

.4 I-

FIG.

ISH

FSH

AcID

Cyclo

7. Effects

of cycloheximide

on the FSH-mediated 1K

a)

total

.3

0

or

protein

cm

0.

D

(100

(200

pg,

(250 whereas

injected

as

30

a single

FSH Cyclo

#g,

of

ip)

was

actinomybefore.

mm

dose

iv

D

synthesis

hormone

ip) was

FSH

ActS

or actinomycin

in testicular

Cycloheximide

before

given

was

FSH

to

FSH

16 day old

rats. After 2 h testes were removed and portions for protein synthesis (Means and Hall, 1967)

SCE

.2

Ac

pg)

increase

ABP.

1 h

administered

E

C

-

assayed or ABP

(Ritzen

et al., 1974).

.1

P C

P

FSH cAMP

6.

8-bromo

Testicular

2 Hr

cyclic

AMP.

hypophysectomized ticularly pg)

was

Day

33.

injected

cytosol

of

on

on

in

in

Rats

which

Day

response

30

FSH

(25

by Ritzen

to cytosol

and

protein

obtained

injection of FSH-mediated and

These

data

continued argues protein.

(50

assayed

was

Such

cycloheximide

FSH

completely

induction also

reveal

of

that

in

a result

transcription the response

of

elongation

of and as de

evidence

can

1

and the

before

inhibits the total protein

of

androgen

h

be

binding

the

stimulation

of

peptide

The

FSH on concentration

abolished the both protein

The

induction

this and

D used

ABP

demonRNA

therefore

turn

decided

to

effect

by FSH by

our

since

these

enzymes

be stimulated FSH (Means,

16 day old of the

II (Means and increased linearly

following

60 mm continued

been

after to

hormone,

this time. These results showing

numbers

newly

of

synthesized

assayed

was

demonstrated

to

testis by that FSH

SCE rats activity

peaked

to

transcription Initially,

results in of RNA

1975). This 1 5 and 60

at 1 to 2 h and

declined. On the other noted in RNA polymerase

maximal at with previous

to be

attention

Tindall, between

FSH. Ribosomal increase for 4

the

D and AMP. We

in normal and immature 1975). It was found

administration to a rapid stimulation polymerase response

had

ABP gene that to

of

appears

actinomycin by cyclic

possible alterations in Sertoli cell in response to this gonadotrophin. nuclear RNA polymerase activity

then rapidly increase was

and

of

an

selective, is inhibitable seems to be mimicked

mm

effect of synthesis

previously messenger

dependent upon on the nucleus.

requires chains

of actinomycin

and be hormone

protein.

for a de novo synthesis of the binding Again, actinomycin D administered 30

mm before gonadotrophin ABP.

was

or

cAMP

the use of inhibitors of protein synthesis. Figure 7 shows that

administration

synthesis

8-Br

ABP

proteins

synthesis.

by acid

nucleic

injected or

FSH

SCE were intrates-

et a!. (1974).

events mediated through translation and thus establish novo

to

were

g)

a 10 p1 volume.

as described

3H-DHT

ABP

was testicular

synthesis (Means, 1975; Means, 1974). synthesis following FSH seems to require transcription. These results would suggest induction of ABP must occur subsequent

C

1 Hr FIG.

in these experiments strated to inhibit

h

RNA and

hand, no I until synthesis was still

data coordinate that the maximal ribosomes

ap-

FSI-1 AND

pear

in

the

cytoplasm

(Means,

1975).

towards

an initial

II activity. transcription decided

to

together

effect

Since of

measure a heterologous

system

at

various

nucleic

ly purified

and

The

wheat

et

injection in an

noted

be

al.,

1975).

the

filter et

system

Figure

testes

this

mm,

30

al.,

1972).

was

chosen

between

a

prevented

by

actinomycin

Thus,

synthesis of Sertoli cell.

FSH

D

but

appears

translatable

2 h

Again, the RNA could not

by

to increase

messenger

RNA

in

RNA

within

activity

is shown

mm

1 h and

(Fig.

cell of

30

and

the

about 2 h. On synthesis is not

until level

on Sertoli induction

that

1 and

messenger

detected

maximal

heterologous protein Marked stimulation is

peaks

and

cell response to binding protein

in Figure 9. It can be seen that the increase in messenger RNA activity and androgen binding protein activity correspond. Increases can be

significantly

partial-

8 shows

respect

response occurs at hand, total protein

technique

3 h has begun to decline. in the activity of messenger

cycloheximide. the the

from

the Sertoli to androgen

of

with

readily

of FSH to 16 day old SCE rats increase in testis messenger RNA

assayed in system.

within

and by increase

injection.

