BIOLOGY
OF
REPRODUCTION
The
14,
Role
30-53
(1976)
of Cyclic
AMP
in Gonadal
JOHN Endocrine The
M. MARSH
Laboratory, Department University of Miami, School Miami,
There
has
been
many
investigators,
which
implicates
functions, role lacking.
one
The
possible
the
of work
the
AMP
a complete
any
in
AMP
deal
over cyclic
but
in
a great
of
THE ROLE OF CYCLIC AMP IN STEROIDOGENESIS IN THE CORPUS LUTEUM
years of is
role
of
stimulation
oidogenesis in ovaries and testis studied the longest and probably thoroughly, but even this action being completely elucidated. The
its
The
still
the
(Haynes et
showed level of
that cyclic
aL,
and Haynes
this
has
ster-
and
in vivo
has been the most is far from first indica-
1959;
ACTH
Berthet, et al.,
increased the in the adrenal
AMP
1962)
Savard, From
1964a; these early
genesis AMP
the field has been
nearly
all
and
some
and
the
Marsh studies
ovary and on
1957; 1960)
has expanded implicated to
actions
of
actions
luteinizing
of
follicle
(Marsh
role
of
this
steroidogenesis testis, since
cyclic in these
reflect contrasting the possible modes the
steroidogenic
‘Dedicated sor of Miami)
nucleotide
hormone
(LH)
stimulating
hor-
the two
corpus gonadal
situations. of action
on
luteum tissues
and seem
Dr.
Karl
in the model
the
of
effect
(Emeritus
species
to be,
is and
by in vitro
at least
report system
LH
on
of several In a series
to some
or other
test
controlling of studies
by Savard et al., 1965) was developed in which progesterone
in incubating
ing hormone 1964a).
synthesis
slices
was
of corpora
lutea
in early pregnancy. The tissue were incubated in buffer in the presence
substances
After
activity
the
initial
on
steroidogenesis lutea slices were logically divided
the
the site pathway;
of the to
nism
and
the
amounts
of
(Mason
studies
on
and the
Savard,
effect
of
LH
in incubating bovine corpora completed, the investigation into two parts: a) a study of
of LH action on and b) an investigation action.
The
site
of
the the
steroidogenic of the mechamajor
effect
of
LH on the steroidogenic pathway has been shown by numerous investigators to be between cholesterol
H. Slotta
in most
luteum
progestins,
progesterone and 2013 hydroxypregn-4-en-3one synthesized were measured. It was found that LH produced a marked increase in progestin synthesis in these incubating slices and this effect was specific for hormones with luteiniz-
pathway.
to
shown
corpus
of
I will also discuss of cyclic AMP on
Profes-
Biochemistry and Medicine of the University on the occasion of his eightieth birthday.
the of
investigations
(reviewed an in vitro
of LH
and
process
ot secretion
obtained from cows slices of corpora lutea Krebs-Ringer bicarbonate
to where cyclic some extent in
in the
been
determined
1964b). steroido-
mone (FSH) in the ovary and the testis. I will attempt to review the studies
and
LH is probably only one hormones (Greep, 1971).
endogenous cortex, and
Savard, gonadal
function
extent, controlled by LH. In some species, such as the cow, the human and the monkey, LH appears to be the only controlling hormone while in others, such as the hamster and the rat,
that exogenous cyclic AMP mimicked the effect of ACTH on steroidogenesis. Guided by this work, other investigators began to study the role of cyclic AMP in the gonadotropic stimulation of steroidogenesis in the testis (Hall and Eik-Nes,
main
synthesis
cyclic of
tion that cyclic AMP was involved with the control of steroidogenesis came from the studies of Haynes and co-workers who in a series of experiments Haynes
33152
gonadal
functions
mediatory
of Biochemistry, of Medicine,
by
10
in several
gonadotropic
Florida
done,
past
understanding these
Steroidogenesis’
and
Hall Hall
and and
Koritz, Young,
The
study
of
pregnenolone 1964; 1968; the
(lchii Koritz Armstrong
mechanism
et al.,
and of
Hall, et al.,
LH
action,
1963; 1965;
1970). on
the other hand, has been largely a study of the possible role of cyclic AMP in this gonadotropic
of
30
CYCLIC
effect
and
Effect
this
under
of Exogenous
Marsh
and that
found
AMP
to
caused [73
the
Cyclic
incubating
slices of
cholesterol
tration
of
AMP
action not
magnitude
amount
of
AMP The this
to
level to an readily
in
The
that
steroid
Marsh
and
acetate
and
a concen-
of
the
cyclic
the
3’-AMP,
effect
by
utiliLH.
5’-AMP
or
by
indicating of this
but
this
of the cells
was
also
a maximal
that action
cyclic AMP far exceeded
tissues,
served to the
of LH
cyclic of LH.
needed for the endoge-
could
have
exogenous or to its
been
nucleotide destruction
(1965a,
at
ment estrous
using cycle
AMP
stimulated
celerating
out
a similar
type
of experi-
bovine corpora lutea and showed that, like progesterone
a step
from the LH, cyclic
synthesis
between
cholesterol
by and
ac-
preg-
nenolone. The progestin
effect of synthesis
tissue
of
(1971)
other
exogenous has been
cyclic assessed
AMP on in luteal
species as well. LeMaire et a stimulation of progesterone
reported
synthesis both in terms of mass and etate incorporation in incubating human
The
corpus effect of
human
chorionic
pattern
of
luteum cyclic
of
the steroids (progesterone, terone, androstenedione duced by the cal in both ness
of
cyclic
AMP
slices. over
[i-’ 4C] acslices of a
(hCG), 4C among 17 and
The
greater
hCG
may
of
but
the
several
of
hydroxyprogesestradiol)
human corpus luteum the hCG and the
tissue
stimulated
al.
of ectopic pregnancy. AMP exceeded that
gonadotropin
distribution
was cyclic
the
the
was tor
AMP
same
the
25
al.,
corpora
adenylate
al.
be able (1972)
for
cyclic
reported
AMP
as for
hCG
on
imidazole, markedly
synthesis These
that proges-
luteum of response
theophylline, phosphodiesterase, of
in the results
the
to by-pass
stimulated
also
a corpus the maximal
effect
cyclase
suggest,
in and
synthesis and that this phosphodiesterase, gesterone incubations.
of
would
also reported that of cyclic nucleotide
tentiated
et
than
experiments
Hermier et cyclic AMP
about
(LeMaire 1974a),
level
these
that block. exogenous
the was
hCG. an
It
inhibipo-
progesterone
a stimulator reduced
control are in
of pro-
and hCG complete
with the proposal that cyclic AMP mediates the action of LH and hCG on progesterone synthesis in corpora lutea. Dorrington and Kilpatrick (1967) reported
that
5 mM
interstitial
and
Jutisz
AMP bating by
serum
external
increased
rabbits
tissue
was
(1969)
of
although
much
showed
to the found
Hermier
3 mM
synthesis ovaries,
rat rats
gonadotropin
its effect
greater. that
immature
medium
pregnant
with
and
of Parlow presence of
was
cyclic
in incuprepared
(PMSG)
procedure that the
the
20a hydroxycorpora lutea
required
hCG
(1968). Ca2 in for
this
effect.
Endogenous
Cyclic
AMP
The results with exogenous compatible with the hypothesis otide was a mediator of the steroidogenesis consideration
tissue. of
to
treatment
according They also the
slightly
stimulated progesterone slices of luteinized
mare
AMP
effective-
AMP
progesterone and in incubating
pseudopregnant
on
would nous
pro-
cyclic
synthesis of pregn-4-en-3-one
identi-
be related
at
synthesis cycle
1’
1973;
cycle. If there is a block in human corpora lutea
cyclic
from
the much smaller concentration (Marsh, 1969). Hall and Koritz
b) carried
synthesis LeMaire,
exogenous
terone menstrual
of pregnancy
and to respond to hCG in cyclase, cyclic AMP accumu-
the menstrual responsiveness as
lutea
capacity to bind hCG (Cole et al.,
accord
by cyclic 3 ,5’ nucleotide phosphodiesterase. In this regard, the dibutyryl derivative of cyclic AMP, which has been reported to be resistant to phosphodiesterase degradation (Posternak et al., 1962), stimulated progesterone synthesis in this system of 0.2 mM
lutea of to hCG activation,
pattern
cholesterol produced
al., 1975) of adenylate
et
of pregnancy
produced
and
produced
of
inability penetrate
LH
as
cyclic AMP, be a mediator
concentration effect, however,
nous due
to
of might
and
1968;
Rao
corpora
smaller LH or
lation
At
that
human
lutea
in the system, which degree of specificity
AMP.
additive
to
[7-3H] that
such
effect a certain
of cyclic
amount
4c]
that much human
terms
corpora
[1-’
equal
nucleotides no
cow
fact
31
1966b) cyclic
of progesterone
about
of [1-’4C] acetate and zation closely resembled ATP had to establish
1964b, exogenous
of
mass,
M the
was
a saturating
Other
AMP
have a labelled
incorporation.
0.02
effect
STEROIDOGENESIS
the
stimulation
in terms
H]
AND
investigation.
Savard (1964a, addition of
a significant
synthesis
by
is still
AMP
much
in the required
bring about concentration Marsh LH
et al.
corpus that
cyclic that action
luteum, but the addition
an increase of cyclic (1966)
AMP were this nudeof LH on
showed
such a of LII
in the endogeAMP in luteal that
2 pg/mI
brought about a striking increase (as as 100 fold) in the level of cyclic AMP in
32
MARSH
incubating
this
slices
response
LH the
as had stimulation
aid
et
of
bovine
showed
the
been of
al.,
1965).
endogenous
of
2 g
of
LH.
At
LH/ml) the
between
and
demonstrated synthesis
Furthermore,
the
AMP
less
the
that
measurements
error
to
(0.2
to
component
cyclic
ered
progesterone synthesis, AMP levels could not This discrepancy was
had small
this
Since
(Beall
1973;
Moyle
shown
that
work and
cyclic
and
indirectly, kinase by
luteum
had
kinase.
Using
the
al.
(1973),
reported, 1972;
concentrations
of cyclic
luteum. In the older relationship (Marsh
study et
looked for of incubation
a change while
measured
after we of
h
(Ling we of
where
modification (Ling
et
course
of cyclic when small
al.,
recent
of
amount
were
there
is still
mum effective steroidogenesis The minimal
and
of
we
have
tropic
hor-
significant
the
of the dose al., 1966) AMP
dose
re-
This
Marsh,
found
response in when large our recent unpublished
that
AMP accumulation amounts of LH
a dissociation doses and effective
tein
the
between
a
time
of
AMP
been
of
and
by
hibits
the
amount corpora
and
Goldstein
of
predominantly
the
in
inhibits
about
25
percent
of LH which will stimulate cyclic AMP accumulation. dose of LH which causes
bound
to
a macromolecule.
of
before, protein
this
pro-
to be effects
of
on steroidogenesis substance had of
which
no
cyclic
indicated
required
not
Marsh
mini-
as mentioned AMP-dependent
LH
cyclic
the
previously
stimulation
slices
will
between
puromycin
lutea
at 5 the
which
stimulatory
stimulation of after the increase
step
increased
the
bovine was a
activity below
which
was
that
kinase
the increase in probably an increase of cyclic AMP.
shown
the
Cor-
different activity
and Marsh, unin terms of dose luteum to LH,
then,
AMP This
on
protein by
LI-I
corpus
the
cyclic 1965).
accumulation
effect,
these
antibiotic
had
synthesis
1973) corpus
slices of that there
(Ling
this in
shown
of
agreement
an
however,
protein
some
good
inhibiting
both LH and (Savard et al., effect,
is nearly are used. In
the
capable
been (1972,
kinase slightly
concentration
synthesis,
had
found
steroidogenesis, kinase activity and effective
attempted
described
in
Puromycin,
was
cyclic AMP by (1970) method
is
cyclic
LH were with this
AMP with the changes
Marsh
in this is even dose
effective
there
the
was
steroidogenesis data). It seems of the bovine
published response
in
It
subunit
in
recently
incubating
which
stimulate
that
levels of associated
the effect on protein
LU
One consid-
that the bovine AMP-dependant
increase
ng LU/mI, minimum
increase protein
response we only
after 15 mm synthesis was
incubation.
measured the Gilman
1973),
in
dose response study, therefore, the progesterone synthesis and cyclic both measured after 2 h of incubapreliminary evidence indicates that
AMP tion.
Our
of
of
LH to increases in AMP in the corpus
in cyclic progesterone
2
concentrations
Dufau,
1973)
low been
procedure
investi-
was measured corpora lutea.
of
had found a maximum cyclic AMP at 15 mm of LH were used. In
experiments data),
and
of steroidogenea detectable change of these reports we
a reinvestigation
begun
because terms amounts
several Catt
changes
It
(1973) a cyclic
Menon
et
regulatory
and
the
cell. be
is the
activity.
Goldstein
in
cyclic
might
to
confined
the
which
the
at
due
was
of cyclic measuring
by
Marsh, inabili-
AMP be
AMP
We have
experimental
was
sponse relationships steroidogenesis and
when have
increases
the
might
be
in cyclic
small
If
subunit.
protein
cyclic
of
cell
an interaction
subunit
used
Ramachandran,
low
the
occur might
detect
in
LU cyclic
kinase.
did they
to needed
increase
compartment
of
sufficient
mones can elicit a stimulation sis in other tissues without in cyclic AMP. In the light
the
to
earlier
Sayers,
and
effective
small
regulatory
of the and
the
the
of
as a compartment
one in view slice technique for
a change
protein
bin
gators
linear
a
to
AMP used
efbe not
data).
that
amount
ng/ml (Ling and It is possible that the
concentration
fact
of
nucleotide.
have
lower
the
significant
100
detect
correlation AMP
first
about
seems
of LU
steroidogenesis
while
the
to
ty
a positive
in endogenous
obscure
cause
AMP is unpublished
concentrations
methodology
AMP
increase
of
10 ng/ml
exthe
to be a serious of the incubating
fact
in
of progesterone synthesis. however, the results were this concentration of LH
usually stimulated fects on cyclic regularly detected. considered variability
to
for (Say-
the
stimulation
be of
was
increase
a
about
for
increase
preceded
higher there
the stimulation 0.02 pg LFI/ml, clear. Although
At
and
synthesis which would AMP were a mediator
in
action
lutea, specificity
previously progesterone
cyclic
progesterone pected if cyclic
corpora same
that
for probably
this in-
steroidogenesis in cyclic AMP. AMP has
(1973) cytosol cytosol The
at
The
produced
been to
in
by
found
be
localized
and
fraction
cyclic corpus
AMP luteum,
also contains a kinase (Goldstein
cyclic and
is
CYCLIC
Marsh,
1972,
this
1973;
enzyme
is
fraction
(Goldstein
probable
that
major
portion
AMP
binding
reported
1973)
located and
this
in
Marsh,
protein
of
the
some
it’s
ability
Menon
stimulate
cyclic
and of
progesterone bovine corpora
lose
its
ability
(Goldstein
to
and
a
from
6 or
observe
be
a
IU)
of
steroid
study, response
so it is not of cyclic
and
corpora
small.
very
have
been
due
when
the
corpora
on steroidogenesis. LU and hCG increase
in cyclic
corpora et al.,
lutea 1966)
incorporation and
also
specific
human
to these
about the same on steroidogenesis
et al., 1965). Corpora cycle were much more in terms
accumulation or steroid lutea of pregnancy 1974a). This diminished
receptor
sites,
and
et al. (1975).
Rao
as reported
Lamprecht corpora lutea
et al. from
luteinized
ovaries
10 pg
rat of
LH/ml
with
of
could quality by Cole
(1973) found pseudopregnant also
(Marsh,
of
AMP
corpora LeMaire, of the
et al. (1973) that
isolated rats or in vitro
in cyclic
stimulating
luteinized rats than
cyclic
AMP
rat ovaries or ovaries ovaries from immature
to
AMP
accumulation from rats.
older,
of
cyclic
Day
chance
and
Cyclic
to
Iutea
selection
were
the 7 it was
et al. (1973)
in corpora
lutea
AMP
response follicles.
however, at
of Mason
of
in
accumulation, but the response of isolated Graafian follicles was more striking. Mason et al. (1973) also found that LH was less effective in
inhibition
activity
be due to a of hormone
responded
an increase
an
cyclase
cyclic
of
may
of ovaries
in this
decline.
in pregnant In fact,
cyclic
nucleotide
phospho-
diesterase and these alternatives gated in homogenates of bovine by assaying the effect of LU
LH
lutea responsive
this
Ph osphodiesterase
sensitivity in this
synthesis than (Marsh and responsiveness
luteum of pregnancy in the number or
to be with
in a lack synthesis.
In fact, the of isolated until
LU
to the
Cyclase
Nucleotide
by
(Marsh
appeared
gonadotropin
gonadotropins
corpus decrease
in human
effect
content
grew
to
measure-
made
their study of corpora rats, found that LU
the
failure
rat or a doses of
No
to correlate with steroid
not
corpora
The increase in endogenous cyclic AMP in luteal tissue could theoretically be brought about by a stimulation of adenylate cyclase or
of mass (Marsh [8-3Hjadenine
[8-3H]AMP
The
LU
a marked
accumulation
cyclic
and showed as the effect
tissue (Savard the menstrual
about
in terms terms of
1974a).
for
activity to hCG
AMP
measured or in into
LeMaire,
brought
Adenylate
LH preparations of action of
lU).
possible AMP
declined
The
We
effect of some the mechanism
(12.5
were
a response
detect
did
in
synthesis
lutea
don’t know what the stimulatory agent is, but we don’t think it is LH. We believe therefore that this direct is not part of
hCG
quickly
they
AMP
a 20 day pregnant treated with low
increased
the
a stimulation
rats,
cyclic
in 1 day old corpora lutea. was much greater than that response
kinase
in
et al. (1974), in from PMSG-treated
markedly
report
pregnant
from been
(8
As
data).
day
ments
Ahren lutea
did
increase
PMSG
accumula-
unpublished
18
an
incubating it did not
protein
authors
accumulation by LU in incubatisolated corpora lutea obtained
lutea obtained rat which had
could
Secondit lost
in but
stimulate
Marsh,
is
a different that only
AMP
synthesis lutea,
AMP of
It
of his protein to the in vitro
come to we found
these
up also
LH
33
although of cyclic ing slices
(1973) of
showed this ability. the LH preparation
tion slices
of
that LH might by a mechanism We have confirmed
but have First of all,
to
cytosl
STEROIDOGENESIS
cyclic
preparations
few batches of LH ly, when we boiled
most
makes
about a direct stimulation preparation when added
this observation interpretation.
and the
macromolecular
enzyme assay. This indicated able to produce its effects independent of cyclic AMP.
AND
1973).
kinase
material.
that
bring kinase
Menon, also
AMP
tion was
the
absence
of detectable accomplished
or
phosphodiesterase
The
complete
phosphodiesterase by using a high
of theophylline period. Under LU/mI adenylate
of
1970a).
were investicorpora lutea on adenylate
(0.04 these
inhibi-
activity concentration
M) and a short conditions,
incubation 0.1 pg
0.01 M NaF significantly cyclase activity. Epinephrine
concentration
of 0.2
mM
also
of
increased at
produced
a
a small,
but statistically significant stimulation of nylate cyclase activity, but LU inactivated hydrogen peroxide, bovine serum albumin,
adeby pro-
lactin,
this
ACTH
and
glucagon
respect. The significance tine is uncertain since one been
concentration carried out
accumulation bated (1970)
slices have
were
and no thorough on its effect on
study has cyclic AMP
corpora
observed
in
of epinephassessed at
or progesterone of
inert
of the effect it was only
synthesis lutea.
a stimulation
in incu-
Fontaine
tine of adenylate cyclase in homogenates whole ovaries obtained from goldfish, and effect
also
required
an
elevated
et
al.
of epineph-
concentration
of this
MARSH
34 of
the
catecholamine
tissue, stimulate Pulsinelli
also
testosterone Nes, plays
mM).
also
has
(Kuehl has
The activity
found et al., cyclic
to 1969; AMP
et al., 1970a). This catebeen found to stimulate
secretion
in
perfused
1971), but it is unknown a physiological role.
testes at this
(Eik-
time
endogenous
AMP
a stimulation by
an
of
in bovine
the
inhibition
the
also
bovine, human and field et al. (1971),
rat
corpora
but
although
The
cyclase
and
was
demonstrated lutea the
localization
in bovine corpora by homogenizing
and
of
in
by Stansproperties
adenylate
lutea has been fractionating
asthe
subcellular components of this tissue by isotonic and hypotonic methods (Sidhu et al., 1975). The clase
fractions activity
Only
were and
assayed standard
5’ nucleotidase
nylate cyclase ly localized
for adenylate cymarker enzymes.
paralleled
of adenylate cyclase hypotonic procedures,
the
in both indicating
distribution isotonic that the
of bovine corpora lutea on the plasma membrane.
g pellet, indicating rat corpora lutea plasma
membrane.
out
on
effect
this
enzyme
and ade-
is primariMenon
bating and
has
LH
adenylate
also
the
plasma
membrane.
shown to stimulate activity of corpora lutea
been
cyclase
activity
the of
of
six
clase lutea
equally or
produce An
significant additivity
cyclase corpora
was
detectable lutea,
but
in homogea very
active
of
incubated et al., effects
on
PGA2,
adenylate
cy-
of bovine corpora and PGE2 were
potent and more effective than PGF2a and PGFIa did not
PGB1.
stimulatory experiment mediatory of LH on an aliquot
a bovine
luteum
corpus
was
effects. carried
incubated
concentration aliquot was
this
concentration third aliquot a fourth aliquot
out
to
role of prostaglandadenylate cyclase. of a homogenate was
maximally stimulatory (100 pg/mI). Another
In of
with
a
of PGE2 incubated
of PGE2 plus LU (10 was incubated with LU, was incubated alone as a
A
If
in
PGE2,
PGF2,)
the possible ins in the action each experiment
and
lutea
rats (Kuehl tested the
(PGE1,
test
with
(Speroff cyclase
corpora
AMP
1971)
and
possible
incorporation
from
prostaglandins
PGA2
LH transmitted cyclase solely
its via
effect PGE2,
on luteal then it
follows that a homogenate of a corpus luteum which was responding maximally to a saturating level of PGE2 should show no further response when LH was added. It was found that LU
cyclase a review rat
the
cyclic
obtained (1970b,
about
as
of bovine 3)
activity of homogenates and found that PGE,
adenylate rized in of
on
they were shown synthesis in incu-
corpora lutea 2) adenylate
and
PGF,
glandins
lutea (summaet al., 1972).
implicated
bovine 1970);
into
ovaries Marsh
PGB,,
responsiveness which of luteal regression. have carried out
correlated with Stansfield and studies on the
of
1970b);
whole 1970b).
and LU the onset co-workers
nates
carried
in the
LU action when 1) progesterone
CI adenine
14
produced
Adenylate
were hormones
Cyclase
in homogenates
other species, such as the rabbit (Anderson et al., 1970) and the pig (Anderson et al., 1974). In the latter investigation, it was found that there was a decline in adenylate cyclase activity
in rat corpora by Stansfield
studies
or other
were
of
slices Ramwell,
control. adenylate
and the plasma membrane marker Na-K-ATPase, indicating that the horreceptor and the adenylate cyclase are on
LH
on Adenylate
Prostaglandins mediators to stimulate:
activity enzyme
localized
No
of
x
enzyme system in be associated with
of Prostaglandins
of LH
pg/mI).
both
this also
system.
