Houston,. Texas. ABSTRACT. Incubation of testis tubules containing predominantly. Sertoli cells ..... 1 h in the presence of. C'4-valine. It can be seen that within. 1 h protein synthesis has been stimu- ...... indebted to. Mr. Charles. R. Mena and.
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
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Press).
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Means,
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French,
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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-
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The
and
in glycoeffect that
other
of
AMP.
(O’Malley
may
and
propranolol,
the
the
could
rate.
these
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optimal
determining what is the best alternais possible that the cyclic nucleotideevents comprise an alternate pathway
that
protein
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that
with
exert absence
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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
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the
effects
GMP
GMP
non-steroidal
protein intermediate
are beginning points (such
cyclic
research of
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mysteries
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that
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cellular
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cyclic
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solve
such in
and
suggest
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tested
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to
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kinase.
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clear
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enzyme
genome.
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stimulate
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affect
AMP
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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.
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