(Means

translation

activity

synthesizing

61

Comparison

synthesis for we

FSI-I

CELLS

FSH

its low endogenous messenger RNA ease of preparation and stability

(Means

activity

RNA

RNA

nitrocellulose

h

its

single results

synthesizing

8 point

on polymerase

A-containing

described germ

because of levels and

after

was extracted

poly

by the

as previously

and

results

protein

times

acid

rats

FSH

messenger

in

of SCE

of

the

this enzyme is responsible unique sequence DNA,

assayed Total

4

between

Taken

SERTOLI

3).

function ABP.

At

at

Thus,

4 h

the

appears any

peak

the other increased is still

effect

of

at FSH

to be a selective rate, these data

establish ABP as a sensitive and specific end point marker by which to assess the biochemical effects of FSH on the Sertoli cell. The effects injection and

of of

can

be

ABP synthesis actinomycin mimicked

can be inhibited D or cycloheximide by

AMP, 8-bromo cyclic hormone induction of

a derivative

by

of cyclic

AMP. Therefore, the ABP involves de novo

synthesis and seems to require event which may be mediated

a transcriptional by cyclic AMP.

Taken together, the data allow us to construct possible sequence of events in the mechanism of

FSI-I

binds

to

adenylate intracellular

action

on

the

membrane cyclase

Sertoli

receptors and

results

accumulation

cell. which

of

in

first

couples an

cyclic

FSH

a

to

increased AMP.

This

cyclic AMP activates protein kinase and also in some unknown way allows the increased activity of RNA polymerase II. This allows the

15

Ac

C,

E 0.

>, >

Ac Ac

z

E

I

2

3

4

TIME AFTER FSH (HR I FIG. 8. Stimulation of translatable mRNA by FSH. Sixteen day old SCE animals were injected with FSH (50 pg, iv) for the times indicated. Total nucleic acid was extracted and a poly A-enriched fraction prepared as previously described (Means et al., 1972). Messenger RNA activity was quantitated in a cell-free system prepared from wheat germ (Means et al., 1975).

2

3

TIME AFTER FSH 1 HR

FIG. 9. Stimulation of translatable mRNA and synthesis as a function of time after administration of FSH (50 pg) to 16 day old SCE rats. ABP

MEANS

62 in the cytoplasm is translated into

accumulation RNA which these action

proteins of FSH

stimulated is androgen

Obviously we still directly demonstrate lates transcription. It one

also

modulates

of messenger protein. One of

very binding

amounts

less)

which

Sertoli

cell

possibility

can

cyclic from

be

activity

AMP

direct

correlation

ACTH

on

AMP

steroid synthesis. It mal steroid hormone

glycogenolysis clear that

(1 pg or

phorylation of gen break-down cyclic 1974). lation is

AMP The

is protein

now

are

now

years

receptors

are

nephrine

in

increases concerns tive. It mediated and

blocked still

can

genolysis

the

no acti-

It is dephos-

hand,

data

when

even

epion

any

big

some

other

direct

glyco-

detectable

problem

then

stimulatory

toxin which recognizes specific the membranes of cells also synthesis.

It

has recently

inhibiting destroying (Pappenheimer

the the

protein activity and

Gill,

of

been

1973).

mones

effect

recepinhibits

hormones.

Finally,

we

specific gene product end in response to FSH) which working

with

peptide

the

same

type

which

of molecular have helped to

of

steroid

hormone The

the

elucidation

of

target

of

by cells.

which

action next

the

peptide

few precise

hor-

This study was supported by NIH grant HD-07503. A. R. Means is the recipient of a Research Career Development Award from the NIH. J. L. Fakunding and D. J. Tindall are postdoctoral fellows of the NIH. NIH-FSH-S-10 and NIH-FSH-S-16 were kindly supplied by the Hormone Distribution officer, NIAMDD, NIH, Bethesda, Maryland 20014. The authors are indebted to Mr. Charles R. Mena and Ms. Elizabeth MacDougall for expert technical assistance.

process factor

REFERENCES Bitensky, M. W. and Gorman, R. E. (1973). Cellular Responses to Cyclic AMP. Prog. Biophys. Mol. Biol. 25, 411-461. Catt, K. J. and Dufau, M. L. (1973). Interactions of LH and hCG with Testicular Gonadotropin Receptors. Adv. Exptl. Med. Biol. 36, 379-418. Fakunding, J. L., Tindall, D. J., Dedman, J. R., Mena, C. R. and Means, A. R. (1975). Biochemical Actions of Follicle Stimulating Hormone in the Sertoli Cell of the Rat Testis. Endocrinology (In F. S. and Ritzen, E. M. (1973). A High Affinity Androgen Binding Protein (ABP) in Rat Testes: Evidence for Secretion into Efferent Duct Fluid and Absorption by Epididymis. Endocrinology 93, 88-95. French, F. S. and Ritzen, E. M. (1973). Androgen Binding Protein in Efferent Duct Fluid of Rat Testis. J. Reprod. Fert. 32, 479-48 3. Gill,

demon-

Alterations

activation

1974).