Role
Action
and Kiburz (1974) have also prepared a membrane fraction from bovine corpora lutea which contains adenylate cyclase, [1251] hCG binding
mone
the
to study. The largest associated with a 700
that might
the
(Marsh,
in bovine tissue were examined, made to assess the effect of LU.
subcellular
cyclase sessed
lutea
phosphodiesterase.
Phosphodiesterase
of this enzyme no attempt was
corpora
adenylate
of
made it difficult of activity was
Possible of
lutea: no change in activity was a variety of experimental condi1970a). These data indicate, LH brings about the in’rease in
cyclic
ATPase amount
if it
effect of LH on phosphodiesterase also was evaluated in homogenates
bovine corpora observed under tions (Marsh, therefore, that
not
In testicular
been
adenylate cyclase (Murad and Eik-Nes, 1970) and
accumulation cholamine
by
(0.05
epinephrine
a clear-cut additive effect by a maximally stimulatory
produced tration
LH
PGE2, probably (Marsh,
Other mental
not
were
cyclase a variety of
Jonsson
using reported
have
format
adenylate
et
al.,
that transmitted
the via
effect of prosta-
1971).
investigators
prostaglandins in
indicating
of
was
above that concen-
in their
additive and
tissues 1972;
a similar that
experi-
LU
and
effects
on
AMP accumulation (Kuehl et al., 1970b;
cyclic
Kolena
and
Channing,
CYCLIC
1972; ever,
et al.,
opposite
conclusion
was
group
of investigators
et
by
another
aL,
1970b)
who
prostaglandin acid, on ins of
evaluated
produced
a subsequent 7-oxa-1 specific
AMP-dependent Marsh and
show
of
They
inhibited [1 4C]
Channing prostynoic
et was also
LU on granulosa was
of the
(1972) also acid inhibited luteinization cells. The
complicated
inhibitor produced
action
of
synthesis (1972)
this out
into
prostaglandin-like idonic acid nates,
and
with
lutea, and inhibition very large they could
competitive
fact
type.
that
added
to
the
suppression. LU can slices of
stimulation
LH
when
stimulate monkey
into
studies
cyclic
AMP
been
synthesis
reported in
incubated
the
LH
to
this
be of
prosta-
administered
in
Tsafriri
et
al.,
antagonists,
synthesis, glandin
and studies of synthesis. Although
have
not
approaches
possibility
glandin
action studies,
yielded
of
a
definitive
majority to an
of the essential
glandins
the
action
prosta-
prostaglandin
inhibitors these
problem, the in opposition in
have
that
of LH on adenylate studies of prosta-
studies
of
prosta-
approaches
answer
to
data appears role for
of
LU
on
the
to be prosta-
adenylate
cyclase.
It
is generally
recognized
suspect adrenal corpus
from the work cortex (Haynes luteum (Marsh
cyclic
AMP
or
cyclic followed carried
ment
Effects
In
in
this
investigation
The
of
of
the
effects
work for
the
of
the
role
of
in the testis to the studies began with the cyclic
exogenous
cyclic
AMP.
AMP
will
be
Cyclic
studies,
last
This
uncovered some in this field, and
has recently investigators
Exogenous
early
the
progressed to the assessment and finally to the measure-
endogenous
problems
of
of
action of
AMP in steroidogenesis a very similar pattern out in the ovary. It
on steroidogenesis, of adenylate cyclase
ade-
synthesis
the
that had gone on in the et al., 1960) and the and Savard, 1964b) that
involved
was
study
be mimDibutyryl
that
of androgens in the testis is under the control of LU (also called interstitial cell stimulating hormone, ICSU), and it was reasonable to
that
intact
the the
this
to stimulate
when
general
study
mediate additivity
these section.
beyond
LH 1972,
glandins cyclase:
gonadotropin.
synthesis in et al., 1972)
it could AMP.
the
reported stimulation
was
al.,
four
to
to
occur
et
summary, used
phase of the new problems
because cyclic
prostaglandin
animal
by
been
1972a).
Graafian follicles (Marsh latter report, however, this
cyclase step, by exogenous also
the
been the
and
have
in inhibiting
inhibitor
(Grinwich
vivo
of
H] arachhomoge-
[3
indicated
prostaglandin ovaries (Wilks
appears
Pharriss of LU
overcame
also
nylate icked
has
this
synthesis
from of ovarian medium
rabbit In the
has
prostaglandin aspirin
THE ROLE OR CYCLIC AMP IN STEROIDOGENESIS IN THE TESTIS
or LU it
the
injected
incubation Other
and incubated et al., 1974).
suppressed
synthesis
of monkey of this effect
Chaslow and the injection
compounds incubations
in
acid AMP
role for prostaglandins in the on adenylate cyclase would stimulation of prostaglandin
rats
that
regard, experi-
3-prostynoic into cyclic
steroidogenesis
glandin
been
found that 7-oxa-13the action of PGE2 or
the
or release. reported that
antiserum
of
compounds
has also inhibiting
in
ovarian
In
not a completely inhibited cyclic
was mixed with prostaglandin severe cellular necrosis.
A mediatory action of LH suggest an LH
LU.
that
1972)
These
be ineffective
ineffective
with some indicated in
al.,
acid.
Indomethacin
therefunctioned
in cultures interpretation by
the cyclic
concluded, receptor in the
effect of LH the antagonist,
it was
of inhibitors as indomethacin,
of cyclic AMP accumulation in mouse or rat ovaries although they a marked inhibition of prostaglandin (Kuehl et al., 1973; Zoretal., 1973).
(Kuehl
of
that
use such
action
to
in incubated slices of bovine corpora although they could demonstrate an
not
the
prosta-
of LH
reported
ments on the effect of 7-oxa-1 on [3 UI adenine incorporation
of the stimulatory concentrations
been
evaluating
mediators
stimulation incubating produced synthesis
protein kinase. In (1974b) carried
LeMaire
as possible
1973).
found that this antagothe effect of PGE1 and
acid but
antagonist,
to
flufenamic
should be viewed the same authors
paper
a
et al.,
synthesis,
LU.
3-prostynoic
glandins
(Kuehl approach
prostaglandin incu-
fore, that a prostaglandin as a necessary intermediate This conclusion reservation, since
ovaries
Another
(Kuehl of
35
3-prostynoic
competitively accumulation
by
how-
effect
7-oxa-1
bated rat ovaries. They nist not only blocked
AMP
reached, the
antagonist,
also of the
STEROIDOGENESIS
1973).
the stimulation by LU and [1 4C] cyclic AMP accumulation
PGE2, but stimulation
AND
mouse
Lamprecht
An
AMP
discussed
later
in
this
AMP
whole
testis
prepara-
MARSH
36 tions
were
were
the
AMP
could used
They
used, to
in
synthesized lesterol
into
[3
AMP
concentration.
confirmed synthesis slices
of
(UalI
and
testosterone
(0.027
an
far in excess
testis.
not
assessed that
of
cyclic AMP on of testosterone
Their
report
about
by
1962)
that
respond
concentrations
an
testicular
cyclic
AMP,
[1-’4C] Eik-Nes
as
acetate (1968)
(107
M),
indicating
that
Other 1972b;
investigators Catt et
(Dufau al., 1972;
1972)
have
demonstrated
et
al., 1971, Rommerts
that
(Catt et stimulation
identical et al.,
The
aL,
dibutyryl
that
produced
nucleotide. using the
cyclic
Van same
effect of steroidogenic
AMP
into
of
pregnenolone.
with
of
U] cholesterol
by cyclic cate that
the
hCG dog gave of
and Eik-Nes demonstrated
These into
3
taken
conversion
luteum cortex
Mieno
et al.
cyclic
AMP
between cholesterol it seems to be.
(Uall and (Karaboyas (1973) into
Koritz, and
found the
systemic
that
1965b)
Koritz, the
AMP
was
was
a
Near-
above
the
but
these
steroidogenesis.
by
is well within the concentration of cells (Rommerts and
Moyle
Leydig
low,
al.
et
cell
(1971)
1974).
exogenous continuous
tumor
using
preparation,
steroidogenesis Moyle et
stimulatory steroidogenesis
of
effect has
a!.,
exogenous thus been
mechanism
by
was
another which
to both LH and cyclic AMP. however, required the usual of cyclic AMP (2 mM to
M) to stimulate Armstrong, 1970;
but the uncertain.
Fritz, of its
to sustain the stimulation The same phenomenon
This high 0.02
(Moyle 1971).
and The
cyclic AMP well established,
which
it works
on
is still
Cyclase
Another approach to the investigation of the of cyclic AMP in testicular steroidogenesis the study of the adenylate cyclase system tissue
and
its response
Murad et al. (1969) FSH would stimulate in whole homogenates of
FSH
of
an
and
the
seminiferous stimulation
of
(Cooke 1974;
effect
et Braun
tubules this
al., and
to gonadotropins.
found that both LU and adenylate cyclase activity of dog and rat testes.
stimulation
of
with
exogenous AMP have
AMP,
cyclic
quite
required
The
of the
with cyclic
concentration
and the
1965).
out
Dorrington
this
in
injection
circulation
1973;
al.,
was sensitive preparation, concentrations
role was
AMP
nucleotides
of
insento very
synthesis.
M cyclic AMP. This of the endogenous AMP in normal Leydig
Although
in this
U] testosterone
AMP (Sandier and Hall, 1966) indithe site of cyclic AMP action in the as
106
et
these
was
found that this preparation of Leydig cells could be stimulated by as little as 5
Adenylate
at steps to the
data
of the
workers tumor
mouse is
the
occurred prior
stimulation
testis is somewhere pregnenolone, just corpus adrenal
AMP pathway
of
Leydig
respond
cyclic
be
and Shin a monofrom a
which
carried dibutyryl
level
to
isolated
in progestin
amounts
caused
(Hookcyclic
(1967) that derived
tumor,
exogenous
used
observed
in vivo also and secretion
der Molen technique
cyclic
together [7o-3
in
would
of the studies AMP or even
of
1972a). of
of
cyclic
in vivo secretion.
considered
studied
cell
ly all cyclic
presence
testosterone (Eik-Nes, 1967, 1969). Again large doses (150-300 pg/mi) of this nucleotide were required, but the effect was specific for the
formation
been
increase
cyclic
time
by
levels
cyclic
cyclic
and its steroidogenesis
testis via the spermatic artery rise to an increased production
that the in the
also
the
production of exogenous
to gonadotropins,
low
x
1972a, et al.,
1972),
of
to
infusion
cyclic (1971)
generally
interstitial
sitive
range
capable of accelerating testosterone in whole testis preparations. This is about 50 times more effective than
AMP of
(Dufau
has
AMP
are
site of testosterone and the effect
physiological
of theophyllmne and testosterone synthesis
nucleotide phosphodiesterase cyclase system in testis tissue.
almost
cells
main 1970)
testicular of
injection
directly into the testis increase in steroid
significant
study
rabbit
of and
the effect it stimulated
corrected
a previous
to
incorporation Connell
the in
there was a cyclic and an adenylate
cyclic course
nucleotide a considerable the er,
in stimulating the
mouse
Eik-Nes,
AMP is production analogue
effective but
Leydig
of [7a-3U] choThe amount
M) was
not
this effect of mass amounts
judged by the into testosterone.
at low
of
was
steroidogenesis,
cell tumor preparations. Shin and Sato (1971) demonstrated layer culture of Leydig cells
would
also found
the
mass
dog
cyclic
production. observed
brought
tissue
(1966)
levels, but the effect was specific, ADP and AMP had no effect at this Connell and Eik-Nes (1968)
of rabbit
impression
Hall
exogenous
the conversion U] testosterone.
used
physiological since ATP,
and
that
testosterone rat testis and
both and
Sandier
show
stimulate teased
increase
cyclic
and
first
appears
to
adenylate and enzyme
1972;
the in
due
to in
LU effect interstitial
Dorrington
Sepsenwol,
be
cyclase
and 1974).
NaF
a the
to a cells Fritz, also
CYCLIC
produced centrations small
a marked stimulation, of epinephrine and
increases
in the
activity
AMP
AND
STEROIDOGENESIS
nated
and high conACTU caused
of this
enzyme
have
by
any
ACTU on
effect
since
it is not
testicular
known
or
spermatogenesis, and there is disagreement on whether or not it has any effect on cyclic AMP accumulation in whole cell testicular preparations (Kuehl et al., 1970a; Cooke et al., 1972). The
epinephrine
effect,
observed by Nes, 1970;
other Kuehl
catecholamine
however,
has
groups (Pulsinelli et al., 1970a)
has
also
been
been
and
found
to
cause
a
when it via the
spermatic
Eik-Nes
(1971) nerve
suggested fibers and
system stimulation rime, tissues whole been
(Eik-Nes,
of
that this effect may the regulation of the
the of
since must testes.
1971).
testis. adenylate
This
might cyclase
reported
in homogenates and
of
corpora
“mitochondrial” were identified
fractions, only by
characteristics. microscopic
No data
enzyme were
but their
assessed the correlation with marker enzymes
branes
(Na-K
dependent
chondria (succinic the cyclase to plasma membranes. also
preparation
which
seems,
was
was
therefore,
that
most
of
the
subcellular
The cyclic
rat
testis
also
contains
two
phosphodiesterase
the
but enzyme com-
isoenzymes desig-
(Uollinger, phosphodi-
the
on only
effect
of gonadotropins LU or 1 ng/ml of
of
been
1972; and
high
(greater hCG) were
than used
found
Catt Dufau,
to
is apparent et al., 1972;
et
Ramachandran, Dorrington
stimulation that prolactin,
al.,
of
1972).
a
AMP in 1970a;
et al., 1972; Dufau 1973; Braun and
A similar effect preparations (Cooke
This in
cause
cyclic et al.,
1974).
cell
gonado-
relatively
of endogenous vitro (Kuehl
Rommerts et al., et aL, 1973; Catt
in endog-
have, however, proposal that
When
repeatedly
(Cooke
effect
its
changes
testis the
steroidogenesis.
increase testis in
In regard It
testis.
nucleotide
mediates
on
the Leydig Sepsenwol,
adenylate
with mitochondria ponents of the of
AMP
tropins
increase cyclic AMP in tions, but, as mentioned act only on the seminiferous
but with
established. membranes, of this
other
on
in the supported
growth hormone do not AMP levels in either type
endo-
1973),
cyclase is located in plasma there may also be an association and
cyclic
tion
life on
and
1974). specific
mito-
enzyme
adenylate
transmitted
Fritz, quite
responsive mitochondrial
of
1970a)
out
AMP
in Leydig
rat’s
rat 80
Endogenous
carried
Spensenwol,
of 5
cyclase.
enous cyclic not completely
have
in
amount increases
of
effect
probably
and
concentrations 10 mg/mI of
Km
found
in
that
of adenylate plasma mem-
free
not
is
adenylate
marked whole
increase to
acute
et al.,
is a high
The testis
et al., 1973; Moyle and Rommerts et al., 1973;
and
relatively
(Kuehi
through
This of the
no
Km
in the
50 of life in the constant to Day
similar
period
has
testis. in the
Cooke 1973;
(Sulimovici et al., possible contamination
membranes
reof the
and found associated with and Lunenfeld
described a hormonally cyclase in a testicular
in this LU
only
c form
to Day essentially
1972).
Christensen is a low
is presumably
the
is very
1970).
is found
or electron Pulsinelli
ATPase)
dehydrogenase) be primarily Sulimovici
plasmic reticulum the degree of plasma
for
20
al.,
cyclase
they
these fractions sedimentation
marker presented.
(1972) cyclase
lutea
(1971) cyclase and
from Day then remains
Studies
obtained
Uollinger (1970) and Eik-Nes ported that most of the adenylate the testis was found in the “nuclear”
(1973) adenylate
involve vascular
explain the by epineph-
whole ovaries et al., 1970).
fold and
et
and The
other parts of phosphodiesterase
production
f form
M) and
the total
Measurement Cyclic AMP
elements of nerve and vascular be included in homogenates of A similar effect of epinephrine has
(Marsh, 1970a), from fish (Fontaine
tubules.
1972;
The M)
10-5
x
steroidogenesis
small increase in testosterone secretion is infused into the dog testis in vivo artery
10-6
x
(6.5
(Monn
et al.,
1973).
(2.5
esterase
Eikthis
and
type type
to
steroidogenesis
f (Monn
Desautel,
seminiferous
whole homogenates of dog testis. Glucagon had no effect. It is difficult to suggest any physiological explanation for the small stimulation produced
c and
and
in
37
increase testicular FSU
and
appears ACTU
was
the
to
be and
cyclic prepara-
found
to
whole testis preparabefore, it appears to tubules and not on
cells (Cooke et aL, 1972; Braun and 1974; Dorrington and Fritz, 1974). to
the
time
course
the rise in endogenous cyclic about 10 mm of incubation
of the
hCG
effect,
AMP appears with hCG,
after while
the increase in testosterone production was not apparent until after 30 to 60 mm of incubation (Rommerts et al., 1972; 1973). This is very similar to the kinetics of the stimulation of cyclic AMP accumulation and progesterone production
by
LU
in
incubating
slices
of
38
MARSH
corpora
lutea
(Marsh
et
1966),
al.,
compatible with the proposal role for cyclic AMP in the steroidogenesis by gonadotropins. When
careful
sponse
of
terone ever,
comparisons
cyclic
AMP
production a dissociation
been tion able
of
change
AMP.
in
This
effect nucleotide
or
of
of
a is
role that
activity terone
this
testos-
out, effects
of
cyclic
since
review
their
level
work
maximum
value
at
0.8
to
hCG and
1.0
enzyme
concentrareached
a the
in this to
report,
but
mm
had
240
(Catt 1-500
et
cyclic reported tion
to
be
in the
more
accumulation
prominent in
in
was
the
amounts
the
testis
found
cyclic of the
been AMP incuba-
tissue
to of
the
medium
was
parallel cyclic
the AMP
AMP comprised cyclic AMP released.
into incorin
accumulated added that
to the newly
a constant
5
released
of was in
to
the
that this phospho-
AMP, or cyclic GMP, are substrates for this 1973).
and isolated in pg
ng
hCG.
Recently
Po-
study carried They
Leydig mouse and
the
a striking
AMP
was the
and for that
this
problem
out by assessed
in
Moyle and the effects
of LH on testosterone accumulation in
after
AMP the
after
tissue.
rat
for
2 10
Again
between
an increase required
an
the
h
of
mm
in
there amount
in testosterone for a stimulation
in incubations of required for a miniwas about one order
(approximately required
of steroidogenesis in the isolated
synthesis
measured
of cyclic AMP accumulation either tissue. The amount mal increase in cyclic AMP that
kinase was reached a
and
corresponding
on
cyclic
difference
magnitude
of prepa-
cell preparation from rat testis Leydig cell tumor preparation.
medium
of LU required production and
protein hCG The
was (1973).
concentrations and cyclic
the stimulation Leydig cell
testosterone
major
testosterone
was
of cyclic AMP steroidogenic
to expect nucleotide
al.,
increase 100-250
other
incubation
above
carried
changes
at
of various synthesis
of
itself
of 14C-ademine in 14C-adenine
when 20-500 ng of hCG/ml were incubation medium. This indicated synthesized proportion
1
In addition, in both of the an assessment of cyclic
incorporation An increase
poration mass
from previously
has also in cyclic
than
out using the cyclic AMP.
studies out
Dufau et al., 1973) with and no transient rises in
et al., 1973). just mentioned,
AMP
time carried
were detected. It the relative changes
medium
(Dufau studies
been
al., 1972; ng hCG/ml
AMP that
appeared
thorough
in the
et
of
to these a small
Dufau (1975) reported that there was dissociation of the effects of hCG on
range for pg of hCG.
both
only
AMP
at
medium
h) and
cyclic
dose-response was 5-100
one
incubated effects
This stimulation by theophylline a detectable increase
acts via cyclic nucleotides
An
isolated and a
the the
levels.
reasonable of cyclic
(Goldberg
The
by
amount in the
maximum
The
(2
of
It is inhibitor
diesterase since both
began at at about only at
period
medium potentiated
by itself release of
of hCG, indicated Dufau, 1973) that
and
amount
medium. classical
other hand, the release of cyclic AMP 1 ng hCG/ml and reached a maximum 100 ng/ml. The cyclic AMP was assessed time
the and
as yet undetectable probably involved
gonadal tissue Ramachadran
rat release
On
into tissue,
ration. detectable
in some
ng/ml.
5 mM theophylline increase in the
testosterone synthesis protein kinase in an
quite testis,
on the release of testosinto the medium during
increased by 0.1 ng/ml
fact that a significant
desta and a similar
compared the effects of hCG (specific
10,000 lU/mg) and cyclic AMP
as
of of
the physio1971). Two
have carried out problem in the
Dufau (1973) concentrations
significantly as low
of
of cyclic AMP in there should be a
producing et al.,
and was
the
one
concentration smallest
The caused
response to low hCG testosterone production also observed without
a detect-
level
a 2 h incubation of intact decapsulated testes. It was found that testosterone was tions
howhas
stimulawith low
without
on the by the
therefore,
Catt and various
dose and
discrepancy,
investigators studies on
and I will, detail.
low concentrations authors (Cart
the
carried two
hCG
the hormone capable of logical response (Robison groups thorough
re-
endogenous
is a crucial
demonstrable this cyclic
testosterone testicular
accumulation
LU
for action
of
brief, a hormonal can be elicited
the
the criteria hormonal
is
of
have been of these
uncovered. In of steroidogenesis
concentrations
and
of a mediatory stimulation
10 a minimal
(approximately Leydig cells.
ng
LU/ml)
stimulation
1 ng LU/mI) A time study of
the changes in cyclic AMP with these rat Leydig cells was carried out and showed that cyclic AMP accumulated progressively from 2.5 to 60 out
only
relatively high concentrations of ng and 5 pg LH/ml). The results of another very interesting
LU
(500
iment, increase
that the relatively
mm,
but
again
this
study
was
carried
with
reported in this paper, indicated in cyclic AMP, observed with
exper-
CYCLIC
high
concentrations
this sis.
gonadotropic It had been
al.,
1971;
cally
to
et
procedure terminate
The
bound
ever,
by
hormone LH, rate
be
the
hand,
it had
cyclic mone
AMP mimicked on steroidogenesis, nucleotide
but
could
be
specifi-
cells,
stimu-
that
a wash-
an the the
On
of the horeffect by the
rapidly
terminated
cells. The interLU was retained
by the cells even after repeated washings, and that the continued presence of the bound LU was required for maintenance of the steroidogenic response. Since the dose response studies just described (Moyle and Ramachandran, 1973)
raised
some
cyclic AMP steroidogenesis, was
in
carried
effect enous
out
role
of
the hormonal stimulation a similar washing experiment
of
by
procedure level of cyclic
concentration in a rapid
of cessation
one
LU (100 of cyclic
ng/ml), AMP
had
high
which had terminated
lower affinity (since by washing) and was
the
increase
cells and
Moyle et al. carbohydrate this hormone
the of
from rat accumulate
the removal resulted in derivative tion
but
in
stimulate
a much
for
mediated
cyclic
(since
AMP
smaller
the on the Leydig
testosterone found that
carbohydrate in the ability
cyclic
AMP
decrease
two
LU. One response
which and
AMP
accumulation.
of hCG of the
accumulain
steroidogenesis. there were
was with
synthesis.
(1975) investigated portion of hCG to stimulate
its
from
it of
the which Further
be
necessary
an increase
to determine but it does seem AMP accumula-
the
testis of
caused LH is
by not
in steroidogenesis. of cyclic
AMP
accumula-
the effects of ACTU on corticoster(Beall and Sayers, 1972; Moyle et Richardson and Schulster, 1973) and
the
droplet
effects
of
formation
TSU
on
thyroid
(Williams,
1972)
colloid have
been
It seems,
then,
that
the
question
of the
role
of cyclic AMP as a mediator of steroidogenesis in testicular tissue is still unanswered. There are several reasons why small changes in cyclic AMP concentration if they are present, evidence level of
this
If
we
not be detected, until there
MODES
AMP
ON
will
remain
there
is cyclic
and of
with carried
be
the site of steroidogenic between al., 1963; and Hall, strong et
that
possibly LU on
cholesterol Hall and
LU
and cyclic has been
mitochondrion,
tion the
cleavage
in
AMP shown
on the to be (Ichii et Koritz Armseveral
by
listed briefly as 1) an increase in as NADPH; 2) an
of the substrate, in the availability
promoting where
system
its transport the
some
restraining
enzyme system, perhaps transport of an end-product pregnenolone, out of the
4)
sidean
of system;
influence by
of into
cholesterol
is located;
or an increase in the synthesis components of the cleavage
decrease
concenwe are
cyclic AMP might bring about of this reaction and these are
the concentration 3) an increase
the
AMP in the in the corpus
Young, 1968; are, however,
shown in Figure 1, and are follows: It could bring about the amount of a cofactor such
cholesterol
considerable
and pregnenolone Koritz, 1964, 1965b;
1965; Hall and al., 1970). There
this
OF
the action of high Leydig cells, then
action of pathway
in
low is
the problem of how this effect out. As mentioned previously,
ways by which the acceleration
increase cholesterol;
even firm
in doubt.
OF ACTION STEROIDOGENESIS
in favor of a role for of LH on steroidogenesis
luteum trations
believe
is
in cyclic AMP at the or another messenger
question
POSSIBLE
evidence action
might but
for a change gonadotropin
discovered,
chain
effec-
Again types
mediated another
tion from oidogenesis al., 1973;
might
the
the effect associated
to
dissociation
confronted
washing) and and another
testes to synthesize cyclic AMP. They
tiveness to stimulate was suggested that receptors steroidogenesis
by
cyclic
of part of the a marked loss to
affect high
from this of receptors
affinity
for
Similar
what
it did result accumulation
LU. It was proposed might be two types which
see
did not a relatively
not be terminated with steroidogenesis,
Recently role of ability
to
required
CYCLIC
have on the endogIt was found that
effect could was involved
major
the
authors
would AMP.
washing procedure response to
caused by the data that there LH:
about
these
this
although the the steroidogenic
for
questions
seem
validity of this suggestion, the large increase in cyclic
reported.
exogenous
effect this
washing the tumor of this data was that
with
would
the that
LU
terminate that
work
39
tion in the Leydig cells of relatively large concentrations
how-
cells
shown the
et
LU
removed,
washed
been
STEROIDOGENESIS
for
the bound steroidogenesis.
and this did of steroidogenesis.
other
by simply pretation
and
could of
required
Leydig
not remove enhanced
AND
steroidogene(Moudgal
that
production, can the
to
not
1971)
tumor
treatment
antiserum accelerated
cyclic
al.,
these
testosterone
ing nor
was
stimulation of shown previously
Moyle
binds
lating
of LU,
AMP
activaone 5) on
of a
this
enhancing the inhibitor, such as mitochondrion. There
40
MARSH
tion
LH
accelerated
in the glucose glucose
/-\ AlP
CYCtIC
AMP
i
(
duction cofactor
I_#{149}.4.
PHOSPHORYLASE
KINASE
]
PRoTEIN
I
.
:
sis, FREE
/ I
,,
CHOLE5ROL
\ “S
0
SYNTHESIS
\
\_#
5
E
STEROL
.
*-
)
the
et
al.,
produced
-H
PREGNENOLONE
FIG. 1. Possible sites of action of cyclic AMP on steroidogenesis in a hypothetical gonadal cell. Solid arrows indicate biochemical reactions, and the large dashed arrows indicate possible effects of cyclic AMP on the steroidogenesis process. Five of the dashed arrows indicate that the possible effects of cyclic AMP might be mediated through protein kinase, but it is also possible that cyclic AMP influences these steps in steroidogenesis by a mechanism not involving protein kinease. substance
The symbol ----p through membranes
supporting
is evidence and
the
at
these proposals it is possible
one
or more
are that
of these
in favor discussed
of (or below
obtained
from
the
time
of it
one of them however, it
that and
The with since tissue
each
present
choose any this regard,
indicates transport of the cell.
these
is not
as the should
to
The
evidence
hypotheses on the
with
In
is data
gonadal
tissue.
results of some important experiments adrenal cortex tissue are also included, the control of steroidogenesis in this has the same general features as that in ovary
and
the
testis.
Via Increased
Haynes (1960) AMP
and
were might
adrenal
Berthet the first accelerate
cortex
of a necessary pathway. Briefly,
by
(1957)
to propose steroidogenesis
increasing
cofactor their
and
the
tissue. in
Marsh and son, 1965; rat ovaries
phosphorylase
system;
2) phosphorylase
prepubertal
the
pathway
(Hal-
NADPH is in
system
such
Estabrook reducing
activity lutea
rat
the
as that
(1969) equivalents
increase
this
activity
(Selstam
is
in vitro
and
vivo will activity
et al.
lutea of
and
(35
phosphorylase ase activity containing enzyme
1964a),
was
was
1973). of LU
It has also into rats in
LU but
and
phosphorylase the luteinized
that
was
have
limited a large
been
number number
cows
were
examined found
a
signifia prep-
ineffective.
to
from was
1965; 1973). lutea,
although
and hCG activity,
however,
obtained
rat
Ahren
hormone
pregnancy percent)
slices Savard,
and Robinson, Ahren et al., bovine corpora
out with only a lutea, and when
Savard,
Ahren,
1971;
reported
appears,
corpora
and
in the or
preparation phosphorylase
of luteinizing study
months small
(1961)
1961;
and Robinluteinized 1965) and
in incubating
Ahren,
type of ovary (Stansfield Seistam and Ahren, 1971; In the experiments on pituitary increased
detected
et al.,
lutea (Marsh and isolated prepubertal
cause an increase in the prepubertal
Williams
a
1971; Ahren et al., has been shown to
Selstam, 1971; Ahren et al., been found that the injection
crude cantly
been
(Selstam
Selstam, LU
of bovine corpora 1964a) and whole ovaries
has
(Williams
ovaries
Ahren and Furthermore,
cyclic in the
activa-
latter in the
cells
and
Savard, 1964a; Stansfield Yunis and Assaf, 1970), (Stansfield and Robinson,
that
1)
cleavage
for this localized
cortex
1961)
and the
corpora
et al.
ACTU increased the synthesis of cyclic AMP in the adrenal cortex, which in turn activated the
adrenal
Phosphorylase
bovine
Uaynes
concentration
of pro-
There is some evidence in support of mechanism such as that suggested by Uaynes and coworkers for the action of LH in ovarian
carried corpora
in the steroidogenic proposal stated that:
phoslevel
side-chain
pentose
organelle.
of of
pentose
increased corticosteroid
system to be
a shuttle
into
aration Cofactors
the
by Simpson to transport
This Action
the
described required
1971; 1973).
exclusive, may act by
mechanisms.
against) these with emphasis
a
proposals, possible
correct one. be recognized
not mutually cyclic AMP
experiments
of
cholesterol
Thus
this
the an
resulting
amounts metabolism
of its role as a required the steps of steroidogene-
1959,
by
cytoplasm. PROGESTEEONE
of
of
kerston
MITOCHONDRION
through
The enzyme has been shown
mitochondria
%
PREGNENOLONE
*-
including
increased 3) the
produced stimulated
by means in many
reaction. reaction
NADPH
breakdown,
of
6-phosphate
phate pathway NADPU, which
I PROTEIN CHOLESTEROL ESTER
glycogen
formation 6-phosphate;
in the
of of first
to produce
significant
6
(Marsh increase
a in
activity. The luteal phosphorylmeasured in an assay medium
5’-AMP, similar
which indicated to the liver enzyme
that this which is
CYCLIC
only
partially
activated
by
the
AMP
AND
nucleotide,
STEROIDOGENESIS
and
differed from skeletal muscle phosphorylase which is completely activated by 5’-AMP. This was confirmed when the luteal enzyme was purified (1970). the
and The
characterized stimulation
incubating
specific
slices
for
lated
an
increase
in these slices. concentration
Assaf in
corpora
lutea
hormones
and
in progesterone
was
not
cause
although
it
a rise did
a small
increase
in progesterone synthesis. One LU and nucleotide incubations
difference was the
sion of medium.
the
0.02 This
M caffeine methylxanthine,
in
in the inclu-
cyclic addition
in
AMP to
its inhibitory effect on cyclic nudeotide phosphodiesterase, has also been reported to stimulate phosphorylase phosphatase (Sutherland, 1951), which may account for the small decrease served
in in
phosphorylase the cyclic
presence
of caffeine
account
for
does
the
phosphorylase
activity actually AMP incubations. not,
lack
of to
system
however,
of
other experiments the omission of
(Marsh, unpublished caffeine from the cyclic
treated
samples
eliminated
activity,
but
still
did
not
A larger has also lutea
exogenous
increase
the
concentration been tested
in the
stimulation an effect
(Savard
et al.,
better
of
synthesis
the
might
stimulating supported
percent
the increase
activity
while
they
LU in synthesis
terms of (Marsh
also
supported
been
to
stimulated us that be
an
corpora exhibited an and by
tested of
finding
of This 10
showed response
in progesterone 1964a). This that
bovine
h
glycogen
Selstam,
and
activity. ovary muscle
in vivo
entirely b form,
The was and
to a shift from to the 5’-AMP
enzyme. at
The
LU of
or
mM
phosphorylase
phosphorylase
it
mM
to
effective
the than
at a concentra-
increased
the
appears
that
LU
and
phosphorylase
percent
similar
situation
Uaynes stimulated
perhaps
in
preparations, but it seems that not be essential for an increase A
of
cyclic
of
a.
stimulate
cortex. ACTU
dependent a form
1-25
more
Epinephrine also
of
dibutyryl
from
FSH.
10
prepubertal rat a rise of ovarian
of
was
of LH
reported to resemble the increase was due
addition
ovaries
et al.,
injection
the 5’-AMP independent
concentrations
incubating
no
Ahren,
Ahren
in vitro incubation of with LU or FSU caused
this that
in
but
concentration.
1971;
the
that the about a
later,
(Selstam
phosphorylase
tion
found caused
activity
4
in
that
prepubertal of skeletal
Kilpatrick, glycogen.
phosphorylase
coworkers
It
has
NADPU
FSH
several
ovarian
this effect may in steroidogene-
exists
in
the
adrenal
(1958) demonstrated phosphorylase activity
been
to (Mason
that in
is
that
the
addition
of
slices of bovine corpora 1962; Savard et al., 1963;
and Casey, 1964; Armstrong, 1966), lutea or interstitial
Mason and Savard, to slices of rabbit tissue (Dorrington
and Kilpatrick, 1966b) or to homogenates of whole rabbit ovaries (Scoon and Major, 1972), caused a marked increase in progestin biosynthesis. strated the
to
found
incubating et al.,
In addition, Savard et al. (1963) that the bovine corpus luteum
concentrations
phosphorylase
a good
increase Savard, the
effect
synthesis. that about
the ovaries or
Savard 1964b; corpora
activate phosof exogenous
essential
reported
lutea
progesterone phosphorylase
lutea
in terms
cyclic
exogenously. to
1973)
(1965) in vivo
ovaries
and
1965), Armstrong, 1965) and
bovine adrenal cortex, but Ferguson (1963) and Kobayashi et al. (1963) found no stimulation by ACTU in rat adrenal cortex tissue even though this hormone produced its usual stimulation of corticosteroidogenesis.
resolved,
intracellular
progesterone by the fact
of or
there
synthesis activity
nucleotide
not
no
little
again
that adding LU to slices may be a much
this inability a concentration
by
activation
never
increasing
AMP which indicated
cyclic
in
suggested lutea
adding
Nevertheless, phorylase
and
Ahren
sis.
activity. (0.02 M) corpora
progesterone phosphorylase
has
been corpora
than
caffeine
1971;
Thus
in
in
changes and
can
1965).
way
AMP
LU was
of of on
discrepancy
but it has incubating
drop
AMP bovine
increase
Ahren
either
Robinson,
and measurable
and Robinson of LU into rats
significant
the
and
(Dorrington little or no
luteinized
to
this for in
nucleotide
phosphorylase
ovaries contain
Stansfield injection
AMP
data) AMP
slight
cyclic
of cyclic on these
absence
was a without This
the
the
rabbit 1967)
obThe seem
response cyclic AMP,
(Stansfield
rat ovaries (Deane, 1952; Stansfield and Robinson,
their
in phosphorylase
cause
lutea
luteinized 1963;
4-fold
corre-
synthesis
The addition of cyclic AMP at a of 2 mM to the incubating slices,
did
activity,
of
LU-containing
with
however,
by Yunis and of phosphorylase
corpora
41
pentose
however,
that
tatively quite first indications the
study
of
of the
NADP
pathway.
It
became
the
of
NADPU
effect
dehydrogenases
different from that of this difference Savard
et al.
demonhad high
(1963)
of
apparent, was
quali-
of LU. The came from where
it was
MARSH
42
found
that
addition
the
incubating
the
in
of
LU
presence
of
of NADPU, caused a further terone synthesis. This should
if the NADPU. also
effect of LU Dorrington observed
effective
be
effects
different
in
LU
of
LU and corpora
and
of
NADPU. to
utilization.
precursor
that for
nearly by
shown that to incubating (10
mm)
free
cholesterol.
either
by
esterase
or
ester action
synthetase, has been
NADPU,
on
the
1964;
into
this
Mason
and
became (1966)
steroid
NADPH
damaged
cells
intact
the
of
adrenal
probably
while
LU
and
it
Armstrong luteum and cortex
acted
only
on
ACTU
acted
on
cells.
In
regard
to
the
endogenous
concentration
of NADP and NADPU and the effect of LH, it has been reported that this gonadotropin has no effect on the concentration of either the reduced
or
nucleotide 1968)
the
or
Denton,
1970).
gators cellular
form
rat
and
that
ovaries, in this
Denton,
that
LU
of
account utilization
the
duced
increase
thereby
concentration seems theoretically more
of
information
had no effect It was concluded
therefore, about its
it
It
Via Increased is
well
known
that it was increase in
not
take
into
of its production, unaltered
how
by the side-chain cleavage impossible to reach a firm matter. Action
on
the produccriticized this
did
NADPU. possible, on
stimulation an
of
inhibition
the of
and evidence presented.
and the
into
come
about
cholesterol
the for
cholesterol both
sites
of
esterase activity No direct effect
in luteinof cyclic
theophylline
mM)
(0.05
mM)
observed, added to
or
however, when the cholesterol have
(1
these esterase
Flint
et al. (1973)
AMP assay rabbit
had no direct effect system for cholesterol ovarian interstitial
also
demonof LH
was
substances were assay mixture.
reported
that
cyclic
when added to the esterase activity in tissue. Another piece
of evidence indicating that LH activates the cholesterol esterase enzyme is that of Behrman et al. (1972), who showed that the administraof an LU
antiserum
in a activity
to pregnant
rats
marked decrease and a reduction
in vivo
in cholesterol in progesteron
LU on cholesterol esterase is secondary to the action of this hormone on steroid synthesis. An enhanced conversion of cholesterol into preg-
rate an
a by
cholesterol could
increased cholesterol ized ovaries of rats.
resulted esterase
and
steady
state
NADPH
(1970)
since
is
is utilized it is this
other
they
injection
of
LH
remove
stimulatory
that
cholesterol effect of observe
ester
depletion by
Flint
feed-
enzyme. Behrman
the
the when
al.
(1973),
The et
LU-induced
action
of
to
LU
a on
steroidogenesis
aminoglutethimide. et
of
ester was not due LU on steroidogenesis,
could
hand,
effect
a hypothetical
of the esterase point is equivocal.
blocked
On working
the with
the same tissue, found that the administration of aminoglutethimide or cycloheximide in vivo blocked the effect of LU on the depletion of cholesterol Goldstein
the
this
concluded of
cholesterol
Substrate that
might
depletion secondary
was
This alternative but until there system in situ, conclusion on
nenolone
that
back inhibition evidence on this al.
that an increased rate of might balance an LU-in-
in the
maintain
ester
effect
Armstrong (1969) vivo administration
tion
investi-
of
but tissue.
that
possibility of NADPH
latter
(Claesson
1967; Armstrong, et al. (1973a) have
secretion. It is possible
1970), brought
saying the
pyridine
be accounted ester
glucose raised the intraof NADPU in slices of
steroidogenesis solely by increasing tion of NADPU. Hall (1971) interpretation
this
corpora lutea (Marsh, rat ovaries (Flint and
Furthermore,
also found concentration
luteinized steroidogenesis (Flint unlikely
oxidized
in bovine luteinized
a and
in
AMP
that
cause
1948; Bell et 1968) and
can
Uerbst, Moyle
This
Behrman strated that
Casey,
Finally,
work corpus
bovine
on
and
1964b). the
increase
but mark[7-3U]cho-
(Savard
from
(1968)
exogenous
not
did
Savard,
apparent on the
Halkerston
hand,
of [114 C] acetate, the incorporation of
the incorporation edly enhanced lesterol
other
decrease
conversion
cholesterol.
U]
will (Claesson
the addition of LU or cyclic AMP Leydig tumor cells causes a rapid
the
[73
rats
in cholesterol
1948, 1953; Furthermore,
[i1 4C] aceextent than
of
and
cholesterol
all of this
LU increased the incorporation of tate into progesterone to a greater incorporation
rabbits
ovarian
a decrease
et al., 1968).
NADPU on incubating lutea also appeared
terms
in
Uillarp, 1947; Levin and Jailer, al., 1964; Herbst, 1967; Armstrong,
maximally
of
into
decrease
solely by (1966b)
mediated Kilpatrick
concentrations
preparations
slices, amounts
increase in progesnot have occurred
was and
additive
The effects of slices of bovine
to luteal optimal
the effect slices of
ester. and
Marsh
(1973)
have
assessed
of LU and cyclic AMP on incubating bovine corpora lutea and found that
CYCLIC
LU
and
cyclic
AMP
treated
AMP
tissues
had
higher values of cholesterol esterase Moreover, homogenates of this tissue
AND
STEROIDOGENESIS
suited
slightly
in
activity labelled
activity. responded
an
These
homogenates
AMP-dependent
(1973a) conversion
of
isolated and
cells
cyclic
might
be on
and
ester AMP in
the
effect
of
esterase
a protein
kinase
as
which
such
1970;
al.
that
triglyceride Corbin
lipase
et
al.,
Tsafriri
follicles
et
al.,
DeAngelo al., 1971;
1972b;
et al., Means
been
lutea 1973)
luteum tionation
purified
from
esterase was soluble fraction
ol esterase cyclic AMP
increase in activity of cyclic AMP activation
of
still
uncertain
the
role
activity, but any further
esterase
AMP
in
the
role the
study
stimulation
ether
plasma kinase adrenal
anesthesia,
in
vitro
cholesterol bovine dependent
also
esterase adrenal upon
pendent partially and
is known
cortex and the presence
protein purified esterase
cyclic
increased
kinase. bovine with
the
preparations
activity from
rat
ATP,
et
inhibition system al.
kinase
and
(1973).
ity
in
of a direct activity by
way
These
ovarian
of
AMP re-
transfer
stimulation cyclic AMP, (Naghshineh (Pittman and
which
in
investigators
LH
could is via
syntheby Flint
showed
that
LU to rabbits ether synthetase
interstitial
steroidogenesis.
onto have
concentrations
tissue
was mimicked by cyclic inhibition of this enzyme cyclic AMP seems to be the
and
in this
vivo activeffect
AMP in vitro. The system by LU or result of an increase
Progesterone are
and
20a
synthesized tissue after
interstitial
hy-
at LU
inor
cyclic AMP treatment and these steroids were found to exert a direct inhibitory effect on the synthetase system. It was also found that if the effect of cyclic AMP on steroidogenesis was inhibited mide then
by cycloheximide the inhibition
also lost. interpretation
This
result that LH
level
and
a cyclic the
the cholesterol ester this has been explored
of
of a choles-
cyclic
of
by
these effects were of cyclic AMP-de-
protein
involves and
to
cortex
group from ATP molecule. There
ester
It seems,
to elevate
Preincubation adrenal cortex
[‘y-32P]
AMP-dependent
the tase
(1974)
adrenal
kinase
possible
cholesterol
progestins.
ACTII concentrations, increased protein and cholesterol esterase activities in rat cortex. The addition of cyclic AMP and
ATP
terol
which
control
synthetase
cholesterol esterase of adrenal cortex tissue has been carried out (Simpson et al., 1972; Boyd and Trzeciak, 1973; Trzeciak and Boyd, 1973, 1974). In brief, it was found that stress induced by
ACTU
been two other reports of cholesterol esterase
1974). other
of 32P fraction.