mechanism

regulate

The

kinase can be used as in assessing the intra-

Press).

on protein fragment (A) thus directly

synthesis elongation

biochemical

hormones

Means,

yield

the

concerning

French,

has yet to be determined. It ruled out that some part of may enter the cell. Indeed,

strated that the inhibitory effect synthesis occurs because a peptide is cleaved off, and enters the cell,

should

also

directly

ACKNOWLEDGMENTS

and Park, the stimu-

effects

The

and

in glycoeffect that

other

of

AMP.

(O’Malley

may

and

propranolol,

the

the

could

rate.

these

investigators

optimal

determining what is the best alternais possible that the cyclic nucleotideevents comprise an alternate pathway

that

protein

of LH

that

with

exert absence

in cyclic

may occur which still has not been peptide hormones diptheria tors on

it had been there was a

allow

of

to find as ABP

out

in

the

effects

GMP

GMP

non-steroidal

protein intermediate

are beginning points (such

cyclic

research of

at a rapid

cAMP-dependent a convenient

mysteries

(Soderling On

that

is proceeding

cellular

to

cyclic

action

solve

such in

and

suggest

of

the that

and

tested

involved

to

mechanism

to apply procedures

(Catt and Dufau, 1973). effect of epinephrine or

kinase.

available

clear

hormones biological

which produce cyclic AMP nor

enzyme

genome.

is

will

the effect

stimulate

the

affect

AMP

and

cells.

the enzymes involved result in a cascade

will first

cyclic

cause

appears to be questionable. phosphorylation and

the

out of

is now obvious that maxisecretion occurs at levels

vate protein kinase Again, the well-known

of

Leydig

formation

of the respective hormone demonstrable increase in

important

the

Thus, that

between

cyclic

ratio

be

also

directly

to be questioned. for several years

the

may

experiments are now being undertaken laboratory. It should also be pointed well established effects of cyclic AMP beginning suggested

with

cannot rule injections

ABP

of testosterone

secretion This

8-bromo

stimulate

AL.

It

not been able to that cyclic AMP stimuis known that testoster-

the

of

the

have

respect to ABP synthesis. We the possibility that intratesticular small

early in protein.

ET

by II in

G.

N.

(1972).

Mechanism

of

ACTH

Action.

Metabolism 23, 571-588. Gilman, A. G. (1970). A Protein Binding Assay for Adenosine 3’, 5’-Cyclic Monophosphates. Proc. NatI. Acad. Sci. USA 67, 305-3 12. Hall, P. F. (1970). Endocrinology in the Testis. In: The Testis (A. D. Johnson, W. R. Gomes, and N. L. VanDemark, eds.) Vol. II, pp. 2-72, Academic Press, New York. Hansson, V., Reusch, E., Trygstad, 0., Jorgensen, 0.,