Boyd of
phosphate esterase
of
is thus
A thorough
by
droxypregn-4-en-3-one creased rates in
fracof
The in
and
protein
esterase
transfer protein
activation
esterase
of a terminal the cholesterol
in purified corpus
muscle protein luteal cholester-
cholesterol time.
et has
Marsh,
sulfate addition
1973). kinase
cholesterol
cholesterol
Trzeciak the
the administration of decreased the cholesterol
corpora
and 1971).
the esterase did not cause
at this cyclic
1973;
al.,
(Goldstein, and protein
luteal
of
et
partially of bovine
skeletal purified
increased or ATP
1973;
bovine
by ammonium 1973). The
AMP dependent to this partially
et al.,
led
that
Khoo, The
1970;
and rat testes (Reddi 1974). This enzyme
1973; Goldstein testes (Reddi et al.,
homogenate (Goldstein,
cyclic kinase
DeAngelo
1974) et al.,
partially
(Menon, and rat
Cholesterol from the
(Lamprecht
data
propose
in
concomitant to the
ATP and protein kinase in idrenal et al., 1974) and adipose tissue
and Huttunen, 1972). Cyclic AMPprotein kinase activity has been in bovine corpora lutea (Goldstein and 1972, 1973; Menon, 1973), rat ovaries
Graafian
also
that
tissue
al.,
et
cholesterol by cyclic
through
adipose et
Moyle
cholesterol
reaction, the
Steinberg dependent detected Marsh,
of
on
mediated
(Uuttunen
of
suggested
AMP
phosphorylation acts
report
a stimulation into cholesterol
Leydig
LU
the
increase
with a phosphate
to cyclic AMP and ATP with a significant increase in cholesterol esterase activity (Goldstein, 1973). This effect of cyclic AMP in and
43
of
esterase ester
the
then,
and
in an
an
production
of
that
LU
increases
the
at
the
expense
of
cholesterol
ester both
was
keeping with the cholesterol ester
rat and activation
inhibition
rabbit gonadal of cholesterol
of
the
cholesterol
synthetase.
The control
importance of this of steroidogenesis
difficult a greater free
is in inhibits
increasing
free
cholesterol tissues by
or aminoglutethithe synthetase
of
to assess. proportion
form,
LU
It is possible of the regulates
steroidogenesis.
On
just a supporting strate when the
effect, steroidogenic
accelerated another corpus cholesterol
by
the
a other
an action
and more luteum, at ester
action of in gonadal
crucial least,
storage
that by cholesterol
keeping in its
limiting
step
in
may
1e
hand,
supplying pathway of the
this
more has
gonadotropin
step. In the effect would
LU in the tissue is
appear
subbeen at
the of
bovine LU on to
be of
MARSH
44 relatively reported
minor importance, that this tissue,
which
since it has is capable
marked steroidogenic response to amounts of free cholesterol and measurable amounts of cholesterol belman Seifart
et al., 1961; and Hansel,
Uafs 1968).
and
been of a
LU, has large small to unester (Zim-
Armstrong,
1968;
been found in the mitochondrial fraction of bovine adrenal cortex al., 1972; 1973). It
Via
Increased
cholesterol
and
side-chain
adrenal
Transport
chondrion, the
system
has
cholesterol been
mitochondria kerston et lutea (Hall 1967),
rat
sky et (Moyle
al., et
side-chain
shown of
to
be
cleavage
localized
in the
bovine adrenal cortex (Ual1959, 1961), bovine corpora Koritz, 1964; Yago et al.,
al., and testis
(Toren
et al.,
1964;
Drosdow-
(Mason and is synthesized
Boyd, and
and since cholesterol outside this organelle,
that
there
be
a migration
mitochondria pregnenolone.
before There
the into
cholesterol
sidechain
the microsomes corpora lutea and
later
The dna and
1971),
of cholesterol
site of tropic there has been very
hormone limited
transport
been
will
be
dis-
into
mitochon-
a limiting step in steroidogenesis could be another candidate
the
has
both
of bovine ovaries (Flint
this
but
in
of
section.
transport
might be therefore
cholesterol
activity
and the mitochondria and rat luteinized in this
into
it can be converted has been a report
cleavage
Armstrong,
cussed
cholesterol
in
a considerable
action. Although experimentation on gonadal
tissues,
amount
of
there
work
done
on this possibility in the study of the mechanism of action of ACTH and cyclic AMP in the adrenal cortex. This possibility was first proposed (1968). such
by
Uechter (1955) In the latter work,
a translocation
volve
the
since
cycloheximide,
of
synthesis
of
AMP, which when
It was enhanced facilitated
a
the and in proposed
protein,
inhibitor
of
that
AC1’H,
the synthesis of cholesterol transport,
cycloheximide
in-
protein
acceleration of steroidoincreased the accumulaextramitochondrial lipid
inhibited
it blocked cholesterol crease in steroidogenesis et al.,
might
regulatory
an
synthesis, blocked genesis by ACTH, tion of cholesterol droplets.
and then by Garren it was suggested that cholesterol
via cyclic a
protein
transport (Garren,
protein and that synthesis
and 1968;
the inGarren
1971).
A heat
stable
cholesterol
binding
protein
Mahafee ACTU
has
et
al.
a
can
direct
of
(1973)
represents
have
a choles-
transport into the
chomito-
evidence
for
this
at this time. This cholesresembles the regulatory
the
hypophysectomized free cholesterol
of this showed
even
when
authors’
at
other AMP
that the
some
later
of
protein
stimulation step
in
block of
the
the
by
synthesis of
in of
AMP
was of
the mitochondrial brought about
this
interpretation
ACTU and cyclic tion of cholesterol
of and
site because promoted this
known to treatment
did not prevent or cholesterol
blocks
of
level
aminoglutathimide
in doses Cycloheximide
indicating
inhibitor genesis
demonstrated treatment
in mitochondria to the stimulation some cyclic
the rats also accumulation
cholesterol
steroid into pregnenothat this stimulation
at dibutyryl
was administered steroidogenesis.
this
have AMP
accumulation not secondary
steroidogenesis ACTU and
ACTU,
of
rats increased the adrenal mitochondria
in
the conversion lone. They also cholesterol probably
level
et al. (1974) and dibutyryl
accumulation for
no
of
protein proposed by Garren et al. (1971), but this hypothesis required that ACTU increase the synthesis of this protein. Ungar et al. (1973) have reported, however, that ACTH does not change binding protein.
of
activity preparation
which cytoplasm
but
Leydig cell tumors and human placenta
must
protein
function is available terol binding protein
1965), mouse al., 1973b) 1971), stored
the
Ungar
this
cytosol (Kan et
Ungar et al., affinity for
cleavage
mitochondria. that
1973; binding
stimulates
terol carrier protein lesterol from the
of
Cholesterol Since
and Ungar, a specific
cholesterol proposed
Action
Kan has
and tissue
steroido-
process. data
The
was
that
increased the accumulathe mitochondria by
increasing esterified
the ratio cholesterol
however, a carrier
that ACTH, via cyclic AMP, activated protein, such as that described by
Ungar
the
free cholesterol to cell. It is possible,
et al. (1973).
A modification al was
of in
suggested
Jefcoate et al. stress increased formation from nal mitochondria
of by
this Simpson
latter
type et al.
of propos(1972)
and
(1974). They found that ether the initial rate of pregnenolone endogenous isolated
cholesterol from these
in adreanimals,
but there was no effect on the initial content of cholesterol in these organelles. The ether stress also induced changes in the type II difference spectra of cytochrome P450 which were inter-
CYCLIC
preted
as indicating
an increased
cholesterol-cytochrome therefore, proposed total mitochondial for
side-chain
ACTU
cleavage,
increases
This
could
cytochrome
be
dial duced
sites. It is also changes may
cholesterol
This
of
inhibited by labile protein port
that
stress
due
to
the
via
is active. transport
side-chain
or
other
possible result
that these stress-inin an increased entry
the
stress
intramitochon-
mitochondria
was
in
found
cycloheximide may be involved
to
be
vivo.
indicating that a with this trans-
on
effect
of
cholesterol
gonadotropins
transport
not been studied tissues. Flint and assay caproic
involving acid
ported side-chain
that
into
microsomes ovaries
was about activity in also
We
have
be no into been
1973).
In
this
however,
hands,
nates as the marker succinic dehydrogenase.
to
this
cholesterol
sidedistribuhomoge-
chrome C reductase. It is unknown at this if this discrepancy between the results of and Armstrong (1971), on the one hand,
time Flint and
methodological the former
Caron (1973) on the other differences, workers did levels they
the endogenous fractions when side-chain
activity.
mitochondria bovine times
corpora as
microsomes
much
not
most other is due to
but it seems that take into account
of cholesterol calculated the have
We
isolated
from
lutea
contain
endogenous (Caron,
and hand,
1973).
found
in these levels of that
the
homogenates more cholesterol If
the
of
than
four as
exogenous
the
be about four incubations Thus
the
reaction
the
would
cleavage microsomes.
in
(1973b) cholesterol
enzyme
and
system
found the
inner
the
enzyme
membrane
activity
was
as localicleavage
cells
limiting availability
step
and
localized
mitochondria,
on while
was associated indicates that
in steroidogenesis of substrate cholesterol
a
could be the in this tissue
as well. Robinson and Stevenson also reported in an abstract that
(1973) have the treatment
immature rats increased in the the mitochondria ovaries. an effect transport
as
cholesterol tumor
all of the cholesterol outer membrane. This
dna remains gonadal tissue, implicate this
times
the
endogenous
essentially with the
the luteinized Thus, although AMP on cholesterol
of
mitochondria
Leydig of the
lower in the
sidechain
determined sidechain
the
from
that
the four
the
the of
amount
of
represent
activity
mitochondria
times than
same
products
Moyle et al. zation of the
of
with hCG transport of obtained
LH into
and cyclic mitochon-
to be examined thoroughly in the studies on the adrenal cortex process in the control of steroido-
genesis.
Action
Via Increase
Cleavage
on
the cyto-
of
activity NADPU
cleavage
Side-Chain
Activity
Cyclic cholesterol
enzyme for mitochondria, There was no correlacleavage enzyme,
in
from
luteabout
confirm
the same of luteal
ones.
radioactivity
Rat
lutea, using an of [414 C] products (Car-
the
microsomal
of superovulated causes a marked cholesterol into
have
is uniformly diluted by then the specific activity
this substrate would in the mitochondrial
as much corpora
and
the data investigators,
the marker
C] isore-
for a transport of in luteal tissue.
chain cleavage activity shows tion in the different fractions
tion between microsomal
to
bovine corpora the conversion labelled steroid our
gonadal using an
activity in microThis suggests that
requirement mitochondria
unable,
distribution in assay involving cholesterol into on,
twice bovine
found
one quarter as much of somes as in mitochondria. there may cholesterol
has
in
as in mitochondria. were
AMP
mitochondria
production of l4 [2614 C] cholesterol,
there cleavage
inized
cyclic
very extensively Armstrong (1971),
the from
lutea
or
45
labelled cholesterol endogenous pools,
in
readily
or binding. The
STEROIDOGENESIS
much in the
cleavage
from
into
effect
of the It was, of the available
which
to the
P-450
amount
fraction
of cholesterol
AND
complex. a fraction was
and
that
increase
binding
of
P-450 that only cholesterol
AMP
the
AMP into
could increase pregnenolone
side-chain
cleavage
activating, or increasing the components of this of
cyclic
stimulation lesterol
AMP of into
the conversion of by a direct action
was
enzyme
system
the synthesis complex. Such proposed
to
the conversion pregnenolone
by
of one of an effect explain
its
of labelled choin various adrenal
cortical mitochondrial preparations (Roberts et al., 1965, 1967; Roberts and Creange, 1968). Koritz et al. (1968), however, were unable to substantiate this proposal and suggested instead that
the
increased
pregnenolone
accumulation
was due to an inhibition of the conversion pregnenolone to progesterone rather than increase in pregnenolone synthesis. t53j3hydoxysteroid dehydrogenase and A53ketosteroid involved both
isomerase in this
the
reaction
mitochondria
enzymes appear and
which
to be present the
microsomes
of an The the are in
46
MARSH
(Sulimovici 1970). This the
and Boyd, inhibitory
pregnenolone
to
confirmed
by
been
1969; McCune effect of cyclic
progesterone several
and and
inhibits nase,
at
tions,
by
Lunenfeld, it seems
that
competing
suggested
that
tory
effect
LU
on
the bovine confirmed later and
1971; the
with
findings have experiments.
al.,
Snnivasan et al., cyclic nucleotide dehydrogefor
the
had
corpus luteum, but this by some of these same
total amount synthesized
stimulasystem
the conversion and inhibited to progesterone.
of
the
side-chain
cleavage
vici and Boyd, 1971; Srinivasan Recently
we
cyclic AMP, reconstituted system from
have
a direct (Caron
system
was
mitochondria and the
solubilized
from
system lesterol
was
sue The
the
other
were tional
arranged to the
present.
When
kinase
preparation,
effect
on a cleavage and have enzyme cleavage
corpora
of The was
adrenal
AMP,
ATP
the chothen
cortex
purified
tis-
reductase). components
and
cyclic
AMP
on
the
a physiobut there of this
in whole
mitochondria
Efflux of Mitochondria
Hall
(1964)
inhibited in
observed
its
an
a protein from
cor-
and
own
extract
since must
the egress of chondria (Koritz
that
preg-
synthesis
from
from
adrenal
mito-
it was believed that leave the mitochondria
pregnenolone and Hall,
the to
(Beyer and that ACTU determining
from 1964;
the Koritz
mitoand
Kumur, 1970). It was also reported that substances which cause mitochondria of steroidogenic tissues to swell stimulated pregnenolone synthesis, decreased itz, 1964,
while ATP which inhibited this stimulation (Uirschfield 1966).
are which
probably
several indicate
not
pieces that
correct.
McCune 1971;
et al., Srinivasan
1970; et
pregnenolone
First
of
all,
mitochondria than that observed cycloheximide-treated
there
was
age the
activity stressed
increased
1972).
to accumulate
cleavage enous
stressed
cholesterol
rats
was
mitochondria even though
side-chain
cleav-
obtained the amount this
in mitochondia, to progesterone with affect the stimulation
activity ACTH
amount isolated
from of
inhibition concentrations
is
mitochondria (Simpson et al., when pregnenolone was made
occur in Fourthly,
conversion it did not
Secondly,
in the rats,
required for the physiologic
in 1969;
that the freshly
in the mitochondria animals. Thirdly,
pregnenolone greater than
is a
present
progesterone.
from
greater from
is
there
and Lunenfeld, which can con-
Simpson et al. (1972) reported of pregnenolone present in adrenal
how-
Boyd,
Sulimovici aL, 1973)
into
swelling and Kor-
of evidence, this hypothesis
5-313-hy droxysteroi d dehydogenase mitochondria (Sulimovici and
which
proporP-450
lutea, were added to this reconstituted cleavage system there was a consistent stimula-
pora
and
cholesterol
vert
lutea
A treatment component was
the activity was of cytochrome
partially
of
ATP
this The
and adenodoxin of these three
cyclic
Konitz
There
isolated cytochrome iron component and of
of
in situ
Increased From
nenolone
Lunenfeld,
chromatography. cleavage activity
so that amount
the
was of the (Sulimo-
components
component
(adrenodoxin proportions
of
from
using this the non-heme
flavoprotein
and
the
phospholipase P450
by column side-chain
reconstituted P450 and
examined
was
effect
de-
indicating involved.
system may represent of the cyclic nucleotide, to be further confirmation
Action Via Pregnenolone
ever,
stimulation et al., 1975).
by cytochrome
isolated
activity
protein kinase and cholesterol side-chain bovine corpora lutea
observed activity
reconstituted logic action will have
of ATP probably
be converted into progesterone Samuels, 1956), it was suggested might control steroidogenesis by
pregnenolone the inhibition dehydrogenase and
vitro
presence
of 20 to 70
completely
of cholesthe converWhen the
of
1969; Sulimovici et al., 1973).
in
order
was
chondria, pregnenolone
compared with that in the controls, however, it was apparent that there was no overall stimulation
This
of the
stimulation
Sulimovici that cyclic
of pregnenolone and progesterone in the cyclic AMP incubations
increased accumulation probably due to A533hydroxysteroid
activity
The
pendent upon the that a phosphorylation
of
could not be authors in a
publication (Yago et al., 1967). Boyd (1968) then reported
of enzyme
enzyme.
a direct cleavage
tion percent.
type of activation and whole cells.
concentra-
NAD
side-chain
AMP both stimulated terol to pregnenolone sion of pregnenolone
using
(Sulimovici 1970; Suli-
been reported in gonalchii et al. (1963)
itself
the
has
investigators
the 5-3(3-hydroxysteroid greater than physiologic
Similar dal tissue
al., on
reactions
adrenal and ovarian preparations and Boyd, 1969; McCune et movici 1973),
et AMP
caused in
vivo
by the (Simpson
by
blocking
its
cyanoketone,
of action et
side-chain of endogal.,
1972).
CYCLIC
Similar
results incubated
using
and
cyclic
were
obtained adrenal
rat
AMP
by Farese sections,
AND
STEROIDOGENESIS
incubated
(1971) ACTU,
and
AMP.
slices
Sato
tumor
ROLE OF PROTEIN IN THE STIMULATION STEROIDOGENESIS
Concomitant
protein
shown to be required LU and cyclic AMP respective
target
on this (1974)
matter, in a
mention
some
on gonadal Hall
SYNTHESIS OF
cells,
tissues.
I will
since recent
it was review
has
of
highlights
the
not
been
go into
detail
the
puromycin stimulatory
were the inhibitors,
and chloramphenicol, effect of LU on
incubating slices have confirmed
of rabbit this effect
inhibitors,
predominantly
using
first such
to as
blocked steroidogenesis
the in
testes. Several groups of protein synthesis puromycin
and
cycloheximide, in testes (Shin and Sato, Moyle et al., 1971), corpora lutea (Savard 1965; Marsh, 1968; Hermier et al.,
1971; et a!., 1971),
Graafian follicles (Tsafrini et al., 1973) and whole ovaries (Gorski and Padnos, 1966). The site of action appears to be after the formation of
cyclic
showed
AMP that
stimulation incubating
in
that
Marsh
puromycin of slices
did
et not
block
(Marsh
and
Uermier action they the They
et
al.
Savard,
1 966a).
(1971)
pinpointed
of these inhibitors even showed that cycloheximide cholesterol incubated
side-chain slices of
the absence found that
of no
presumably the side-chain
due
could
not
indicated
02 for 90 progesterone
side-chain
by
cleavage
step
or
since
progesterone
beyond
if it did, synthesis
LI-I
is
whole
process.
There indicates stimulation
the
for the
on
the
of
uncertain.
RNA
synthesis Savard
et
in the al.
(1965),
in-
data).
decide,
at
this
time,
of action of is the correct
limiting but once other
mediates tropic or
parts
action necessary
the
ONS
body AMP
adrenal of
the
of evidence plays a role
cortex.
which in the
to
that
amounts
on steroidogenesis,
one
of the cyclic steroidogenic
cyclic
AMP
of
steroidogenic
the
to
limiting
of
normally
detect
this of
methodological
in
is possible
investigators
small
is due
AMP
It
concentration
when
used,
aspects just
become
of cyclic AMP at to accelerate the
but a final answer to this await further results. In regard to the possible
be
might
the
the effect of small concentrahormones on steroidogenesis
inability
in
nucleotide are
step before steroidogenesis
of gonadal steroidogenesis. This is true in the case of the corpus There is considerable doubt, however,
a
cyclic
hormones
deficiencies,
problem
will
modes
have
to
of action
of
there nucleotide pathway,
is evidence at several and it may
accelerates process
several rather
than
step.
have second
ACKNOWLEDGMENT I would
role
the
concerted may be
for an effect steps in the
point
it would
of
cyclic
This
the
be begins,
is a large that cyclic
change
incubation.
The
several
that
act
unpublished to
accelerate
A sites
testis
in
lutea,
is experimental data in support of of them is mutually exclusive and it
to
limiting.
the
that in
starts
it
when before
of rabbit
corpora
have shown that this stimulation by actinomycin
possible
may
that tions
of
reaction. rat ovary
not
pathway stimulation
the
site
on
hand,
that actinostimulation
slices
studies
cyclic
other
may be that LU and cyclic AMP have pleiotropic types of effects stimulating many aspects of the steroidogenic pathway. One part of the
luteum.
cycloheximide.
incubated
(Marsh,
There None
particularly
then incubated was a rapid this synthesis
did
one. each.
using actino-
LI-I and
the
of the possible mechanisms AMP on steroidogenesis
exogenous this same
with LH and was synthesized,
cycloheximide
steroidogenesis,
blocked
in but it
mm
were there and
which cyclic
that
of
CONCLUSI
LH
to the lack of 02 needed cleavage reaction steps. When
be inhibited that
the
further, acted
cleavage luteinized
slices of luteinized ovary in the presence of 02 synthesis of progesterone
on
of in
D is inconsistent
(1966)
cyclic AMP accumulation of bovine corpora lutea,
did block the stimulatory effect cyclic AMP on steroidogenesis tissue
al.
in
not
(1973),
On
experiments of the LH is
a!.
action
to the
Shin Leydig
found
(1966) reported effect on the LH
In regard
It
tissues.
the
steroidogenesis.
further hibition
lutea, mouse
et
follicles,
steroidogenesis
studies
corpora
incubated
Tsafriri
and Padnos D had no
ovary.
by Wicks will just
I
in
of
and
D blocked on
Gorski mycin
of ACTH, in their
covered but
and Eik-Nes (1962) that protein synthesis
show
mycin
synthesis
bovine
using
Graafian
AMP
for the actions on steroidogenesis
of
(1971),
isolated THE
47
action
of using
several of the
like
to
acknowledge
the
collaboration
colleagues who worked with me on the studies role of cyclic AMP in the bovine corpus luteum,
of
MARSH
48
namely Dr. Kenneth Savard, Dr. Steven Goldstein, Dr. Marc G. Caron, Dr. Kirpal S. Sidhu and Dr. William V. Ling. I would also like to thank Mrs. Nieves Cerver for the preparation of this manuscript.
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esterase
stimulation
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luteinizing
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(1971). Specific binding of Iuteinizing hormone to Leydig tumor cells. J. Biol. Chem. 246, 498 3-4986. Moyle, W. R. and Armstrong, D. T. (1970). Stimulation of testosterone biosynthesis by luteinizing hormone in transplantable mouse Leydig cell nimors. Steroids 15, 681-693. Moyle, W. R., Moudgal, N. R. and Greep, R. 0. (1971). Cessation of steroidogenesis in Leydig cell tumors after removal of luteinizing hormone and adenosine cyclic 3’,5’-monophosphate. J. Biol. Chem.
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Moyle, W. R., Jungas, R. L. and Greep, R. 0. (1973a). Influence of luteinizing hormone and adenosine 3’,5’-cyclic monophosphate on the metabolism of free and esterified cholesterol in mouse Leydig-cell tumors. Biochem. J. 134, 407-413. Moyle, W. R., Jungas, R. L. and Greep, R. 0. (1973b). Metabolism of free and sterified cholesterol by Leydig-cell tumour mitochondria. Biochem. J. 134, 41 5-424. Moyle, W. R. and Ramachandran, J. (1973). Effect of LH on steroidogenesis and cyclic AMP accumulation in rat Leydig cell preparations and mouse tumor Leydig cells. Endocrinology 93, 127-134. Moyle, W. R., BahI, 0. P. and Marz, L. (1975). Carbohydrate Biol.
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and
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Naghshineh,
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of
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918
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(Abstract
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3828).
cyclase testis
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in
effect
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hCG and cyclic AMP on testicular cholesterol demolase. Diss. Abstr. 32, 6823-B. Rao, Ch. V., Griffin, L. P., Carman, F. R., Yussman, M. A. and Queenan, J. T. (1975). Search for gonadotropin receptors in human corpus luteum of pregnancy.
Reddi, C.
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372-3
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OF
REPRODUCTION
The
14,
Role
30-53
(1976)
of Cyclic
AMP
in Gonadal
JOHN Endocrine The
M. MARSH
Laboratory, Department University of Miami, School Miami,
There
has
been
many
investigators,
which
implicates
functions, role lacking.
one
The
possible
the
of work
the
AMP
a complete
any
in
AMP
deal
over cyclic
but
in
a great
of
THE ROLE OF CYCLIC AMP IN STEROIDOGENESIS IN THE CORPUS LUTEUM
years of is
role
of
stimulation
oidogenesis in ovaries and testis studied the longest and probably thoroughly, but even this action being completely elucidated. The
its
The
still
the
(Haynes et
showed level of
that cyclic
aL,
and Haynes
this
has
ster-
and
in vivo
has been the most is far from first indica-
1959;
ACTH
Berthet, et al.,
increased the in the adrenal
AMP
1962)
Savard, From
1964a; these early
genesis AMP
the field has been
nearly
all
and
some
and
the
Marsh studies
ovary and on
1957; 1960)
has expanded implicated to
actions
of
actions
luteinizing
of
follicle
(Marsh
role
of
this
steroidogenesis testis, since
cyclic in these
reflect contrasting the possible modes the
steroidogenic
‘Dedicated sor of Miami)
nucleotide
hormone
(LH)
stimulating
hor-
the two
corpus gonadal
situations. of action
on
luteum tissues
and seem
Dr.
Karl
in the model
the
of
effect
(Emeritus
species
to be,
is and
by in vitro
at least
report system
LH
on
of several In a series
to some
or other
test
controlling of studies
by Savard et al., 1965) was developed in which progesterone
in incubating
ing hormone 1964a).
synthesis
slices
was
of corpora
lutea
in early pregnancy. The tissue were incubated in buffer in the presence
substances
After
activity
the
initial
on
steroidogenesis lutea slices were logically divided
the
the site pathway;
of the to
nism
and
the
amounts
of
(Mason
studies
on
and the
Savard,
effect
of
LH
in incubating bovine corpora completed, the investigation into two parts: a) a study of
of LH action on and b) an investigation action.
The
site
of
the the
steroidogenic of the mechamajor
effect
of
LH on the steroidogenic pathway has been shown by numerous investigators to be between cholesterol
H. Slotta
in most
luteum
progestins,
progesterone and 2013 hydroxypregn-4-en-3one synthesized were measured. It was found that LH produced a marked increase in progestin synthesis in these incubating slices and this effect was specific for hormones with luteiniz-
pathway.
to
shown
corpus
of
I will also discuss of cyclic AMP on
Profes-
Biochemistry and Medicine of the University on the occasion of his eightieth birthday.
the of
investigations
(reviewed an in vitro
of LH
and
process
ot secretion
obtained from cows slices of corpora lutea Krebs-Ringer bicarbonate
to where cyclic some extent in
in the
been
determined
1964b). steroido-
mone (FSH) in the ovary and the testis. I will attempt to review the studies
and
LH is probably only one hormones (Greep, 1971).
endogenous cortex, and
Savard, gonadal
function
extent, controlled by LH. In some species, such as the cow, the human and the monkey, LH appears to be the only controlling hormone while in others, such as the hamster and the rat,
that exogenous cyclic AMP mimicked the effect of ACTH on steroidogenesis. Guided by this work, other investigators began to study the role of cyclic AMP in the gonadotropic stimulation of steroidogenesis in the testis (Hall and Eik-Nes,
main
synthesis
cyclic of
tion that cyclic AMP was involved with the control of steroidogenesis came from the studies of Haynes and co-workers who in a series of experiments Haynes
33152
gonadal
functions
mediatory
of Biochemistry, of Medicine,
by
10
in several
gonadotropic
Florida
done,
past
understanding these
Steroidogenesis’
and
Hall Hall
and and
Koritz, Young,
The
study
of
pregnenolone 1964; 1968; the
(lchii Koritz Armstrong
mechanism
et al.,
and of
Hall, et al.,
LH
action,
1963; 1965;
1970). on
the other hand, has been largely a study of the possible role of cyclic AMP in this gonadotropic
of
30
CYCLIC
effect
and
Effect
this
under
of Exogenous
Marsh
and that
found
AMP
to
caused [73
the
Cyclic
incubating
slices of
cholesterol
tration
of
AMP
action not
magnitude
amount
of
AMP The this
to
level to an readily
in
The
that
steroid
Marsh
and
acetate
and
a concen-
of
the
cyclic
the
3’-AMP,
effect
by
utiliLH.
5’-AMP
or
by
indicating of this
but
this
of the cells
was
also
a maximal
that action
cyclic AMP far exceeded
tissues,
served to the
of LH
cyclic of LH.
needed for the endoge-
could
have
exogenous or to its
been
nucleotide destruction
(1965a,
at
ment estrous
using cycle
AMP
stimulated
celerating
out
a similar
type
of experi-
bovine corpora lutea and showed that, like progesterone
a step
from the LH, cyclic
synthesis
between
cholesterol
by and
ac-
preg-
nenolone. The progestin
effect of synthesis
tissue
of
(1971)
other
exogenous has been
cyclic assessed
AMP on in luteal
species as well. LeMaire et a stimulation of progesterone
reported
synthesis both in terms of mass and etate incorporation in incubating human
The
corpus effect of
human
chorionic
pattern
of
luteum cyclic
of
the steroids (progesterone, terone, androstenedione duced by the cal in both ness
of
cyclic
AMP
slices. over
[i-’ 4C] acslices of a
(hCG), 4C among 17 and
The
greater
hCG
may
of
but
the
several
of
hydroxyprogesestradiol)
human corpus luteum the hCG and the
tissue
stimulated
al.
of ectopic pregnancy. AMP exceeded that
gonadotropin
distribution
was cyclic
the
the
was tor
AMP
same
the
25
al.,
corpora
adenylate
al.
be able (1972)
for
cyclic
reported
AMP
as for
hCG
on
imidazole, markedly
synthesis These
that proges-
luteum of response
theophylline, phosphodiesterase, of
in the results
the
to by-pass
stimulated
also
a corpus the maximal
effect
cyclase
suggest,
in and
synthesis and that this phosphodiesterase, gesterone incubations.
of
would
also reported that of cyclic nucleotide
tentiated
et
than
experiments
Hermier et cyclic AMP
about
(LeMaire 1974a),
level
these
that block. exogenous
the was
hCG. an
It
inhibipo-
progesterone
a stimulator reduced
control are in
of pro-
and hCG complete
with the proposal that cyclic AMP mediates the action of LH and hCG on progesterone synthesis in corpora lutea. Dorrington and Kilpatrick (1967) reported
that
5 mM
interstitial
and
Jutisz
AMP bating by
serum
external
increased
rabbits
tissue
was
(1969)
of
although
much
showed
to the found
Hermier
3 mM
synthesis ovaries,
rat rats
gonadotropin
its effect
greater. that
immature
medium
pregnant
with
and
of Parlow presence of
was
cyclic
in incuprepared
(PMSG)
procedure that the
the
20a hydroxycorpora lutea
required
hCG
(1968). Ca2 in for
this
effect.
Endogenous
Cyclic
AMP
The results with exogenous compatible with the hypothesis otide was a mediator of the steroidogenesis consideration
tissue. of
to
treatment
according They also the
slightly
stimulated progesterone slices of luteinized
mare
AMP
effective-
AMP
progesterone and in incubating
pseudopregnant
on
would nous
pro-
cyclic
synthesis of pregn-4-en-3-one
identi-
be related
at
synthesis cycle
1’
1973;
cycle. If there is a block in human corpora lutea
cyclic
from
the much smaller concentration (Marsh, 1969). Hall and Koritz
b) carried
synthesis LeMaire,
exogenous
terone menstrual
of pregnancy
and to respond to hCG in cyclase, cyclic AMP accumu-
the menstrual responsiveness as
lutea
capacity to bind hCG (Cole et al.,
accord
by cyclic 3 ,5’ nucleotide phosphodiesterase. In this regard, the dibutyryl derivative of cyclic AMP, which has been reported to be resistant to phosphodiesterase degradation (Posternak et al., 1962), stimulated progesterone synthesis in this system of 0.2 mM
lutea of to hCG activation,
pattern
cholesterol produced
al., 1975) of adenylate
et
of pregnancy
produced
and
produced
of
inability penetrate
LH
as
cyclic AMP, be a mediator
concentration effect, however,
nous due
to
of might
and
1968;
Rao
corpora
smaller LH or
lation
At
that
human
lutea
in the system, which degree of specificity
AMP.
additive
to
[7-3H] that
such
effect a certain
of cyclic
amount
4c]
that much human
terms
corpora
[1-’
equal
nucleotides no
cow
fact
31
1966b) cyclic
of progesterone
about
of [1-’4C] acetate and zation closely resembled ATP had to establish
1964b, exogenous
of
mass,
M the
was
a saturating
Other
AMP
have a labelled
incorporation.
0.02
effect
STEROIDOGENESIS
the
stimulation
in terms
H]
AND
investigation.
Savard (1964a, addition of
a significant
synthesis
by
is still
AMP
much
in the required
bring about concentration Marsh LH
et al.
corpus that
cyclic that action
luteum, but the addition
an increase of cyclic (1966)
AMP were this nudeof LH on
showed
such a of LII
in the endogeAMP in luteal that
2 pg/mI
brought about a striking increase (as as 100 fold) in the level of cyclic AMP in
32
MARSH
incubating
this
slices
response
LH the
as had stimulation
aid
et
of
bovine
showed
the
been of
al.,
1965).
endogenous
of
2 g
of
LH.
At
LH/ml) the
between
and
demonstrated synthesis
Furthermore,
the
AMP
less
the
that
measurements
error
to
(0.2
to
component
cyclic
ered
progesterone synthesis, AMP levels could not This discrepancy was
had small
this
Since
(Beall
1973;
Moyle
shown
that
work and
cyclic
and
indirectly, kinase by
luteum
had
kinase.
Using
the
al.
(1973),
reported, 1972;
concentrations
of cyclic
luteum. In the older relationship (Marsh
study et
looked for of incubation
a change while
measured
after we of
h
(Ling we of
where
modification (Ling
et
course
of cyclic when small
al.,
recent
of
amount
were
there
is still
mum effective steroidogenesis The minimal
and
of
we
have
tropic
hor-
significant
the
of the dose al., 1966) AMP
dose
re-
This
Marsh,
found
response in when large our recent unpublished
that
AMP accumulation amounts of LH
a dissociation doses and effective
tein
the
between
a
time
of
AMP
been
of
and
by
hibits
the
amount corpora
and
Goldstein
of
predominantly
the
in
inhibits
about
25
percent
of LH which will stimulate cyclic AMP accumulation. dose of LH which causes
bound
to
a macromolecule.
of
before, protein
this
pro-
to be effects
of
on steroidogenesis substance had of
which
no
cyclic
indicated
required
not
Marsh
mini-
as mentioned AMP-dependent
LH
cyclic
the
previously
stimulation
slices
will
between
puromycin
lutea
at 5 the
which
stimulatory
stimulation of after the increase
step
increased
the
bovine was a
activity below
which
was
that
kinase
the increase in probably an increase of cyclic AMP.
shown
the
Cor-
different activity
and Marsh, unin terms of dose luteum to LH,
then,
AMP This
on
protein by
LI-I
corpus
the
cyclic 1965).
accumulation
effect,
these
antibiotic
had
synthesis
1973) corpus
slices of that there
(Ling
this in
shown
of
agreement
an
however,
protein
some
good
inhibiting
both LH and (Savard et al., effect,
is nearly are used. In
the
capable
been (1972,
kinase slightly
concentration
synthesis,
had
found
steroidogenesis, kinase activity and effective
attempted
described
in
Puromycin,
was
cyclic AMP by (1970) method
is
cyclic
LH were with this
AMP with the changes
Marsh
in this is even dose
effective
there
the
was
steroidogenesis data). It seems of the bovine
published response
in
It
subunit
in
recently
incubating
which
stimulate
that
levels of associated
the effect on protein
LU
One consid-
that the bovine AMP-dependant
increase
ng LU/mI, minimum
increase protein
response we only
after 15 mm synthesis was
incubation.
measured the Gilman
1973),
in
dose response study, therefore, the progesterone synthesis and cyclic both measured after 2 h of incubapreliminary evidence indicates that
AMP tion.
Our
of
of
LH to increases in AMP in the corpus
in cyclic progesterone
2
concentrations
Dufau,
1973)
low been
procedure
investi-
was measured corpora lutea.
of
had found a maximum cyclic AMP at 15 mm of LH were used. In
experiments data),
and
of steroidogenea detectable change of these reports we
a reinvestigation
begun
because terms amounts
several Catt
changes
It
(1973) a cyclic
Menon
et
regulatory
and
the
cell. be
is the
activity.
Goldstein
in
cyclic
might
to
confined
the
which
the
at
due
was
of cyclic measuring
by
Marsh, inabili-
AMP be
AMP
We have
experimental
was
sponse relationships steroidogenesis and
when have
increases
the
might
be
in cyclic
small
If
subunit.
protein
cyclic
of
cell
an interaction
subunit
used
Ramachandran,
low
the
occur might
detect
in
LU cyclic
kinase.
did they
to needed
increase
compartment
of
sufficient
mones can elicit a stimulation sis in other tissues without in cyclic AMP. In the light
the
to
earlier
Sayers,
and
effective
small
regulatory
of the and
the
the
of
as a compartment
one in view slice technique for
a change
protein
bin
gators
linear
a
to
AMP used
efbe not
data).
that
amount
ng/ml (Ling and It is possible that the
concentration
fact
of
nucleotide.
have
lower
the
significant
100
detect
correlation AMP
first
about
seems
of LU
steroidogenesis
while
the
to
ty
a positive
in endogenous
obscure
cause
AMP is unpublished
concentrations
methodology
AMP
increase
of
10 ng/ml
exthe
to be a serious of the incubating
fact
in
of progesterone synthesis. however, the results were this concentration of LH
usually stimulated fects on cyclic regularly detected. considered variability
to
for (Say-
the
stimulation
be of
was
increase
a
about
for
increase
preceded
higher there
the stimulation 0.02 pg LFI/ml, clear. Although
At
and
synthesis which would AMP were a mediator
in
action
lutea, specificity
previously progesterone
cyclic
progesterone pected if cyclic
corpora same
that
for probably
this in-
steroidogenesis in cyclic AMP. AMP has
(1973) cytosol cytosol The
at
The
produced
been to
in
by
found
be
localized
and
fraction
cyclic corpus
AMP luteum,
also contains a kinase (Goldstein
cyclic and
is
CYCLIC
Marsh,
1972,
this
1973;
enzyme
is
fraction
(Goldstein
probable
that
major
portion
AMP
binding
reported
1973)
located and
this
in
Marsh,
protein
of
the
some
it’s
ability
Menon
stimulate
cyclic
and of
progesterone bovine corpora
lose
its
ability
(Goldstein
to
and
a
from
6 or
observe
be
a
IU)
of
steroid
study, response
so it is not of cyclic
and
corpora
small.
very
have
been
due
when
the
corpora
on steroidogenesis. LU and hCG increase
in cyclic
corpora et al.,
lutea 1966)
incorporation and
also
specific
human
to these
about the same on steroidogenesis
et al., 1965). Corpora cycle were much more in terms
accumulation or steroid lutea of pregnancy 1974a). This diminished
receptor
sites,
and
et al. (1975).
Rao
as reported
Lamprecht corpora lutea
et al. from
luteinized
ovaries
10 pg
rat of
LH/ml
with
of
could quality by Cole
(1973) found pseudopregnant also
(Marsh,
of
AMP
corpora LeMaire, of the
et al. (1973) that
isolated rats or in vitro
in cyclic
stimulating
luteinized rats than
cyclic
AMP
rat ovaries or ovaries ovaries from immature
to
AMP
accumulation from rats.
older,
of
cyclic
Day
chance
and
Cyclic
to
Iutea
selection
were
the 7 it was
et al. (1973)
in corpora
lutea
AMP
response follicles.
however, at
of Mason
of
in
accumulation, but the response of isolated Graafian follicles was more striking. Mason et al. (1973) also found that LH was less effective in
inhibition
activity
be due to a of hormone
responded
an increase
an
cyclase
cyclic
of
may
of ovaries
in this
decline.
in pregnant In fact,
cyclic
nucleotide
phospho-
diesterase and these alternatives gated in homogenates of bovine by assaying the effect of LU
LH
lutea responsive
this
Ph osphodiesterase
sensitivity in this
synthesis than (Marsh and responsiveness
luteum of pregnancy in the number or
to be with
in a lack synthesis.
In fact, the of isolated until
LU
to the
Cyclase
Nucleotide
by
(Marsh
appeared
gonadotropin
gonadotropins
corpus decrease
in human
effect
content
grew
to
measure-
made
their study of corpora rats, found that LU
the
failure
rat or a doses of
No
to correlate with steroid
not
corpora
The increase in endogenous cyclic AMP in luteal tissue could theoretically be brought about by a stimulation of adenylate cyclase or
of mass (Marsh [8-3Hjadenine
[8-3H]AMP
The
LU
a marked
accumulation
cyclic
and showed as the effect
tissue (Savard the menstrual
about
in terms terms of
1974a).
for
activity to hCG
AMP
measured or in into
LeMaire,
brought
Adenylate
LH preparations of action of
lU).
possible AMP
declined
The
We
effect of some the mechanism
(12.5
were
a response
detect
did
in
synthesis
lutea
don’t know what the stimulatory agent is, but we don’t think it is LH. We believe therefore that this direct is not part of
hCG
quickly
they
AMP
a 20 day pregnant treated with low
increased
the
a stimulation
rats,
cyclic
in 1 day old corpora lutea. was much greater than that response
kinase
in
et al. (1974), in from PMSG-treated
markedly
report
pregnant
from been
(8
As
data).
day
ments
Ahren lutea
did
increase
PMSG
accumula-
unpublished
18
an
incubating it did not
protein
authors
accumulation by LU in incubatisolated corpora lutea obtained
lutea obtained rat which had
could
Secondit lost
in but
stimulate
Marsh,
is
a different that only
AMP
synthesis lutea,
AMP of
It
of his protein to the in vitro
come to we found
these
up also
LH
33
although of cyclic ing slices
(1973) of
showed this ability. the LH preparation
tion slices
of
that LH might by a mechanism We have confirmed
but have First of all,
to
cytosl
STEROIDOGENESIS
cyclic
preparations
few batches of LH ly, when we boiled
most
makes
about a direct stimulation preparation when added
this observation interpretation.
and the
macromolecular
enzyme assay. This indicated able to produce its effects independent of cyclic AMP.
AND
1973).
kinase
material.
that
bring kinase
Menon, also
AMP
tion was
the
absence
of detectable accomplished
or
phosphodiesterase
The
complete
phosphodiesterase by using a high
of theophylline period. Under LU/mI adenylate
of
1970a).
were investicorpora lutea on adenylate
(0.04 these
inhibi-
activity concentration
M) and a short conditions,
incubation 0.1 pg
0.01 M NaF significantly cyclase activity. Epinephrine
concentration
of 0.2
mM
also
of
increased at
produced
a
a small,
but statistically significant stimulation of nylate cyclase activity, but LU inactivated hydrogen peroxide, bovine serum albumin,
adeby pro-
lactin,
this
ACTH
and
glucagon
respect. The significance tine is uncertain since one been
concentration carried out
accumulation bated (1970)
slices have
were
and no thorough on its effect on
study has cyclic AMP
corpora
observed
in
of epinephassessed at
or progesterone of
inert
of the effect it was only
synthesis lutea.
a stimulation
in incu-
Fontaine
tine of adenylate cyclase in homogenates whole ovaries obtained from goldfish, and effect
also
required
an
elevated
et
al.
of epineph-
concentration
of this
MARSH
34 of
the
catecholamine
tissue, stimulate Pulsinelli
also
testosterone Nes, plays
mM).
also
has
(Kuehl has
The activity
found et al., cyclic
to 1969; AMP
et al., 1970a). This catebeen found to stimulate
secretion
in
perfused
1971), but it is unknown a physiological role.
testes at this
(Eik-
time
endogenous
AMP
a stimulation by
an
of
in bovine
the
inhibition
the
also
bovine, human and field et al. (1971),
rat
corpora
but
although
The
cyclase
and
was
demonstrated lutea the
localization
in bovine corpora by homogenizing
and
of
in
by Stansproperties
adenylate
lutea has been fractionating
asthe
subcellular components of this tissue by isotonic and hypotonic methods (Sidhu et al., 1975). The clase
fractions activity
Only
were and
assayed standard
5’ nucleotidase
nylate cyclase ly localized
for adenylate cymarker enzymes.
paralleled
of adenylate cyclase hypotonic procedures,
the
in both indicating
distribution isotonic that the
of bovine corpora lutea on the plasma membrane.
g pellet, indicating rat corpora lutea plasma
membrane.
out
on
effect
this
enzyme
and ade-
is primariMenon
bating and
has
LH
adenylate
also
the
plasma
membrane.
shown to stimulate activity of corpora lutea
been
cyclase
activity
the of
of
six
clase lutea
equally or
produce An
significant additivity
cyclase corpora
was
detectable lutea,
but
in homogea very
active
of
incubated et al., effects
on
PGA2,
adenylate
cy-
of bovine corpora and PGE2 were
potent and more effective than PGF2a and PGFIa did not
PGB1.
stimulatory experiment mediatory of LH on an aliquot
a bovine
luteum
corpus
was
effects. carried
incubated
concentration aliquot was
this
concentration third aliquot a fourth aliquot
out
to
role of prostaglandadenylate cyclase. of a homogenate was
maximally stimulatory (100 pg/mI). Another
In of
with
a
of PGE2 incubated
of PGE2 plus LU (10 was incubated with LU, was incubated alone as a
A
If
in
PGE2,
PGF2,)
the possible ins in the action each experiment
and
lutea
rats (Kuehl tested the
(PGE1,
test
with
(Speroff cyclase
corpora
AMP
1971)
and
possible
incorporation
from
prostaglandins
PGA2
LH transmitted cyclase solely
its via
effect PGE2,
on luteal then it
follows that a homogenate of a corpus luteum which was responding maximally to a saturating level of PGE2 should show no further response when LH was added. It was found that LU
cyclase a review rat
the
cyclic
obtained (1970b,
about
as
of bovine 3)
activity of homogenates and found that PGE,
adenylate rized in of
on
they were shown synthesis in incu-
corpora lutea 2) adenylate
and
PGF,
glandins
lutea (summaet al., 1972).
implicated
bovine 1970);
into
ovaries Marsh
PGB,,
responsiveness which of luteal regression. have carried out
correlated with Stansfield and studies on the
of
1970b);
whole 1970b).
and LU the onset co-workers
nates
carried
in the
LU action when 1) progesterone
CI adenine
14
produced
Adenylate
were hormones
Cyclase
in homogenates
other species, such as the rabbit (Anderson et al., 1970) and the pig (Anderson et al., 1974). In the latter investigation, it was found that there was a decline in adenylate cyclase activity
in rat corpora by Stansfield
studies
or other
were
of
slices Ramwell,
control. adenylate
and the plasma membrane marker Na-K-ATPase, indicating that the horreceptor and the adenylate cyclase are on
LH
on Adenylate
Prostaglandins mediators to stimulate:
activity enzyme
localized
No
of
x
enzyme system in be associated with
of Prostaglandins
of LH
pg/mI).
both
this also
system.