FSI-I

AND

SERTOLI

Riczen, E. M. and French, F. 5. (1973). FSH Stimulation of Testicular Androgen Binding Protein. Nature New Biol. 246, (150)56-58. Krebs, E. G. (1972). Protein Kinases. In: Current Topics in Cellular Regulation (B. L. Horecker and E. R. Stadtman, eds.) Vol. V, pp. 99-133, Academic Press, New York. Means, A. R. (1973). Specific Interaction of 3H-FSH with Rat Testis Binding Sites. Ads’. Exptl. Med. Biol. 36, 431-448. Means, A. R. (1974). Early Sequence of Biochemical Events in the Action of Follicle Stimulating Hormone on the Testis. Life Sci. 15, 371-389. Means, A. R. (1974). Concerning the Mechanism of FSI-I Action: Rapid Stimulation of Testicular Synthesis of Nuclear RNA. Endocrinology 89, 98 1-989. Means, A. R. (1975). Biochemical Effects of FollicleStimulating Hormone on the Testis. In: Handbook of Physiology, Endocrinology Vol. V. (R. 0. Greep and D. Hamilton, eds.) pp. 203-21 8. Means, A. R., Comstock, J. P., Rosenfeld, G. C. and O’Malley, B. W. (1972). Ovalbumin Messenger RNA of Chick Oviduct: Partial Characterization, Estrogen Dependence and Translation In Vitro. Proc. NatI. Acad. Sci. USA 69, 1146-1150. Means, A. R. and Hall, P. F. (1967). Effect of FSH on Protein Biosynthesis in the Testes of the Immature Rat. Endocrinology 81, 1151-1160. Means, A. R. and Hall, P. F. (1969). Protein Biosynthesis in the Testis. V. The Nature of Stimulation by Follicle-Stimulating Hormone. Biochemistry 8, 429 3-4298. Means, A. R. and Huckins, C. (1974). Coupled Events in the Early Biochemical Actions of FSH on the Sertoli Cells of the Testis. In: Hormone Binding and Target Cell Activation in Testis. (M. L. Dufau and A. R. Means, eds.), pp.. 145-165, Plenum Press, New York. Means, A. R., MacDougall, E., Solerling, T. R. and Corbin, J. D. (1974). Testicular Adenosine 3’,5’Monophosphate-Dependent Protein Kinases: Regulation by Follicle Stimulating Hormone. J. Biol. Chem. 249, 1231-1238. Means, A. R. and Tindall, D. J. (1975). FSH-lnduction of Androgen Binding Protein in Testes of Sertoli Cell-Only Rats. In: Hormone Regulation of Spermatogenesis (F. French, V. Hansson, S. Nayfeh, and M. Ritzen, eds.) Plenum Press, New York (In Press). Means, A. R. and Vaitukaitis, J. (1972). Peptide Hormone Receptors: Specific Binding of H-FSH to Testis. Endocrinology 90, 39-46. Means, A. R., Woo, S. L. C., Harris, S. E. and

CELLS

63

O’Malley, B. W. (1975). Estrogen Induction of Ovalbumin mRNA: Evidence for Transcription Control. Cell. Molec. Biochem. 7, 33-42. Muneyama, K., Bauer, R. J., Shuman, D. A., Robins, R. K. and Limon, L. N. (1971). Chemical Synthesis and Biological Activity of 8-Substituted Adenosine 3’,5’-Cyclic Monophosphate Derivatives. Biochemistry 10, 2390-2395. Muneyama, K., Shuman, D. A., Boswell, K. H., Robins, R. K., Simon, L. N. and Miller, J. P. (1974). Synthesis and Biological Activity of 8-Haloadenosine 3’,S’-Cyclic Phosphates. J. Carbohydrates Nucleosides Nucleotide 1, (1)55-60. O’Malley, B. W. and Means, A. R. (1974). Female Steroid Hormones and Target Cell Nuclei. Science 83, 610-620. Pappenheimer, A. M., Jr. and Gill, D. M. (1973). Diptheria. Science 182, 353-358. Ritzen, E. M., French, F. S., Weddington, S. C., Nayfeh, S. N. and Hansson, V. (1974). Steroid Binding in Polyacrylamide Gels: Quantitation at Steady-State Conditions. J. Biol. Chem. 249 (20)6597-6604. Sanborn, B. M., Elkington, S. H., Chowdhury, M., Tcholakian, R. K. and Steinberger, E. (1975). Hormonal Influences on the Level of Testicular Androgen Binding Activity: Effect of FSH Following Hypophysectomy. Endocrinology 96, 304-312. Soderling, T. R., Corbin, J. D. and Park, C. R. (1973). Regulation of Adenosine 3’,5’-Monophosphate-Dependent Protein Kinase. 11. Hormonal Regulation of Adipose Tissue Enzyme. J. Biol. Chem. 248, 1822-1 829. Soderling, T. R. and Park, C. R. (1974). Recent Advances in Glycogen Metabolism. In: Advances in Cyclic Nucleotide Research. 4, 283-334. Tindall, D. J., Schrader, W. T. and Means, A. R. (1974). The Production of Androgen Binding Protein by Sertoli Cells. In: Hormone Binding and Target Cell Activating in Testis (M. L. Dufau and A. R. Means, eds.) pp. 167-175, Plenum Press, New York. Tindall, D. J., Vitale, R. and Means, A. R. (1975). Androgen Binding Protein as a Biochemical Marker of Formation of the Blood-Testis Barrier. Endocrinology 97 (3)636-648. Vernon, R. G., Kopec, B. and Fritz, I. B. (1974). Observations on the Binding of Androgens by Rat Testis Seminiferous Tubules and Testis Extracts. Mol. Cell. Endocrinol. 1, 167-187. Wastila, W. B., Stull, J. T., Mayer, S. E. and Walsh, D. A. (1971). Measurement of Cyclic 3’,5’-Adenosine Monophosphates by Activation of Skeletal Muscle Protein Kinase. J. Biol. Chem. 246, 1996-2003.