Role
Action
and Kiburz (1974) have also prepared a membrane fraction from bovine corpora lutea which contains adenylate cyclase, [1251] hCG binding
mone
the
to study. The largest associated with a 700
that might
the
(Marsh,
in bovine tissue were examined, made to assess the effect of LU.
subcellular
cyclase sessed
lutea
phosphodiesterase.
Phosphodiesterase
of this enzyme no attempt was
corpora
adenylate
of
made it difficult of activity was
Possible of
lutea: no change in activity was a variety of experimental condi1970a). These data indicate, LH brings about the in’rease in
cyclic
ATPase amount
if it
effect of LH on phosphodiesterase also was evaluated in homogenates
bovine corpora observed under tions (Marsh, therefore, that
not
In testicular
been
adenylate cyclase (Murad and Eik-Nes, 1970) and
accumulation cholamine
by
(0.05
epinephrine
a clear-cut additive effect by a maximally stimulatory
produced tration
LH
PGE2, probably (Marsh,
Other mental
not
were
cyclase a variety of
Jonsson
using reported
have
format
adenylate
et
al.,
that transmitted
the via
effect of prosta-
1971).
investigators
prostaglandins in
indicating
of
was
above that concen-
in their
additive and
tissues 1972;
a similar that
experi-
LU
and
effects
on
AMP accumulation (Kuehl et al., 1970b;
cyclic
Kolena
and
Channing,
CYCLIC
1972; ever,
et al.,
opposite
conclusion
was
group
of investigators
et
by
another
aL,
1970b)
who
prostaglandin acid, on ins of
evaluated
produced
a subsequent 7-oxa-1 specific
AMP-dependent Marsh and
show
of
They
inhibited [1 4C]
Channing prostynoic
et was also
LU on granulosa was
of the
(1972) also acid inhibited luteinization cells. The
complicated
inhibitor produced
action
of
synthesis (1972)
this out
into
prostaglandin-like idonic acid nates,
and
with
lutea, and inhibition very large they could
competitive
fact
type.
that
added
to
the
suppression. LU can slices of
stimulation
LH
when
stimulate monkey
into
studies
cyclic
AMP
been
synthesis
reported in
incubated
the
LH
to
this
be of
prosta-
administered
in
Tsafriri
et
al.,
antagonists,
synthesis, glandin
and studies of synthesis. Although
have
not
approaches
possibility
glandin
action studies,
yielded
of
a
definitive
majority to an
of the essential
glandins
the
action
prosta-
prostaglandin
inhibitors these
problem, the in opposition in
have
that
of LH on adenylate studies of prosta-
studies
of
prosta-
approaches
answer
to
data appears role for
of
LU
on
the
to be prosta-
adenylate
cyclase.
It
is generally
recognized
suspect adrenal corpus
from the work cortex (Haynes luteum (Marsh
cyclic
AMP
or
cyclic followed carried
ment
Effects
In
in
this
investigation
The
of
of
the
effects
work for
the
of
the
role
of
in the testis to the studies began with the cyclic
exogenous
cyclic
AMP.
AMP
will
be
Cyclic
studies,
last
This
uncovered some in this field, and
has recently investigators
Exogenous
early
the
progressed to the assessment and finally to the measure-
endogenous
problems
of
of
action of
AMP in steroidogenesis a very similar pattern out in the ovary. It
on steroidogenesis, of adenylate cyclase
ade-
synthesis
the
that had gone on in the et al., 1960) and the and Savard, 1964b) that
involved
was
study
be mimDibutyryl
that
of androgens in the testis is under the control of LU (also called interstitial cell stimulating hormone, ICSU), and it was reasonable to
that
intact
the the
this
to stimulate
when
general
study
mediate additivity
these section.
beyond
LH 1972,
glandins cyclase:
gonadotropin.
synthesis in et al., 1972)
it could AMP.
the
reported stimulation
was
al.,
four
to
to
occur
et
summary, used
phase of the new problems
because cyclic
prostaglandin
animal
by
been
1972a).
Graafian follicles (Marsh latter report, however, this
cyclase step, by exogenous also
the
been the
and
have
in inhibiting
inhibitor
(Grinwich
vivo
of
H] arachhomoge-
[3
indicated
prostaglandin ovaries (Wilks
appears
Pharriss of LU
overcame
also
nylate icked
has
this
synthesis
from of ovarian medium
rabbit In the
has
prostaglandin aspirin
THE ROLE OR CYCLIC AMP IN STEROIDOGENESIS IN THE TESTIS
or LU it
the
injected
incubation Other
and incubated et al., 1974).
suppressed
synthesis
of monkey of this effect
Chaslow and the injection
compounds incubations
in
acid AMP
role for prostaglandins in the on adenylate cyclase would stimulation of prostaglandin
rats
that
regard, experi-
3-prostynoic into cyclic
steroidogenesis
glandin
been
found that 7-oxa-13the action of PGE2 or
the
or release. reported that
antiserum
of
compounds
has also inhibiting
in
ovarian
In
not a completely inhibited cyclic
was mixed with prostaglandin severe cellular necrosis.
A mediatory action of LH suggest an LH
LU.
that
1972)
These
be ineffective
ineffective
with some indicated in
al.,
acid.
Indomethacin
therefunctioned
in cultures interpretation by
the cyclic
concluded, receptor in the
effect of LH the antagonist,
it was
of inhibitors as indomethacin,
of cyclic AMP accumulation in mouse or rat ovaries although they a marked inhibition of prostaglandin (Kuehl et al., 1973; Zoretal., 1973).
(Kuehl
of
that
use such
action
to
in incubated slices of bovine corpora although they could demonstrate an
not
the
prosta-
of LH
reported
ments on the effect of 7-oxa-1 on [3 UI adenine incorporation
of the stimulatory concentrations
been
evaluating
mediators
stimulation incubating produced synthesis
protein kinase. In (1974b) carried
LeMaire
as possible
1973).
found that this antagothe effect of PGE1 and
acid but
antagonist,
to
flufenamic
should be viewed the same authors
paper
a
et al.,
synthesis,
LU.
3-prostynoic
glandins
(Kuehl approach
prostaglandin incu-
fore, that a prostaglandin as a necessary intermediate This conclusion reservation, since
ovaries
Another
(Kuehl of
35
3-prostynoic
competitively accumulation
by
how-
effect
7-oxa-1
bated rat ovaries. They nist not only blocked
AMP
reached, the
antagonist,
also of the
STEROIDOGENESIS
1973).
the stimulation by LU and [1 4C] cyclic AMP accumulation
PGE2, but stimulation
AND
mouse
Lamprecht
An
AMP
discussed
later
in
this
AMP
whole
testis
prepara-
MARSH
36 tions
were
were
the
AMP
could used
They
used, to
in
synthesized lesterol
into
[3
AMP
concentration.
confirmed synthesis slices
of
(UalI
and
testosterone
(0.027
an
far in excess
testis.
not
assessed that
of
cyclic AMP on of testosterone
Their
report
about
by
1962)
that
respond
concentrations
an
testicular
cyclic
AMP,
[1-’4C] Eik-Nes
as
acetate (1968)
(107
M),
indicating
that
Other 1972b;
investigators Catt et
(Dufau al., 1972;
1972)
have
demonstrated
et
al., 1971, Rommerts
that
(Catt et stimulation
identical et al.,
The
aL,
dibutyryl
that
produced
nucleotide. using the
cyclic
Van same
effect of steroidogenic
AMP
into
of
pregnenolone.
with
of
U] cholesterol
by cyclic cate that
the
hCG dog gave of
and Eik-Nes demonstrated
These into
3
taken
conversion
luteum cortex
Mieno
et al.
cyclic
AMP
between cholesterol it seems to be.
(Uall and (Karaboyas (1973) into
Koritz, and
found the
systemic
that
1965b)
Koritz, the
AMP
was
was
a
Near-
above
the
but
these
steroidogenesis.
by
is well within the concentration of cells (Rommerts and
Moyle
Leydig
low,
al.
et
cell
(1971)
1974).
exogenous continuous
tumor
using
preparation,
steroidogenesis Moyle et
stimulatory steroidogenesis
of
effect has
a!.,
exogenous thus been
mechanism
by
was
another which
to both LH and cyclic AMP. however, required the usual of cyclic AMP (2 mM to
M) to stimulate Armstrong, 1970;
but the uncertain.
Fritz, of its
to sustain the stimulation The same phenomenon
This high 0.02
(Moyle 1971).
and The
cyclic AMP well established,
which
it works
on
is still
Cyclase
Another approach to the investigation of the of cyclic AMP in testicular steroidogenesis the study of the adenylate cyclase system tissue
and
its response
Murad et al. (1969) FSH would stimulate in whole homogenates of
FSH
of
an
and
the
seminiferous stimulation
of
(Cooke 1974;
effect
et Braun
tubules this
al., and
to gonadotropins.
found that both LU and adenylate cyclase activity of dog and rat testes.
stimulation
of
with
exogenous AMP have
AMP,
cyclic
quite
required
The
of the
with cyclic
concentration
and the
1965).
out
Dorrington
this
in
injection
circulation
1973;
al.,
was sensitive preparation, concentrations
role was
AMP
nucleotides
of
insento very
synthesis.
M cyclic AMP. This of the endogenous AMP in normal Leydig
Although
in this
U] testosterone
AMP (Sandier and Hall, 1966) indithe site of cyclic AMP action in the as
106
et
these
was
found that this preparation of Leydig cells could be stimulated by as little as 5
Adenylate
at steps to the
data
of the
workers tumor
mouse is
the
occurred prior
stimulation
testis is somewhere pregnenolone, just corpus adrenal
AMP pathway
of
Leydig
respond
cyclic
be
and Shin a monofrom a
which
carried dibutyryl
level
to
isolated
in progestin
amounts
caused
(Hookcyclic
(1967) that derived
tumor,
exogenous
used
observed
in vivo also and secretion
der Molen technique
cyclic
together [7o-3
in
would
of the studies AMP or even
of
1972a). of
of
cyclic
in vivo secretion.
considered
studied
cell
ly all cyclic
presence
testosterone (Eik-Nes, 1967, 1969). Again large doses (150-300 pg/mi) of this nucleotide were required, but the effect was specific for the
formation
been
increase
cyclic
time
by
levels
cyclic
cyclic
and its steroidogenesis
testis via the spermatic artery rise to an increased production
that the in the
also
the
production of exogenous
to gonadotropins,
low
x
1972a, et al.,
1972),
of
to
infusion
cyclic (1971)
generally
interstitial
sitive
range
capable of accelerating testosterone in whole testis preparations. This is about 50 times more effective than
AMP of
(Dufau
has
AMP
are
site of testosterone and the effect
physiological
of theophyllmne and testosterone synthesis
nucleotide phosphodiesterase cyclase system in testis tissue.
almost
cells
main 1970)
testicular of
injection
directly into the testis increase in steroid
significant
study
rabbit
of and
the effect it stimulated
corrected
a previous
to
incorporation Connell
the in
there was a cyclic and an adenylate
cyclic course
nucleotide a considerable the er,
in stimulating the
mouse
Eik-Nes,
AMP is production analogue
effective but
Leydig
of [7a-3U] choThe amount
M) was
not
this effect of mass amounts
judged by the into testosterone.
at low
of
was
steroidogenesis,
cell tumor preparations. Shin and Sato (1971) demonstrated layer culture of Leydig cells
would
also found
the
mass
dog
cyclic
production. observed
brought
tissue
(1966)
levels, but the effect was specific, ADP and AMP had no effect at this Connell and Eik-Nes (1968)
of rabbit
impression
Hall
exogenous
the conversion U] testosterone.
used
physiological since ATP,
and
that
testosterone rat testis and
both and
Sandier
show
stimulate teased
increase
cyclic
and
first
appears
to
adenylate and enzyme
1972;
the in
due
to in
LU effect interstitial
Dorrington
Sepsenwol,
be
cyclase
and 1974).
NaF
a the
to a cells Fritz, also
CYCLIC
produced centrations small
a marked stimulation, of epinephrine and
increases
in the
activity
AMP
AND
STEROIDOGENESIS
nated
and high conACTU caused
of this
enzyme
have
by
any
ACTU on
effect
since
it is not
testicular
known
or
spermatogenesis, and there is disagreement on whether or not it has any effect on cyclic AMP accumulation in whole cell testicular preparations (Kuehl et al., 1970a; Cooke et al., 1972). The
epinephrine
effect,
observed by Nes, 1970;
other Kuehl
catecholamine
however,
has
groups (Pulsinelli et al., 1970a)
has
also
been
been
and
found
to
cause
a
when it via the
spermatic
Eik-Nes
(1971) nerve
suggested fibers and
system stimulation rime, tissues whole been
(Eik-Nes,
of
that this effect may the regulation of the
the of
since must testes.
1971).
testis. adenylate
This
might cyclase
reported
in homogenates and
of
corpora
“mitochondrial” were identified
fractions, only by
characteristics. microscopic
No data
enzyme were
but their
assessed the correlation with marker enzymes
branes
(Na-K
dependent
chondria (succinic the cyclase to plasma membranes. also
preparation
which
seems,
was
was
therefore,
that
most
of
the
subcellular
The cyclic
rat
testis
also
contains
two
phosphodiesterase
the
but enzyme com-
isoenzymes desig-
(Uollinger, phosphodi-
the
on only
effect
of gonadotropins LU or 1 ng/ml of
of
been
1972; and
high
(greater hCG) were
than used
found
Catt Dufau,
to
is apparent et al., 1972;
et
Ramachandran, Dorrington
stimulation that prolactin,
al.,
of
1972).
a
AMP in 1970a;
et al., 1972; Dufau 1973; Braun and
A similar effect preparations (Cooke
This in
cause
cyclic et al.,
1974).
cell
gonado-
relatively
of endogenous vitro (Kuehl
Rommerts et al., et aL, 1973; Catt
in endog-
have, however, proposal that
When
repeatedly
(Cooke
effect
its
changes
testis the
steroidogenesis.
increase testis in
In regard It
testis.
nucleotide
mediates
on
the Leydig Sepsenwol,
adenylate
with mitochondria ponents of the of
AMP
tropins
increase cyclic AMP in tions, but, as mentioned act only on the seminiferous
but with
established. membranes, of this
other
on
in the supported
growth hormone do not AMP levels in either type
endo-
1973),
cyclase is located in plasma there may also be an association and
cyclic
tion
life on
and
1974). specific
mito-
enzyme
adenylate
transmitted
Fritz, quite
responsive mitochondrial
of
1970a)
out
AMP
in Leydig
rat’s
rat 80
Endogenous
carried
Spensenwol,
of 5
cyclase.
enous cyclic not completely
have
in
amount increases
of
effect
probably
and
concentrations 10 mg/mI of
Km
found
in
that
of adenylate plasma mem-
free
not
is
adenylate
marked whole
increase to
acute
et al.,
is a high
The testis
et al., 1973; Moyle and Rommerts et al., 1973;
and
relatively
(Kuehi
through
This of the
no
Km
in the
50 of life in the constant to Day
similar
period
has
testis. in the
Cooke 1973;
(Sulimovici et al., possible contamination
membranes
reof the
and found associated with and Lunenfeld
described a hormonally cyclase in a testicular
in this LU
only
c form
to Day essentially
1972).
Christensen is a low
is presumably
the
is very
1970).
is found
or electron Pulsinelli
ATPase)
dehydrogenase) be primarily Sulimovici
plasmic reticulum the degree of plasma
for
20
al.,
cyclase
they
these fractions sedimentation
marker presented.
(1972) cyclase
lutea
(1971) cyclase and
from Day then remains
Studies
obtained
Uollinger (1970) and Eik-Nes ported that most of the adenylate the testis was found in the “nuclear”
(1973) adenylate
involve vascular
explain the by epineph-
whole ovaries et al., 1970).
fold and
et
and The
other parts of phosphodiesterase
production
f form
M) and
the total
Measurement Cyclic AMP
elements of nerve and vascular be included in homogenates of A similar effect of epinephrine has
(Marsh, 1970a), from fish (Fontaine
tubules.
1972;
The M)
10-5
x
steroidogenesis
small increase in testosterone secretion is infused into the dog testis in vivo artery
10-6
x
(6.5
(Monn
et al.,
1973).
(2.5
esterase
Eikthis
and
type type
to
steroidogenesis
f (Monn
Desautel,
seminiferous
whole homogenates of dog testis. Glucagon had no effect. It is difficult to suggest any physiological explanation for the small stimulation produced
c and
and
in
37
increase testicular FSU
and
appears ACTU
was
the
to
be and
cyclic prepara-
found
to
whole testis preparabefore, it appears to tubules and not on
cells (Cooke et aL, 1972; Braun and 1974; Dorrington and Fritz, 1974). to
the
time
course
the rise in endogenous cyclic about 10 mm of incubation
of the
hCG
effect,
AMP appears with hCG,
after while
the increase in testosterone production was not apparent until after 30 to 60 mm of incubation (Rommerts et al., 1972; 1973). This is very similar to the kinetics of the stimulation of cyclic AMP accumulation and progesterone production
by
LU
in
incubating
slices
of
38
MARSH
corpora
lutea
(Marsh
et
1966),
al.,
compatible with the proposal role for cyclic AMP in the steroidogenesis by gonadotropins. When
careful
sponse
of
terone ever,
comparisons
cyclic
AMP
production a dissociation
been tion able
of
change
AMP.
in
This
effect nucleotide
or
of
of
a is
role that
activity terone
this
testos-
out, effects
of
cyclic
since
review
their
level
work
maximum
value
at
0.8
to
hCG and
1.0
enzyme
concentrareached
a the
in this to
report,
but
mm
had
240
(Catt 1-500
et
cyclic reported tion
to
be
in the
more
accumulation
prominent in
in
was
the
amounts
the
testis
found
cyclic of the
been AMP incuba-
tissue
to of
the
medium
was
parallel cyclic
the AMP
AMP comprised cyclic AMP released.
into incorin
accumulated added that
to the newly
a constant
5
released
of was in
to
the
that this phospho-
AMP, or cyclic GMP, are substrates for this 1973).
and isolated in pg
ng
hCG.
Recently
Po-
study carried They
Leydig mouse and
the
a striking
AMP
was the
and for that
this
problem
out by assessed
in
Moyle and the effects
of LH on testosterone accumulation in
after
AMP the
after
tissue.
rat
for
2 10
Again
between
an increase required
an
the
h
of
mm
in
there amount
in testosterone for a stimulation
in incubations of required for a miniwas about one order
(approximately required
of steroidogenesis in the isolated
synthesis
measured
of cyclic AMP accumulation either tissue. The amount mal increase in cyclic AMP that
kinase was reached a
and
corresponding
on
cyclic
difference
magnitude
of prepa-
cell preparation from rat testis Leydig cell tumor preparation.
medium
of LU required production and
protein hCG The
was (1973).
concentrations and cyclic
the stimulation Leydig cell
testosterone
major
testosterone
was
of cyclic AMP steroidogenic
to expect nucleotide
al.,
increase 100-250
other
incubation
above
carried
changes
at
of various synthesis
of
itself
of 14C-ademine in 14C-adenine
when 20-500 ng of hCG/ml were incubation medium. This indicated synthesized proportion
1
In addition, in both of the an assessment of cyclic
incorporation An increase
poration mass
from previously
has also in cyclic
than
out using the cyclic AMP.
studies out
Dufau et al., 1973) with and no transient rises in
et al., 1973). just mentioned,
AMP
time carried
were detected. It the relative changes
medium
(Dufau studies
been
al., 1972; ng hCG/ml
AMP that
appeared
thorough
in the
et
of
to these a small
Dufau (1975) reported that there was dissociation of the effects of hCG on
range for pg of hCG.
both
only
AMP
at
medium
h) and
cyclic
dose-response was 5-100
one
incubated effects
This stimulation by theophylline a detectable increase
acts via cyclic nucleotides
An
isolated and a
the the
levels.
reasonable of cyclic
(Goldberg
The
by
amount in the
maximum
The
(2
of
It is inhibitor
diesterase since both
began at at about only at
period
medium potentiated
by itself release of
of hCG, indicated Dufau, 1973) that
and
amount
medium. classical
other hand, the release of cyclic AMP 1 ng hCG/ml and reached a maximum 100 ng/ml. The cyclic AMP was assessed time
the and
as yet undetectable probably involved
gonadal tissue Ramachadran
rat release
On
into tissue,
ration. detectable
in some
ng/ml.
5 mM theophylline increase in the
testosterone synthesis protein kinase in an
quite testis,
on the release of testosinto the medium during
increased by 0.1 ng/ml
fact that a significant
desta and a similar
compared the effects of hCG (specific
10,000 lU/mg) and cyclic AMP
as
of of
the physio1971). Two
have carried out problem in the
Dufau (1973) concentrations
significantly as low
of
of cyclic AMP in there should be a
producing et al.,
and was
the
one
concentration smallest
The caused
response to low hCG testosterone production also observed without
a detect-
level
a 2 h incubation of intact decapsulated testes. It was found that testosterone was tions
howhas
stimulawith low
without
on the by the
therefore,
Catt and various
dose and
discrepancy,
investigators studies on
and I will, detail.
low concentrations authors (Cart
the
carried two
hCG
the hormone capable of logical response (Robison groups thorough
re-
endogenous
is a crucial
demonstrable this cyclic
testosterone testicular
accumulation
LU
for action
of
brief, a hormonal can be elicited
the
the criteria hormonal
is
of
have been of these
uncovered. In of steroidogenesis
concentrations
and
of a mediatory stimulation
10 a minimal
(approximately Leydig cells.
ng
LU/ml)
stimulation
1 ng LU/mI) A time study of
the changes in cyclic AMP with these rat Leydig cells was carried out and showed that cyclic AMP accumulated progressively from 2.5 to 60 out
only
relatively high concentrations of ng and 5 pg LH/ml). The results of another very interesting
LU
(500
iment, increase
that the relatively
mm,
but
again
this
study
was
carried
with
reported in this paper, indicated in cyclic AMP, observed with
exper-
CYCLIC
high
concentrations
this sis.
gonadotropic It had been
al.,
1971;
cally
to
et
procedure terminate
The
bound
ever,
by
hormone LH, rate
be
the
hand,
it had
cyclic mone
AMP mimicked on steroidogenesis, nucleotide
but
could
be
specifi-
cells,
stimu-
that
a wash-
an the the
On
of the horeffect by the
rapidly
terminated
cells. The interLU was retained
by the cells even after repeated washings, and that the continued presence of the bound LU was required for maintenance of the steroidogenic response. Since the dose response studies just described (Moyle and Ramachandran, 1973)
raised
some
cyclic AMP steroidogenesis, was
in
carried
effect enous
out
role
of
the hormonal stimulation a similar washing experiment
of
by
procedure level of cyclic
concentration in a rapid
of cessation
one
LU (100 of cyclic
ng/ml), AMP
had
high
which had terminated
lower affinity (since by washing) and was
the
increase
cells and
Moyle et al. carbohydrate this hormone
the of
from rat accumulate
the removal resulted in derivative tion
but
in
stimulate
a much
for
mediated
cyclic
(since
AMP
smaller
the on the Leydig
testosterone found that
carbohydrate in the ability
cyclic
AMP
decrease
two
LU. One response
which and
AMP
accumulation.
of hCG of the
accumulain
steroidogenesis. there were
was with
synthesis.
(1975) investigated portion of hCG to stimulate
its
from
it of
the which Further
be
necessary
an increase
to determine but it does seem AMP accumula-
the
testis of
caused LH is
by not
in steroidogenesis. of cyclic
AMP
accumula-
the effects of ACTU on corticoster(Beall and Sayers, 1972; Moyle et Richardson and Schulster, 1973) and
the
droplet
effects
of
formation
TSU
on
thyroid
(Williams,
1972)
colloid have
been
It seems,
then,
that
the
question
of the
role
of cyclic AMP as a mediator of steroidogenesis in testicular tissue is still unanswered. There are several reasons why small changes in cyclic AMP concentration if they are present, evidence level of
this
If
we
not be detected, until there
MODES
AMP
ON
will
remain
there
is cyclic
and of
with carried
be
the site of steroidogenic between al., 1963; and Hall, strong et
that
possibly LU on
cholesterol Hall and
LU
and cyclic has been
mitochondrion,
tion the
cleavage
in
AMP shown
on the to be (Ichii et Koritz Armseveral
by
listed briefly as 1) an increase in as NADPH; 2) an
of the substrate, in the availability
promoting where
system
its transport the
some
restraining
enzyme system, perhaps transport of an end-product pregnenolone, out of the
4)
sidean
of system;
influence by
of into
cholesterol
is located;
or an increase in the synthesis components of the cleavage
decrease
concenwe are
cyclic AMP might bring about of this reaction and these are
the concentration 3) an increase
the
AMP in the in the corpus
Young, 1968; are, however,
shown in Figure 1, and are follows: It could bring about the amount of a cofactor such
cholesterol
considerable
and pregnenolone Koritz, 1964, 1965b;
1965; Hall and al., 1970). There
this
OF
the action of high Leydig cells, then
action of pathway
in
low is
the problem of how this effect out. As mentioned previously,
ways by which the acceleration
increase cholesterol;
even firm
in doubt.
OF ACTION STEROIDOGENESIS
in favor of a role for of LH on steroidogenesis
luteum trations
believe
is
in cyclic AMP at the or another messenger
question
POSSIBLE
evidence action
might but
for a change gonadotropin
discovered,
chain
effec-
Again types
mediated another
tion from oidogenesis al., 1973;
might
the
the effect associated
to
dissociation
confronted
washing) and and another
testes to synthesize cyclic AMP. They
tiveness to stimulate was suggested that receptors steroidogenesis
by
cyclic
of part of the a marked loss to
affect high
from this of receptors
affinity
for
Similar
what
it did result accumulation
LU. It was proposed might be two types which
see
did not a relatively
not be terminated with steroidogenesis,
Recently role of ability
to
required
CYCLIC
have on the endogIt was found that
effect could was involved
major
the
authors
would AMP.
washing procedure response to
caused by the data that there LH:
about
these
this
although the the steroidogenic
for
questions
seem
validity of this suggestion, the large increase in cyclic
reported.
exogenous
effect this
washing the tumor of this data was that
with
would
the that
LU
terminate that
work
39
tion in the Leydig cells of relatively large concentrations
how-
cells
shown the
et
LU
removed,
washed
been
STEROIDOGENESIS
for
the bound steroidogenesis.
and this did of steroidogenesis.
other
by simply pretation
and
could of
required
Leydig
not remove enhanced
AND
steroidogene(Moudgal
that
production, can the
to
not
1971)
tumor
treatment
antiserum accelerated
cyclic
al.,
these
testosterone
ing nor
was
stimulation of shown previously
Moyle
binds
lating
of LU,
AMP
activaone 5) on
of a
this
enhancing the inhibitor, such as mitochondrion. There
40
MARSH
tion
LH
accelerated
in the glucose glucose
/-\ AlP
CYCtIC
AMP
i
(
duction cofactor
I_#{149}.4.
PHOSPHORYLASE
KINASE
]
PRoTEIN
I
.
:
sis, FREE
/ I
,,
CHOLE5ROL
\ “S
0
SYNTHESIS
\
\_#
5
E
STEROL
.
*-
)
the
et
al.,
produced
-H
PREGNENOLONE
FIG. 1. Possible sites of action of cyclic AMP on steroidogenesis in a hypothetical gonadal cell. Solid arrows indicate biochemical reactions, and the large dashed arrows indicate possible effects of cyclic AMP on the steroidogenesis process. Five of the dashed arrows indicate that the possible effects of cyclic AMP might be mediated through protein kinase, but it is also possible that cyclic AMP influences these steps in steroidogenesis by a mechanism not involving protein kinease. substance
The symbol ----p through membranes
supporting
is evidence and
the
at
these proposals it is possible
one
or more
are that
of these
in favor discussed
of (or below
obtained
from
the
time
of it
one of them however, it
that and
The with since tissue
each
present
choose any this regard,
indicates transport of the cell.
these
is not
as the should
to
The
evidence
hypotheses on the
with
In
is data
gonadal
tissue.
results of some important experiments adrenal cortex tissue are also included, the control of steroidogenesis in this has the same general features as that in ovary
and
the
testis.
Via Increased
Haynes (1960) AMP
and
were might
adrenal
Berthet the first accelerate
cortex
of a necessary pathway. Briefly,
by
(1957)
to propose steroidogenesis
increasing
cofactor their
and
the
tissue. in
Marsh and son, 1965; rat ovaries
phosphorylase
system;
2) phosphorylase
prepubertal
the
pathway
(Hal-
NADPH is in
system
such
Estabrook reducing
activity lutea
rat
the
as that
(1969) equivalents
increase
this
activity
(Selstam
is
in vitro
and
vivo will activity
et al.
lutea of
and
(35
phosphorylase ase activity containing enzyme
1964a),
was
was
1973). of LU
It has also into rats in
LU but
and
phosphorylase the luteinized
that
was
have
limited a large
been
number number
cows
were
examined found
a
signifia prep-
ineffective.
to
from was
1965; 1973). lutea,
although
and hCG activity,
however,
obtained
rat
Ahren
hormone
pregnancy percent)
slices Savard,
and Robinson, Ahren et al., bovine corpora
out with only a lutea, and when
Savard,
Ahren,
1971;
reported
appears,
corpora
and
in the or
preparation phosphorylase
of luteinizing study
months small
(1961)
1961;
and Robinluteinized 1965) and
in incubating
Ahren,
type of ovary (Stansfield Seistam and Ahren, 1971; In the experiments on pituitary increased
detected
et al.,
lutea (Marsh and isolated prepubertal
cause an increase in the prepubertal
Williams
a
1971; Ahren et al., has been shown to
Selstam, 1971; Ahren et al., been found that the injection
crude cantly
been
(Selstam
Selstam, LU
of bovine corpora 1964a) and whole ovaries
has
(Williams
ovaries
Ahren and Furthermore,
cyclic in the
activa-
latter in the
cells
and
Savard, 1964a; Stansfield Yunis and Assaf, 1970), (Stansfield and Robinson,
that
1)
cleavage
for this localized
cortex
1961)
and the
corpora
et al.
ACTU increased the synthesis of cyclic AMP in the adrenal cortex, which in turn activated the
adrenal
Phosphorylase
bovine
Uaynes
concentration
of pro-
There is some evidence in support of mechanism such as that suggested by Uaynes and coworkers for the action of LH in ovarian
carried corpora
in the steroidogenic proposal stated that:
phoslevel
side-chain
pentose
organelle.
of of
pentose
increased corticosteroid
system to be
a shuttle
into
aration Cofactors
the
by Simpson to transport
This Action
the
described required
1971; 1973).
exclusive, may act by
mechanisms.
against) these with emphasis
a
proposals, possible
correct one. be recognized
not mutually cyclic AMP
experiments
of
cholesterol
Thus
this
the an
resulting
amounts metabolism
of its role as a required the steps of steroidogene-
1959,
by
cytoplasm. PROGESTEEONE
of
of
kerston
MITOCHONDRION
through
The enzyme has been shown
mitochondria
%
PREGNENOLONE
*-
including
increased 3) the
produced stimulated
by means in many
reaction. reaction
NADPH
breakdown,
of
6-phosphate
phate pathway NADPU, which
I PROTEIN CHOLESTEROL ESTER
glycogen
formation 6-phosphate;
in the
of of first
to produce
significant
6
(Marsh increase
a in
activity. The luteal phosphorylmeasured in an assay medium
5’-AMP, similar
which indicated to the liver enzyme
that this which is
CYCLIC
only
partially
activated
by
the
AMP
AND
nucleotide,
STEROIDOGENESIS
and
differed from skeletal muscle phosphorylase which is completely activated by 5’-AMP. This was confirmed when the luteal enzyme was purified (1970). the
and The
characterized stimulation
incubating
specific
slices
for
lated
an
increase
in these slices. concentration
Assaf in
corpora
lutea
hormones
and
in progesterone
was
not
cause
although
it
a rise did
a small
increase
in progesterone synthesis. One LU and nucleotide incubations
difference was the
sion of medium.
the
0.02 This
M caffeine methylxanthine,
in
in the inclu-
cyclic addition
in
AMP to
its inhibitory effect on cyclic nudeotide phosphodiesterase, has also been reported to stimulate phosphorylase phosphatase (Sutherland, 1951), which may account for the small decrease served
in in
phosphorylase the cyclic
presence
of caffeine
account
for
does
the
phosphorylase
activity actually AMP incubations. not,
lack
of to
system
however,
of
other experiments the omission of
(Marsh, unpublished caffeine from the cyclic
treated
samples
eliminated
activity,
but
still
did
not
A larger has also lutea
exogenous
increase
the
concentration been tested
in the
stimulation an effect
(Savard
et al.,
better
of
synthesis
the
might
stimulating supported
percent
the increase
activity
while
they
LU in synthesis
terms of (Marsh
also
supported
been
to
stimulated us that be
an
corpora exhibited an and by
tested of
finding
of This 10
showed response
in progesterone 1964a). This that
bovine
h
glycogen
Selstam,
and
activity. ovary muscle
in vivo
entirely b form,
The was and
to a shift from to the 5’-AMP
enzyme. at
The
LU of
or
mM
phosphorylase
phosphorylase
it
mM
to
effective
the than
at a concentra-
increased
the
appears
that
LU
and
phosphorylase
percent
similar
situation
Uaynes stimulated
perhaps
in
preparations, but it seems that not be essential for an increase A
of
cyclic
of
a.
stimulate
cortex. ACTU
dependent a form
1-25
more
Epinephrine also
of
dibutyryl
from
FSH.
10
prepubertal rat a rise of ovarian
of
was
of LH
reported to resemble the increase was due
addition
ovaries
et al.,
injection
the 5’-AMP independent
concentrations
incubating
no
Ahren,
Ahren
in vitro incubation of with LU or FSU caused
this that
in
but
concentration.
1971;
the
that the about a
later,
(Selstam
phosphorylase
tion
found caused
activity
4
in
that
prepubertal of skeletal
Kilpatrick, glycogen.
phosphorylase
coworkers
It
has
NADPU
FSH
several
ovarian
this effect may in steroidogene-
exists
in
the
adrenal
(1958) demonstrated phosphorylase activity
been
to (Mason
that in
is
that
the
addition
of
slices of bovine corpora 1962; Savard et al., 1963;
and Casey, 1964; Armstrong, 1966), lutea or interstitial
Mason and Savard, to slices of rabbit tissue (Dorrington
and Kilpatrick, 1966b) or to homogenates of whole rabbit ovaries (Scoon and Major, 1972), caused a marked increase in progestin biosynthesis. strated the
to
found
incubating et al.,
In addition, Savard et al. (1963) that the bovine corpus luteum
concentrations
phosphorylase
a good
increase Savard, the
effect
synthesis. that about
the ovaries or
Savard 1964b; corpora
activate phosof exogenous
essential
reported
lutea
progesterone phosphorylase
lutea
in terms
cyclic
exogenously. to
1973)
(1965) in vivo
ovaries
and
1965), Armstrong, 1965) and
bovine adrenal cortex, but Ferguson (1963) and Kobayashi et al. (1963) found no stimulation by ACTU in rat adrenal cortex tissue even though this hormone produced its usual stimulation of corticosteroidogenesis.
resolved,
intracellular
progesterone by the fact
of or
there
synthesis activity
nucleotide
not
no
little
again
that adding LU to slices may be a much
this inability a concentration
by
activation
never
increasing
AMP which indicated
cyclic
in
suggested lutea
adding
Nevertheless, phorylase
and
Ahren
sis.
activity. (0.02 M) corpora
progesterone phosphorylase
has
been corpora
than
caffeine
1971;
Thus
in
in
changes and
can
1965).
way
AMP
LU was
of of on
discrepancy
but it has incubating
drop
AMP bovine
increase
Ahren
either
Robinson,
and measurable
and Robinson of LU into rats
significant
the
and
(Dorrington little or no
luteinized
to
this for in
nucleotide
phosphorylase
ovaries contain
Stansfield injection
AMP
data) AMP
slight
cyclic
of cyclic on these
absence
was a without This
the
the
rabbit 1967)
obThe seem
response cyclic AMP,
(Stansfield
rat ovaries (Deane, 1952; Stansfield and Robinson,
their
in phosphorylase
cause
lutea
luteinized 1963;
4-fold
corre-
synthesis
The addition of cyclic AMP at a of 2 mM to the incubating slices,
did
activity,
of
LU-containing
with
however,
by Yunis and of phosphorylase
corpora
41
pentose
however,
that
tatively quite first indications the
study
of
of the
NADP
pathway.
It
became
the
of
NADPU
effect
dehydrogenases
different from that of this difference Savard
et al.
demonhad high
(1963)
of
apparent, was
quali-
of LU. The came from where
it was
MARSH
42
found
that
addition
the
incubating
the
in
of
LU
presence
of
of NADPU, caused a further terone synthesis. This should
if the NADPU. also
effect of LU Dorrington observed
effective
be
effects
different
in
LU
of
LU and corpora
and
of
NADPU. to
utilization.
precursor
that for
nearly by
shown that to incubating (10
mm)
free
cholesterol.
either
by
esterase
or
ester action
synthetase, has been
NADPU,
on
the
1964;
into
this
Mason
and
became (1966)
steroid
NADPH
damaged
cells
intact
the
of
adrenal
probably
while
LU
and
it
Armstrong luteum and cortex
acted
only
on
ACTU
acted
on
cells.
In
regard
to
the
endogenous
concentration
of NADP and NADPU and the effect of LH, it has been reported that this gonadotropin has no effect on the concentration of either the reduced
or
nucleotide 1968)
the
or
Denton,
1970).
gators cellular
form
rat
and
that
ovaries, in this
Denton,
that
LU
of
account utilization
the
duced
increase
thereby
concentration seems theoretically more
of
information
had no effect It was concluded
therefore, about its
it
It
Via Increased is
well
known
that it was increase in
not
take
into
of its production, unaltered
how
by the side-chain cleavage impossible to reach a firm matter. Action
on
the produccriticized this
did
NADPU. possible, on
stimulation an
of
inhibition
the of
and evidence presented.
and the
into
come
about
cholesterol
the for
cholesterol both
sites
of
esterase activity No direct effect
in luteinof cyclic
theophylline
mM)
(0.05
mM)
observed, added to
or
however, when the cholesterol have
(1
these esterase
Flint
et al. (1973)
AMP assay rabbit
had no direct effect system for cholesterol ovarian interstitial
also
demonof LH
was
substances were assay mixture.
reported
that
cyclic
when added to the esterase activity in tissue. Another piece
of evidence indicating that LH activates the cholesterol esterase enzyme is that of Behrman et al. (1972), who showed that the administraof an LU
antiserum
in a activity
to pregnant
rats
marked decrease and a reduction
in vivo
in cholesterol in progesteron
LU on cholesterol esterase is secondary to the action of this hormone on steroid synthesis. An enhanced conversion of cholesterol into preg-
rate an
a by
cholesterol could
increased cholesterol ized ovaries of rats.
resulted esterase
and
steady
state
NADPH
(1970)
since
is
is utilized it is this
other
they
injection
of
LH
remove
stimulatory
that
cholesterol effect of observe
ester
depletion by
Flint
feed-
enzyme. Behrman
the
the when
al.
(1973),
The et
LU-induced
action
of
to
LU
a on
steroidogenesis
aminoglutethimide. et
of
ester was not due LU on steroidogenesis,
could
hand,
effect
a hypothetical
of the esterase point is equivocal.
blocked
On working
the with
the same tissue, found that the administration of aminoglutethimide or cycloheximide in vivo blocked the effect of LU on the depletion of cholesterol Goldstein
the
this
concluded of
cholesterol
Substrate that
might
depletion secondary
was
This alternative but until there system in situ, conclusion on
nenolone
that
back inhibition evidence on this al.
that an increased rate of might balance an LU-in-
in the
maintain
ester
effect
Armstrong (1969) vivo administration
tion
investi-
of
but tissue.
that
possibility of NADPH
latter
(Claesson
1967; Armstrong, et al. (1973a) have
secretion. It is possible
1970), brought
saying the
pyridine
be accounted ester
glucose raised the intraof NADPU in slices of
steroidogenesis solely by increasing tion of NADPU. Hall (1971) interpretation
this
corpora lutea (Marsh, rat ovaries (Flint and
Furthermore,
also found concentration
luteinized steroidogenesis (Flint unlikely
oxidized
in bovine luteinized
a and
in
AMP
that
cause
1948; Bell et 1968) and
can
Uerbst, Moyle
This
Behrman strated that
Casey,
Finally,
work corpus
bovine
on
and
1964b). the
increase
but mark[7-3U]cho-
(Savard
from
(1968)
exogenous
not
did
Savard,
apparent on the
Halkerston
hand,
of [114 C] acetate, the incorporation of
the incorporation edly enhanced lesterol
other
decrease
conversion
cholesterol.
U]
will (Claesson
the addition of LU or cyclic AMP Leydig tumor cells causes a rapid
the
[73
rats
in cholesterol
1948, 1953; Furthermore,
[i1 4C] aceextent than
of
and
cholesterol
all of this
LU increased the incorporation of tate into progesterone to a greater incorporation
rabbits
ovarian
a decrease
et al., 1968).
NADPU on incubating lutea also appeared
terms
in
Uillarp, 1947; Levin and Jailer, al., 1964; Herbst, 1967; Armstrong,
maximally
of
into
decrease
solely by (1966b)
mediated Kilpatrick
concentrations
preparations
slices, amounts
increase in progesnot have occurred
was and
additive
The effects of slices of bovine
to luteal optimal
the effect slices of
ester. and
Marsh
(1973)
have
assessed
of LU and cyclic AMP on incubating bovine corpora lutea and found that
CYCLIC
LU
and
cyclic
AMP
treated
AMP
tissues
had
higher values of cholesterol esterase Moreover, homogenates of this tissue
AND
STEROIDOGENESIS
suited
slightly
in
activity labelled
activity. responded
an
These
homogenates
AMP-dependent
(1973a) conversion
of
isolated and
cells
cyclic
might
be on
and
ester AMP in
the
effect
of
esterase
a protein
kinase
as
which
such
1970;
al.
that
triglyceride Corbin
lipase
et
al.,
Tsafriri
follicles
et
al.,
DeAngelo al., 1971;
1972b;
et al., Means
been
lutea 1973)
luteum tionation
purified
from
esterase was soluble fraction
ol esterase cyclic AMP
increase in activity of cyclic AMP activation
of
still
uncertain
the
role
activity, but any further
esterase
AMP
in
the
role the
study
stimulation
ether
plasma kinase adrenal
anesthesia,
in
vitro
cholesterol bovine dependent
also
esterase adrenal upon
pendent partially and
is known
cortex and the presence
protein purified esterase
cyclic
increased
kinase. bovine with
the
preparations
activity from
rat
ATP,
et
inhibition system al.
kinase
and
(1973).
ity
in
of a direct activity by
way
These
ovarian
of
AMP re-
transfer
stimulation cyclic AMP, (Naghshineh (Pittman and
which
in
investigators
LH
could is via
syntheby Flint
showed
that
LU to rabbits ether synthetase
interstitial
steroidogenesis.
onto have
concentrations
tissue
was mimicked by cyclic inhibition of this enzyme cyclic AMP seems to be the
and
in this
vivo activeffect
AMP in vitro. The system by LU or result of an increase
Progesterone are
and
20a
synthesized tissue after
interstitial
hy-
at LU
inor
cyclic AMP treatment and these steroids were found to exert a direct inhibitory effect on the synthetase system. It was also found that if the effect of cyclic AMP on steroidogenesis was inhibited mide then
by cycloheximide the inhibition
also lost. interpretation
This
result that LH
level
and
a cyclic the
the cholesterol ester this has been explored
of
of a choles-
cyclic
of
by
these effects were of cyclic AMP-de-
protein
involves and
to
cortex
group from ATP molecule. There
ester
It seems,
to elevate
Preincubation adrenal cortex
[‘y-32P]
AMP-dependent
the tase
(1974)
adrenal
kinase
possible
cholesterol
progestins.
ACTII concentrations, increased protein and cholesterol esterase activities in rat cortex. The addition of cyclic AMP and
ATP
terol
which
control
synthetase
cholesterol esterase of adrenal cortex tissue has been carried out (Simpson et al., 1972; Boyd and Trzeciak, 1973; Trzeciak and Boyd, 1973, 1974). In brief, it was found that stress induced by
ACTU
been two other reports of cholesterol esterase
1974). other
of 32P fraction.
Boyd of
phosphate esterase
of
is thus
A thorough
by
droxypregn-4-en-3-one creased rates in
fracof
The in
and
protein
esterase
transfer protein
activation
esterase
of a terminal the cholesterol
in purified corpus
muscle protein luteal cholester-
cholesterol time.
et has
Marsh,
sulfate addition
1973). kinase
cholesterol
cholesterol
Trzeciak the
the administration of decreased the cholesterol
corpora
and 1971).
the esterase did not cause
at this cyclic
1973;
al.,
(Goldstein, and protein
luteal
of
et
partially of bovine
skeletal purified
increased or ATP
1973;
bovine
by ammonium 1973). The
AMP dependent to this partially
et al.,
led
that
Khoo, The
1970;
and rat testes (Reddi 1974). This enzyme
1973; Goldstein testes (Reddi et al.,
homogenate (Goldstein,
cyclic kinase
DeAngelo
1974) et al.,
partially
(Menon, and rat
Cholesterol from the
(Lamprecht
data
propose
in
concomitant to the
ATP and protein kinase in idrenal et al., 1974) and adipose tissue
and Huttunen, 1972). Cyclic AMPprotein kinase activity has been in bovine corpora lutea (Goldstein and 1972, 1973; Menon, 1973), rat ovaries
Graafian
also
that
tissue
al.,
et
cholesterol by cyclic
through
adipose et
Moyle
cholesterol
reaction, the
Steinberg dependent detected Marsh,
of
on
mediated
(Uuttunen
of
suggested
AMP
phosphorylation acts
report
a stimulation into cholesterol
Leydig
LU
the
increase
with a phosphate
to cyclic AMP and ATP with a significant increase in cholesterol esterase activity (Goldstein, 1973). This effect of cyclic AMP in and
43
of
esterase ester
the
then,
and
in an
an
production
of
that
LU
increases
the
at
the
expense
of
cholesterol
ester both
was
keeping with the cholesterol ester
rat and activation
inhibition
rabbit gonadal of cholesterol
of
the
cholesterol
synthetase.
The control
importance of this of steroidogenesis
difficult a greater free
is in inhibits
increasing
free
cholesterol tissues by
or aminoglutethithe synthetase
of
to assess. proportion
form,
LU
It is possible of the regulates
steroidogenesis.
On
just a supporting strate when the
effect, steroidogenic
accelerated another corpus cholesterol
by
the
a other
an action
and more luteum, at ester
action of in gonadal
crucial least,
storage
that by cholesterol
keeping in its
limiting
step
in
may
1e
hand,
supplying pathway of the
this
more has
gonadotropin
step. In the effect would
LU in the tissue is
appear
subbeen at
the of
bovine LU on to
be of
MARSH
44 relatively reported
minor importance, that this tissue,
which
since it has is capable
marked steroidogenic response to amounts of free cholesterol and measurable amounts of cholesterol belman Seifart
et al., 1961; and Hansel,
Uafs 1968).
and
been of a
LU, has large small to unester (Zim-
Armstrong,
1968;
been found in the mitochondrial fraction of bovine adrenal cortex al., 1972; 1973). It
Via
Increased
cholesterol
and
side-chain
adrenal
Transport
chondrion, the
system
has
cholesterol been
mitochondria kerston et lutea (Hall 1967),
rat
sky et (Moyle
al., et
side-chain
shown of
to
be
cleavage
localized
in the
bovine adrenal cortex (Ual1959, 1961), bovine corpora Koritz, 1964; Yago et al.,
al., and testis
(Toren
et al.,
1964;
Drosdow-
(Mason and is synthesized
Boyd, and
and since cholesterol outside this organelle,
that
there
be
a migration
mitochondria pregnenolone.
before There
the into
cholesterol
sidechain
the microsomes corpora lutea and
later
The dna and
1971),
of cholesterol
site of tropic there has been very
hormone limited
transport
been
will
be
dis-
into
mitochon-
a limiting step in steroidogenesis could be another candidate
the
has
both
of bovine ovaries (Flint
this
but
in
of
section.
transport
might be therefore
cholesterol
activity
and the mitochondria and rat luteinized in this
into
it can be converted has been a report
cleavage
Armstrong,
cussed
cholesterol
in
a considerable
action. Although experimentation on gonadal
tissues,
amount
of
there
work
done
on this possibility in the study of the mechanism of action of ACTH and cyclic AMP in the adrenal cortex. This possibility was first proposed (1968). such
by
Uechter (1955) In the latter work,
a translocation
volve
the
since
cycloheximide,
of
synthesis
of
AMP, which when
It was enhanced facilitated
a
the and in proposed
protein,
inhibitor
of
that
AC1’H,
the synthesis of cholesterol transport,
cycloheximide
in-
protein
acceleration of steroidoincreased the accumulaextramitochondrial lipid
inhibited
it blocked cholesterol crease in steroidogenesis et al.,
might
regulatory
an
synthesis, blocked genesis by ACTH, tion of cholesterol droplets.
and then by Garren it was suggested that cholesterol
via cyclic a
protein
transport (Garren,
protein and that synthesis
and 1968;
the inGarren
1971).
A heat
stable
cholesterol
binding
protein
Mahafee ACTU
has
et
al.
a
can
direct
of
(1973)
represents
have
a choles-
transport into the
chomito-
evidence
for
this
at this time. This cholesresembles the regulatory
the
hypophysectomized free cholesterol
of this showed
even
when
authors’
at
other AMP
that the
some
later
of
protein
stimulation step
in
block of
the
the
by
synthesis of
in of
AMP
was of
the mitochondrial brought about
this
interpretation
ACTU and cyclic tion of cholesterol
of and
site because promoted this
known to treatment
did not prevent or cholesterol
blocks
of
level
aminoglutathimide
in doses Cycloheximide
indicating
inhibitor genesis
demonstrated treatment
in mitochondria to the stimulation some cyclic
the rats also accumulation
cholesterol
steroid into pregnenothat this stimulation
at dibutyryl
was administered steroidogenesis.
this
have AMP
accumulation not secondary
steroidogenesis ACTU and
ACTU,
of
rats increased the adrenal mitochondria
in
the conversion lone. They also cholesterol probably
level
et al. (1974) and dibutyryl
accumulation for
no
of
protein proposed by Garren et al. (1971), but this hypothesis required that ACTU increase the synthesis of this protein. Ungar et al. (1973) have reported, however, that ACTH does not change binding protein.
of
activity preparation
which cytoplasm
but
Leydig cell tumors and human placenta
must
protein
function is available terol binding protein
1965), mouse al., 1973b) 1971), stored
the
Ungar
this
cytosol (Kan et
Ungar et al., affinity for
cleavage
mitochondria. that
1973; binding
stimulates
terol carrier protein lesterol from the
of
Cholesterol Since
and Ungar, a specific
cholesterol proposed
Action
Kan has
and tissue
steroido-
process. data
The
was
that
increased the accumulathe mitochondria by
increasing esterified
the ratio cholesterol
however, a carrier
that ACTH, via cyclic AMP, activated protein, such as that described by
Ungar
the
free cholesterol to cell. It is possible,
et al. (1973).
A modification al was
of in
suggested
Jefcoate et al. stress increased formation from nal mitochondria
of by
this Simpson
latter
type et al.
of propos(1972)
and
(1974). They found that ether the initial rate of pregnenolone endogenous isolated
cholesterol from these
in adreanimals,
but there was no effect on the initial content of cholesterol in these organelles. The ether stress also induced changes in the type II difference spectra of cytochrome P450 which were inter-
CYCLIC
preted
as indicating
an increased
cholesterol-cytochrome therefore, proposed total mitochondial for
side-chain
ACTU
cleavage,
increases
This
could
cytochrome
be
dial duced
sites. It is also changes may
cholesterol
This
of
inhibited by labile protein port
that
stress
due
to
the
via
is active. transport
side-chain
or
other
possible result
that these stress-inin an increased entry
the
stress
intramitochon-
mitochondria
was
in
found
cycloheximide may be involved
to
be
vivo.
indicating that a with this trans-
on
effect
of
cholesterol
gonadotropins
transport
not been studied tissues. Flint and assay caproic
involving acid
ported side-chain
that
into
microsomes ovaries
was about activity in also
We
have
be no into been
1973).
In
this
however,
hands,
nates as the marker succinic dehydrogenase.
to
this
cholesterol
sidedistribuhomoge-
chrome C reductase. It is unknown at this if this discrepancy between the results of and Armstrong (1971), on the one hand,
time Flint and
methodological the former
Caron (1973) on the other differences, workers did levels they
the endogenous fractions when side-chain
activity.
mitochondria bovine times
corpora as
microsomes
much
not
most other is due to
but it seems that take into account
of cholesterol calculated the have
We
isolated
from
lutea
contain
endogenous (Caron,
and hand,
1973).
found
in these levels of that
the
homogenates more cholesterol If
the
of
than
four as
exogenous
the
be about four incubations Thus
the
reaction
the
would
cleavage microsomes.
in
(1973b) cholesterol
enzyme
and
system
found the
inner
the
enzyme
membrane
activity
was
as localicleavage
cells
limiting availability
step
and
localized
mitochondria,
on while
was associated indicates that
in steroidogenesis of substrate cholesterol
a
could be the in this tissue
as well. Robinson and Stevenson also reported in an abstract that
(1973) have the treatment
immature rats increased in the the mitochondria ovaries. an effect transport
as
cholesterol tumor
all of the cholesterol outer membrane. This
dna remains gonadal tissue, implicate this
times
the
endogenous
essentially with the
the luteinized Thus, although AMP on cholesterol
of
mitochondria
Leydig of the
lower in the
sidechain
determined sidechain
the
from
that
the four
the
the of
amount
of
represent
activity
mitochondria
times than
same
products
Moyle et al. zation of the
of
with hCG transport of obtained
LH into
and cyclic mitochon-
to be examined thoroughly in the studies on the adrenal cortex process in the control of steroido-
genesis.
Action
Via Increase
Cleavage
on
the cyto-
of
activity NADPU
cleavage
Side-Chain
Activity
Cyclic cholesterol
enzyme for mitochondria, There was no correlacleavage enzyme,
in
from
luteabout
confirm
the same of luteal
ones.
radioactivity
Rat
lutea, using an of [414 C] products (Car-
the
microsomal
of superovulated causes a marked cholesterol into
have
is uniformly diluted by then the specific activity
this substrate would in the mitochondrial
as much corpora
and
the data investigators,
the marker
C] isore-
for a transport of in luteal tissue.
chain cleavage activity shows tion in the different fractions
tion between microsomal
to
bovine corpora the conversion labelled steroid our
gonadal using an
activity in microThis suggests that
requirement mitochondria
unable,
distribution in assay involving cholesterol into on,
twice bovine
found
one quarter as much of somes as in mitochondria. there may cholesterol
has
in
as in mitochondria. were
AMP
mitochondria
production of l4 [2614 C] cholesterol,
there cleavage
inized
cyclic
very extensively Armstrong (1971),
the from
lutea
or
45
labelled cholesterol endogenous pools,
in
readily
or binding. The
STEROIDOGENESIS
much in the
cleavage
from
into
effect
of the It was, of the available
which
to the
P-450
amount
fraction
of cholesterol
AND
complex. a fraction was
and
that
increase
binding
of
P-450 that only cholesterol
AMP
the
AMP into
could increase pregnenolone
side-chain
cleavage
activating, or increasing the components of this of
cyclic
stimulation lesterol
AMP of into
the conversion of by a direct action
was
enzyme
system
the synthesis complex. Such proposed
to
the conversion pregnenolone
by
of one of an effect explain
its
of labelled choin various adrenal
cortical mitochondrial preparations (Roberts et al., 1965, 1967; Roberts and Creange, 1968). Koritz et al. (1968), however, were unable to substantiate this proposal and suggested instead that
the
increased
pregnenolone
accumulation
was due to an inhibition of the conversion pregnenolone to progesterone rather than increase in pregnenolone synthesis. t53j3hydoxysteroid dehydrogenase and A53ketosteroid involved both
isomerase in this
the
reaction
mitochondria
enzymes appear and
which
to be present the
microsomes
of an The the are in
46
MARSH
(Sulimovici 1970). This the
and Boyd, inhibitory
pregnenolone
to
confirmed
by
been
1969; McCune effect of cyclic
progesterone several
and and
inhibits nase,
at
tions,
by
Lunenfeld, it seems
that
competing
suggested
that
tory
effect
LU
on
the bovine confirmed later and
1971; the
with
findings have experiments.
al.,
Snnivasan et al., cyclic nucleotide dehydrogefor
the
had
corpus luteum, but this by some of these same
total amount synthesized
stimulasystem
the conversion and inhibited to progesterone.
of
the
side-chain
cleavage
vici and Boyd, 1971; Srinivasan Recently
we
cyclic AMP, reconstituted system from
have
a direct (Caron
system
was
mitochondria and the
solubilized
from
system lesterol
was
sue The
the
other
were tional
arranged to the
present.
When
kinase
preparation,
effect
on a cleavage and have enzyme cleavage
corpora
of The was
adrenal
AMP,
ATP
the chothen
cortex
purified
tis-
reductase). components
and
cyclic
AMP
on
the
a physiobut there of this
in whole
mitochondria
Efflux of Mitochondria
Hall
(1964)
inhibited in
observed
its
an
a protein from
cor-
and
own
extract
since must
the egress of chondria (Koritz
that
preg-
synthesis
from
from
adrenal
mito-
it was believed that leave the mitochondria
pregnenolone and Hall,
the to
(Beyer and that ACTU determining
from 1964;
the Koritz
mitoand
Kumur, 1970). It was also reported that substances which cause mitochondria of steroidogenic tissues to swell stimulated pregnenolone synthesis, decreased itz, 1964,
while ATP which inhibited this stimulation (Uirschfield 1966).
are which
probably
several indicate
not
pieces that
correct.
McCune 1971;
et al., Srinivasan
1970; et
pregnenolone
First
of
all,
mitochondria than that observed cycloheximide-treated
there
was
age the
activity stressed
increased
1972).
to accumulate
cleavage enous
stressed
cholesterol
rats
was
mitochondria even though
side-chain
cleav-
obtained the amount this
in mitochondia, to progesterone with affect the stimulation
activity ACTH
amount isolated
from of
inhibition concentrations
is
mitochondria (Simpson et al., when pregnenolone was made
occur in Fourthly,
conversion it did not
Secondly,
in the rats,
required for the physiologic
in 1969;
that the freshly
in the mitochondria animals. Thirdly,
pregnenolone greater than
is a
present
progesterone.
from
greater from
is
there
and Lunenfeld, which can con-
Simpson et al. (1972) reported of pregnenolone present in adrenal
how-
Boyd,
Sulimovici aL, 1973)
into
swelling and Kor-
of evidence, this hypothesis
5-313-hy droxysteroi d dehydogenase mitochondria (Sulimovici and
which
proporP-450
lutea, were added to this reconstituted cleavage system there was a consistent stimula-
pora
and
cholesterol
vert
lutea
A treatment component was
the activity was of cytochrome
partially
of
ATP
this The
and adenodoxin of these three
cyclic
Konitz
There
isolated cytochrome iron component and of
of
in situ
Increased From
nenolone
Lunenfeld,
chromatography. cleavage activity
so that amount
the
was of the (Sulimo-
components
component
(adrenodoxin proportions
of
from
using this the non-heme
flavoprotein
and
the
phospholipase P450
by column side-chain
reconstituted P450 and
examined
was
effect
de-
indicating involved.
system may represent of the cyclic nucleotide, to be further confirmation
Action Via Pregnenolone
ever,
stimulation et al., 1975).
by cytochrome
isolated
activity
protein kinase and cholesterol side-chain bovine corpora lutea
observed activity
reconstituted logic action will have
of ATP probably
be converted into progesterone Samuels, 1956), it was suggested might control steroidogenesis by
pregnenolone the inhibition dehydrogenase and
vitro
presence
of 20 to 70
completely
of cholesthe converWhen the
of
1969; Sulimovici et al., 1973).
in
order
was
chondria, pregnenolone
compared with that in the controls, however, it was apparent that there was no overall stimulation
This
of the
stimulation
Sulimovici that cyclic
of pregnenolone and progesterone in the cyclic AMP incubations
increased accumulation probably due to A533hydroxysteroid
activity
The
pendent upon the that a phosphorylation
of
could not be authors in a
publication (Yago et al., 1967). Boyd (1968) then reported
of enzyme
enzyme.
a direct cleavage
tion percent.
type of activation and whole cells.
concentra-
NAD
side-chain
AMP both stimulated terol to pregnenolone sion of pregnenolone
using
(Sulimovici 1970; Suli-
been reported in gonalchii et al. (1963)
itself
the
has
investigators
the 5-3(3-hydroxysteroid greater than physiologic
Similar dal tissue
al., on
reactions
adrenal and ovarian preparations and Boyd, 1969; McCune et movici 1973),
et AMP
caused in
vivo
by the (Simpson
by
blocking
its
cyanoketone,
of action et
side-chain of endogal.,
1972).
CYCLIC
Similar
results incubated
using
and
cyclic
were
obtained adrenal
rat
AMP
by Farese sections,
AND
STEROIDOGENESIS
incubated
(1971) ACTU,
and
AMP.
slices
Sato
tumor
ROLE OF PROTEIN IN THE STIMULATION STEROIDOGENESIS
Concomitant
protein
shown to be required LU and cyclic AMP respective
target
on this (1974)
matter, in a
mention
some
on gonadal Hall
SYNTHESIS OF
cells,
tissues.
I will
since recent
it was review
has
of
highlights
the
not
been
go into
detail
the
puromycin stimulatory
were the inhibitors,
and chloramphenicol, effect of LU on
incubating slices have confirmed
of rabbit this effect
inhibitors,
predominantly
using
first such
to as
blocked steroidogenesis
the in
testes. Several groups of protein synthesis puromycin
and
cycloheximide, in testes (Shin and Sato, Moyle et al., 1971), corpora lutea (Savard 1965; Marsh, 1968; Hermier et al.,
1971; et a!., 1971),
Graafian follicles (Tsafrini et al., 1973) and whole ovaries (Gorski and Padnos, 1966). The site of action appears to be after the formation of
cyclic
showed
AMP that
stimulation incubating
in
that
Marsh
puromycin of slices
did
et not
block
(Marsh
and
Uermier action they the They
et
al.
Savard,
1 966a).
(1971)
pinpointed
of these inhibitors even showed that cycloheximide cholesterol incubated
side-chain slices of
the absence found that
of no
presumably the side-chain
due
could
not
indicated
02 for 90 progesterone
side-chain
by
cleavage
step
or
since
progesterone
beyond
if it did, synthesis
LI-I
is
whole
process.
There indicates stimulation
the
for the
on
the
of
uncertain.
RNA
synthesis Savard
et
in the al.
(1965),
in-
data).
decide,
at
this
time,
of action of is the correct
limiting but once other
mediates tropic or
parts
action necessary
the
ONS
body AMP
adrenal of
the
of evidence plays a role
cortex.
which in the
to
that
amounts
on steroidogenesis,
one
of the cyclic steroidogenic
cyclic
AMP
of
steroidogenic
the
to
limiting
of
normally
detect
this of
methodological
in
is possible
investigators
small
is due
AMP
It
concentration
when
used,
aspects just
become
of cyclic AMP at to accelerate the
but a final answer to this await further results. In regard to the possible
be
might
the
the effect of small concentrahormones on steroidogenesis
inability
in
nucleotide are
step before steroidogenesis
of gonadal steroidogenesis. This is true in the case of the corpus There is considerable doubt, however,
a
cyclic
hormones
deficiencies,
problem
will
modes
have
to
of action
of
there nucleotide pathway,
is evidence at several and it may
accelerates process
several rather
than
step.
have second
ACKNOWLEDGMENT I would
role
the
concerted may be
for an effect steps in the
point
it would
of
cyclic
This
the
be begins,
is a large that cyclic
change
incubation.
The
several
that
act
unpublished to
accelerate
A sites
testis
in
lutea,
is experimental data in support of of them is mutually exclusive and it
to
limiting.
the
that in
starts
it
when before
of rabbit
corpora
have shown that this stimulation by actinomycin
possible
may
that tions
of
reaction. rat ovary
not
pathway stimulation
the
site
on
hand,
that actinostimulation
slices
studies
cyclic
other
may be that LU and cyclic AMP have pleiotropic types of effects stimulating many aspects of the steroidogenic pathway. One part of the
luteum.
cycloheximide.
incubated
(Marsh,
There None
particularly
then incubated was a rapid this synthesis
did
one. each.
using actino-
LI-I and
the
of the possible mechanisms AMP on steroidogenesis
exogenous this same
with LH and was synthesized,
cycloheximide
steroidogenesis,
blocked
in but it
mm
were there and
which cyclic
that
of
CONCLUSI
LH
to the lack of 02 needed cleavage reaction steps. When
be inhibited that
the
further, acted
cleavage luteinized
slices of luteinized ovary in the presence of 02 synthesis of progesterone
on
of in
D is inconsistent
(1966)
cyclic AMP accumulation of bovine corpora lutea,
did block the stimulatory effect cyclic AMP on steroidogenesis tissue
al.
in
not
(1973),
On
experiments of the LH is
a!.
action
to the
Shin Leydig
found
(1966) reported effect on the LH
In regard
It
tissues.
the
steroidogenesis.
further hibition
lutea, mouse
et
follicles,
steroidogenesis
studies
corpora
incubated
Tsafriri
and Padnos D had no
ovary.
by Wicks will just
I
in
of
and
D blocked on
Gorski mycin
of ACTH, in their
covered but
and Eik-Nes (1962) that protein synthesis
show
mycin
synthesis
bovine
using
Graafian
AMP
for the actions on steroidogenesis
of
(1971),
isolated THE
47
action
of using
several of the
like
to
acknowledge
the
collaboration
colleagues who worked with me on the studies role of cyclic AMP in the bovine corpus luteum,
of
MARSH
48
namely Dr. Kenneth Savard, Dr. Steven Goldstein, Dr. Marc G. Caron, Dr. Kirpal S. Sidhu and Dr. William V. Ling. I would also like to thank Mrs. Nieves Cerver for the preparation of this manuscript.
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